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Sample records for acyl coa oxidase

  1. Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids.

    PubMed

    Oakes, Ken D; Sibley, Paul K; Martin, Jon W; MacLean, Dan D; Solomon, Keith R; Mabury, Scott A; Van Der Kraak, Glen J

    2005-05-01

    This study investigated the effects of exposure to waterborne perfluorooctane sulfonate (PFOS) on oxidative stress and reproductive endpoints in fish. Exposures utilized species commonly used in toxicological testing, including the fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), as well as relatively insensitive taxa such as creek chub (Semotilus atromaculatus), spottail shiner (Notropis hudsonius), and white sucker (Catostomus commersoni). In all fish species, short-term (14-28 d) exposure to PFOS produced only modest mortality at concentrations consistent with environmental spill scenarios. However, PFOS consistently increased hepatic fatty acyl-CoA oxidase activity and increased oxidative damage, as quantified using the 2-thiobarbituric acid-reactive substances assay. Plasma testosterone, 11-ketotestosterone, and 17beta-estradiol titers were often elevated with PFOS exposure. Vitellogenin, the egg yolk precursor protein, was occasionally altered in the plasma with PFOS exposure, but responses varied with maturity. Oviposition frequency and egg deposition in fathead minnow were not significantly impaired with PFOS exposure, despite a trend toward progressive impairment with increasing exposure concentrations. Although short-term PFOS exposure produced significant impacts on biochemical and reproductive endpoints in fish at concentrations consistent with environmental spills, the impact of long-term exposure to environmentally relevant concentrations of PFOS is unclear.

  2. Bitter gourd (Momordica charantia) extract activates peroxisome proliferator-activated receptors and upregulates the expression of the acyl CoA oxidase gene in H4IIEC3 hepatoma cells.

    PubMed

    Chao, Che-Yi; Huang, Ching-jang

    2003-01-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-dependent transcription factor that regulates the expression of genes involved in lipid metabolism and transport. Ligands/activators of PPARalpha, like fibrate-type drugs, may have hypolipidemic effects. To identify food that contains activators of PPARalpha, a transactivation assay employing a clone of CHO-K1 cells stably transfected with a (UAS)(4)-tk-alkaline phosphatase reporter and a chimeric receptor of Gal4-rPPARalpha LBD was used to screen ethyl acetate (EA) extracts of a large variety of food materials. It was found that the EA extract of bitter gourd (Momordica charantia), a common oriental vegetable, activated PPARalpha to an extent that was equivalent to or even higher than 10 microM Wy-14643, a known ligand of PPARalpha. This extract also activated PPARgamma to a significant extent which was comparable to 0.5 microM BRL-49653. The activity toward PPARalpha was mainly in the soluble fraction of the organic solvent. The EA extract prepared from the whole fruit showed significantly higher activity than that from seeds or flesh alone. The bitter gourd EA extract was then incorporated into the medium for treatment of a peroxisome proliferator-responsive murine hepatoma cell line, H4IIEC3, for 72 h. Treated cells showed significantly higher activity of acyl CoA oxidase and higher expressions of mRNA of this enzyme and fatty acid-binding protein, indicating that the bitter gourd EA extract was able to act on a natural PPARalpha signaling pathway in this cell line. It is thus worth further investigating the PPAR-associated health benefits of bitter gourd.

  3. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    PubMed

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

  4. Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy.

    PubMed

    Baertling, Fabian; A M van den Brand, Mariel; Hertecant, Jozef L; Al-Shamsi, Aisha; P van den Heuvel, Lambert; Distelmaier, Felix; Mayatepek, Ertan; Smeitink, Jan A; Nijtmans, Leo G J; Rodenburg, Richard J T

    2015-01-01

    COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.

  5. Purification and Characterization of a Novel Pumpkin Short-Chain Acyl-Coenzyme A Oxidase with Structural Similarity to Acyl-Coenzyme A Dehydrogenases

    PubMed Central

    De Bellis, Luigi; Gonzali, Silvia; Alpi, Amedeo; Hayashi, Hiroshi; Hayashi, Makoto; Nishimura, Mikio

    2000-01-01

    A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs. PMID:10806249

  6. Acyl CoA Binding Proteins are Required for Cuticle Formation and Plant Responses to Microbes.

    PubMed

    Xia, Ye; Yu, Keshun; Gao, Qing-Ming; Wilson, Ella V; Navarre, Duroy; Kachroo, Pradeep; Kachroo, Aardra

    2012-01-01

    Fatty acids (FA) and lipids are well known regulators of plant defense. Our previous studies have shown that components of prokaryotic (plastidal) FA biosynthesis pathway regulate various aspects of plant defense. Here, we investigated the defense related roles of the soluble acyl CoA binding proteins (ACBPs), which are thought to facilitate the intracellular transport of FA/lipids. We show that ACBP3 and 4 are required for maintaining normal lipid levels and that ACBP3 contributes to the lipid flux between the prokaryotic and eukaryotic pathways. We also show that loss of ACBP3, 4, or 6 impair normal development of the cuticle and affect both basal and resistance protein-mediated defense against bacterial and fungal pathogens. Loss of ACBP3, 4, or 6 also inhibits the induction of systemic acquired resistance (SAR) due to the plants inability to generate SAR inducing signal(s). Together, these data show that ACBP3, ACBP4, and ACBP6 are required for cuticle development as well as defense against microbial pathogens.

  7. Cooperation between COA6 and SCO2 in COX2 maturation during cytochrome c oxidase assembly links two mitochondrial cardiomyopathies.

    PubMed

    Pacheu-Grau, David; Bareth, Bettina; Dudek, Jan; Juris, Lisa; Vögtle, F-Nora; Wissel, Mirjam; Leary, Scot C; Dennerlein, Sven; Rehling, Peter; Deckers, Markus

    2015-06-02

    Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency.

  8. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

  9. COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency

    PubMed Central

    Martinez Lyons, Anabel; Ardissone, Anna; Reyes, Aurelio; Robinson, Alan J; Moroni, Isabella; Fernandez-Vizarra, Erika; Zeviani, Massimo

    2016-01-01

    Background Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. Methods Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. Results We found compound heterozygous mutations in COA7: a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. Conclusions We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested. PMID:27683825

  10. Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily

    PubMed Central

    Daubner, S. Colette; Gadda, Giovanni; Valley, Michael P.; Fitzpatrick, Paul F.

    2002-01-01

    The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes. PMID:11867731

  11. Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis.

    PubMed

    Swarbrick, Crystall M D; Bythrow, Glennon V; Aragao, David; Germain, Gabrielle A; Quadri, Luis E N; Forwood, Jade K

    2017-03-14

    Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

  12. Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans.

    PubMed

    Zhang, Xinxing; Feng, Likui; Chinta, Satya; Singh, Prashant; Wang, Yuting; Nunnery, Joshawna K; Butcher, Rebecca A

    2015-03-31

    Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions.

  13. Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans

    PubMed Central

    Zhang, Xinxing; Feng, Likui; Chinta, Satya; Singh, Prashant; Wang, Yuting; Nunnery, Joshawna K.; Butcher, Rebecca A.

    2015-01-01

    Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions. PMID:25775534

  14. Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

    PubMed Central

    Schürmann, Marc; Demming, Rebecca Michaela; Krewing, Marco; Rose, Judith; Wübbeler, Jan Hendrik

    2014-01-01

    In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7T (AcdDPN7) during degradation of 3,3′-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). AcdDPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. AcdS110, AcdB4, AcdH16, and AcdKT2440 were able to dehydrogenate isobutyryl-CoA. AcdKT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas AcdDSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only AcdTBEA6, AcdN-1, and AcdLB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for AcdBL21 and AcdWT001. Spectrophotometric assays provided apparent Km and Vmax values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of

  15. Identification of 3-sulfinopropionyl coenzyme A (CoA) desulfinases within the Acyl-CoA dehydrogenase superfamily.

    PubMed

    Schürmann, Marc; Demming, Rebecca Michaela; Krewing, Marco; Rose, Judith; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2014-02-01

    In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7(T) (AcdDPN7) during degradation of 3,3'-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). AcdDPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. AcdS110, AcdB4, AcdH16, and AcdKT2440 were able to dehydrogenate isobutyryl-CoA. AcdKT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas AcdDSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only AcdTBEA6, AcdN-1, and AcdLB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for AcdBL21 and AcdWT001. Spectrophotometric assays provided apparent Km and Vmax values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of

  16. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain...

  17. Copper supplementation restores cytochrome c oxidase assembly defect in a mitochondrial disease model of COA6 deficiency

    PubMed Central

    Ghosh, Alok; Trivedi, Prachi P.; Timbalia, Shrishiv A.; Griffin, Aaron T.; Rahn, Jennifer J.; Chan, Sherine S. L.; Gohil, Vishal M.

    2014-01-01

    Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx9CxnCx10C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx9CxnCx10C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient. PMID:24549041

  18. Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester.

    PubMed

    Ikegawa, S; Goto, T; Mano, N; Goto, J

    1998-11-01

    The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

  19. Effect of various eicosanoid products of arachidonic acid on the acyl CoA: Cholesterol acyl transferase activity in three different mammalian cell lines

    SciTech Connect

    Malo, P.El.

    1988-01-01

    Acylcoenzyme A:cholesterol acyltransferase (ACAT) catalyzes cholesterol ester synthesis intracellularly and has been implicated in the development of atherosclerosis. An in vitro assay has been adapted for determining ACAT activity from rat FU5AH hepatoma, Chinese hamster ovary (CHO) and rat thoracic aortic smooth muscle (RSM) cells. Formation of {sup 14}C-labelled cholesteryl oleate at 0 to 60 min {plus minus} cholesterol was determined; in the presence of exogenous cholesterol, ACAT activity was approximately linear and surpassed the plateau observed in ACAT activity without cholesterol. Increasing exogenous cholesterol concentration, the amount of oleoyl CoA or the amount of microsomal protein produced a corresponding increase in ACAT activity, while ester formation was slightly increased by decreasing the ratio of Triton WR-1339 to cholesterol. Both the thromboxane A{sub 2} (TxA{sub 2}) mimic, U-44069, and the inflammatory lipoxygenase product, LTB{sub 4}, decreased optimal in vitro microsomal ACAT activity from RSM, but not form FU5AH, while CHO ACAT activity was suppressed by LTB{sub r} only. PGI{sub 2}, PGE{sub 2} and PGF{sub 2{alpha}} had minimal effects for each cell type.

  20. The Inflammatory Response in Acyl-CoA Oxidase 1 Deficiency (Pseudoneonatal Adrenoleukodystrophy)

    PubMed Central

    El Hajj, H. I.; Vluggens, A.; Andreoletti, P.; Ragot, K.; Mandard, S.; Kersten, S.; Waterham, H. R.; Lizard, G.; Wanders, R. J. A.; Reddy, J. K.

    2012-01-01

    Among several peroxisomal neurodegenerative disorders, the pseudoneonatal adrenoleukodystrophy (P-NALD) is characterized by the acyl-coenzyme A oxidase 1 (ACOX1) deficiency, which leads to the accumulation of very-long-chain fatty acids (VLCFA) and inflammatory demyelination. However, the components of this inflammatory process in P-NALD remain elusive. In this study, we used transcriptomic profiling and PCR array analyses to explore inflammatory gene expression in patient fibroblasts. Our results show the activation of IL-1 inflammatory pathway accompanied by the increased secretion of two IL-1 target genes, IL-6 and IL-8 cytokines. Human fibroblasts exposed to very-long-chain fatty acids exhibited increased mRNA expression of IL-1α and IL-1β cytokines. Furthermore, expression of IL-6 and IL-8 cytokines in patient fibroblasts was down-regulated by MAPK, p38MAPK, and Jun N-terminal kinase inhibitors. Thus, the absence of acyl-coenzyme A oxidase 1 activity in P-NALD fibroblasts triggers an inflammatory process, in which the IL-1 pathway seems to be central. The use of specific kinase inhibitors may permit the modulation of the enhanced inflammatory status. PMID:22508517

  1. Sudden unexpected infant death (SUDI) in a newborn due to medium chain acyl CoA dehydrogenase (MCAD) deficiency with an unusual severe genotype

    PubMed Central

    2012-01-01

    Medium chain acyl CoA dehydrogenase deficiency (MCAD) is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23) mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI) and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death. PMID:23095120

  2. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    PubMed Central

    Bowman, Thomas A.; O'Keeffe, Kayleigh R.; D'Aquila, Theresa; Yan, Qing Wu; Griffin, John D.; Killion, Elizabeth A.; Salter, Deanna M.; Mashek, Douglas G.; Buhman, Kimberly K.; Greenberg, Andrew S.

    2016-01-01

    Objective The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform. Methods To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5−/−). Results Ablation of ACSL5 reduced ACSL activity by ∼80% in jejunal mucosa, ∼50% in liver, and ∼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5−/−, as compared to control ACSL5loxP/loxP, mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5−/− had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5−/− mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (∼16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (∼13-fold) were increased in ACSL5−/− as compared to ACSL5loxP/loxP. Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5−/− mice as compared to control mice. Conclusions In summary, ACSL5−/− mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies

  3. Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica.

    PubMed

    Mlícková, Katerina; Roux, Emeline; Athenstaedt, Karin; d'Andrea, Sabine; Daum, Günther; Chardot, Thierry; Nicaud, Jean-Marc

    2004-07-01

    Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.

  4. ACX3, a Novel Medium-Chain Acyl-Coenzyme A Oxidase from Arabidopsis

    PubMed Central

    Froman, Byron E.; Edwards, Patricia C.; Bursch, Adam G.; Dehesh, Katayoon

    2000-01-01

    In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0- to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene. PMID:10859203

  5. Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

    PubMed Central

    Waché, Yves; Laroche, Céline; Bergmark, Karin; Møller-Andersen, Charlotte; Aguedo, Mario; Le Dall, Marie-Thérèse; Wang, Huijie; Nicaud, Jean-Marc; Belin, Jean-Marc

    2000-01-01

    We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation. PMID:10698800

  6. Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica.

    PubMed

    Waché, Y; Laroche, C; Bergmark, K; Møller-Andersen, C; Aguedo, M; Le Dall, M T; Wang, H; Nicaud, J M; Belin, J M

    2000-03-01

    We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140-5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h. The Deltapox2 Deltapox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.

  7. Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans.

    PubMed

    Zhang, Xinxing; Li, Kunhua; Jones, Rachel A; Bruner, Steven D; Butcher, Rebecca A

    2016-09-06

    Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state.

  8. Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

    PubMed Central

    Zhang, Xinxing; Jones, Rachel A.; Bruner, Steven D.; Butcher, Rebecca A.

    2016-01-01

    Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state. PMID:27551084

  9. Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

    PubMed Central

    Wadum, Majken C T; Villadsen, Jens K; Feddersen, Søren; Møller, Rikke S; Neergaard, Thomas B F; Kragelund, Birthe B; Højrup, Peter; Faergeman, Nils J; Knudsen, Jens

    2002-01-01

    Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters. PMID:12071849

  10. Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis.

    PubMed

    Lü, Shiyou; Song, Tao; Kosma, Dylan K; Parsons, Eugene P; Rowland, Owen; Jenks, Matthew A

    2009-08-01

    Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

  11. Unraveling Cholesterol Catabolism in Mycobacterium tuberculosis: ChsE4-ChsE5 α2β2 Acyl-CoA Dehydrogenase Initiates β-Oxidation of 3-Oxo-cholest-4-en-26-oyl CoA

    PubMed Central

    2016-01-01

    The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first

  12. Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

    PubMed

    Nchoutmboube, Jules A; Viktorova, Ekaterina G; Scott, Alison J; Ford, Lauren A; Pei, Zhengtong; Watkins, Paul A; Ernst, Robert K; Belov, George A

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

  13. A Rare Case of Short-Chain Acyl-COA Dehydrogenase Deficiency: The Apparent Rarity of the Disorder Results in Under Diagnosis.

    PubMed

    Reddy, G Shilpa; Sujatha, M

    2011-07-01

    Short-chain acyl-CoA dehydrogenase (ACAD) deficiency is an extremely rare inherited mitochondrial disorder of fat metabolism. This belongs to a group of diseases known as fatty acid oxidation disorders. Screening programmes have provided evidence that all the fatty acid oxidation disorders combined are among the most common inborn errors of metabolism. Mitochondrial beta oxidation of fatty acids is an essential energy producing pathway. It is a particularly important pathway during prolonged periods of starvation and during periods of reduced caloric intake due to gastrointestinal illness or increased energy expenditure during febrile illness. The most common presentation is an acute episode of life threatening coma and hypoglycemia induced by a period of fasting due to defective hepatic ketogenesis. Here, the case of a 4 month old female patient who had seizures since the third day of her birth and persistent hypoglycemia is described. She was born to parents of second degree consanguinity after 10 years of infertility treatment. There was history of delayed cry after birth. Metabolic screening for TSH, galactosemia, 17-OHP, G6PD, cystic fibrosis, biotinidase were normal. Tandem mass spectrometric (TMS) screening for blood amino acids, organic acids, fatty acids showed elevated butyryl carnitine (C4) as 3.40 μmol/L (normal <2.00 μmol/L), hexanoyl carnitine (C6) as 0.92 μmol/L (normal <0.72 μmol/L), C4/C3 as 2.93 μmol/L (normal <1.18 μmol/L). The child was started immediately on carnitor syrup (carnitine) 1/2 ml twice daily. Limitation of fasting stress and dietary fat was advised. Baby responded well by gaining weight and seizures were controlled. Until now, less than 25 patients have been reported worldwide. The limited number of patients diagnosed until now is due to the rarity of the disorder resulting in under diagnosis.

  14. COAs: Behind the Masks.

    ERIC Educational Resources Information Center

    Birke, Szifra

    1993-01-01

    Provides information on alcoholism and codependency to help teachers identify and respond to children of alcoholics (COAs). Discusses characteristics of alcoholic homes and problems encountered by children and adult COAs. Examines survival "masks" of COAs, including hero, rebel, adjustor, clown, and caretaker. Lists organizational,…

  15. Differential induction of stearoyl-CoA desaturase and acyl-CoA oxidase genes by fibrates in HepG2 cells.

    PubMed

    Rodríguez, C; Cabrero, A; Roglans, N; Adzet, T; Sánchez, R M; Vázquez, M; Ciudad, C J; Laguna, J C

    2001-02-01

    We studied whether two typical effects of fibrates, induction of stearoyl-CoA desaturase (EC 1.14.99.5) and peroxisome proliferation, are related. The effect of bezafibrate on the activity and mRNA of stearoyl-CoA desaturase and acyl-CoA oxidase in the liver and epididymal white adipose tissue of male Sprague-Dawley rats was determined. The same parameters were measured in HepG2 cells, a cell line resistant to peroxisome proliferation, following incubation with ciprofibrate. Bezafibrate increased the hepatic mRNA levels (14.5-fold on day 7) and activity (9.3-fold on day 15) of acyl-CoA oxidase. Stearoyl-CoA desaturase mRNA levels were transiently increased (2.7-fold on day 7), while its activity remained increased at the end of the treatment (2.4-fold). In white adipose tissue, bezafibrate increased the mRNA (5-fold) and activity (1.9-fold) of acyl-CoA oxidase, while stearoyl-CoA desaturase was not modified. Ciprofibrate addition to HepG2 cells cultured in 7% fetal bovine serum (FBS) only increased the stearoyl-CoA desaturase mRNA (1.9-fold). When cells were cultured in 0.5% FBS, ciprofibrate increased acyl-CoA oxidase mRNA (2.2-fold), while the increase in stearoyl-CoA desaturase mRNA was identical (1.9-fold). Further, its activity was also increased (1.5-fold). Incubation of HepG2 cells in the presence of cycloheximide did not alter the capacity of ciprofibrate to induce stearoyl-CoA desaturase mRNA, whereas the presence of actinomycin abolished the induction. In addition, preincubation of HepG2 cells with ciprofibrate increased the rate of stearoyl-CoA desaturase mRNA degradation. The results presented in this study suggest that fibrates induce stearoyl-CoA desaturase activity and mRNA levels independently of peroxisome proliferation.

  16. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  17. Down-regulation of acyl-CoA oxidase gene expression and increased NF-kappaB activity in etomoxir-induced cardiac hypertrophy.

    PubMed

    Cabrero, Agatha; Merlos, Manuel; Laguna, Juan C; Carrera, Manuel Vázquez

    2003-02-01

    Activation of nuclear factor-kappaB (NF-kappaB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor alpha (PPARalpha) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal beta-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-kappaB was increased in heart after 10 days of etomoxir treatment. Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-kappaB activation.

  18. Synthesis of coenzyme A thioesters using methyl acyl phosphates in an aqueous medium.

    PubMed

    Pal, Mohan; Bearne, Stephen L

    2014-12-28

    Regioselective S-acylation of coenzyme A (CoA) is achieved under aqueous conditions using various aliphatic and aromatic carboxylic acids activated as their methyl acyl phosphate monoesters. Unlike many hydrophobic activating groups, the anionic methyl acyl phosphate mixed anhydride is more compatible with aqueous solvents, making it useful for conducting acylation reactions in an aqueous medium.

  19. Bioconversion of α-linolenic acid to n-3 LCPUFA and expression of PPAR-alpha, acyl Coenzyme A oxidase 1 and carnitine acyl transferase I are incremented after feeding rats with α-linolenic acid-rich oils.

    PubMed

    González-Mañán, Daniel; Tapia, Gladys; Gormaz, Juan Guillermo; D'Espessailles, Amanda; Espinosa, Alejandra; Masson, Lilia; Varela, Patricia; Valenzuela, Alfonso; Valenzuela, Rodrigo

    2012-07-01

    High dietary intake of n-6 fatty acids in relation to n-3 fatty acids may generate health disorders, such as cardiovascular and other chronic diseases. Fish consumption rich in n-3 fatty acids is low in Latin America, it being necessary to seek other alternatives to provide α-linolenic acid (ALA), precursor of n-3 LCPUFA (EPA and DHA). Two innovative oils were assayed, chia (Salvia hispanica) and rosa mosqueta (Rosa rubiginosa). This study evaluated hepatic bioconversion of ALA to EPA and DHA, expression of PPAR-α, acyl-Coenzyme A oxidase 1 (ACOX1) and carnitine acyltransferase I (CAT-I), and accumulation of EPA and DHA in plasma and adipose tissue in Sprague-Dawley rats. Three experimental groups were fed 21 days: sunflower oil (SFO, control); chia oil (CO); rosa mosqueta oil (RMO). Fatty acid composition of total lipids and phospholipids from plasma, hepatic and adipose tissue was assessed by gas-liquid chromatography and TLC. Expression of PPAR-α (RT-PCR) and ACOX1 and CAT-I (Western blot). CO and RMO increased plasma, hepatic and adipose tissue levels of ALA, EPA and DHA and decreased n-6:n-3 ratio compared to SFO (p < 0.05, One-way ANOVA and Newman-Keuls test). CO increased levels of ALA and EPA compared to RMO (p < 0.05). No significant differences were observed for DHA levels. CO also increased the expression of PPAR-α, ACOX1 and CAT-I. Only CAT-I levels were increased by RO. CO and RMO may be a nutritional alternative to provide ALA for its bioconversion to EPA and DHA, and to increase the expression of PPAR-α, ACOX1 and CAT-I, especially CO-oil.

  20. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    SciTech Connect

    Nemazanyy, Ivan . E-mail: nemazanyy@imbg.org.ua; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T. . E-mail: i.gout@ucl.ac.uk

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation.

  1. Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

    ERIC Educational Resources Information Center

    Thomas, Jacob

    2009-01-01

    Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

  2. Mitochondrial disease genes COA6, COX6B and SCO2 have overlapping roles in COX2 biogenesis

    PubMed Central

    Ghosh, Alok; Pratt, Anthony T.; Soma, Shivatheja; Theriault, Sarah G.; Griffin, Aaron T.; Trivedi, Prachi P.; Gohil, Vishal M.

    2016-01-01

    Biogenesis of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, is a complex process facilitated by several assembly factors. Pathogenic mutations were recently reported in one such assembly factor, COA6, and our previous work linked Coa6 function to mitochondrial copper metabolism and expression of Cox2, a copper-containing subunit of CcO. However, the precise role of Coa6 in Cox2 biogenesis remained unknown. Here we show that yeast Coa6 is an orthologue of human COA6, and like Cox2, is regulated by copper availability, further implicating it in copper delivery to Cox2. In order to place Coa6 in the Cox2 copper delivery pathway, we performed a comprehensive genetic epistasis analysis in the yeast Saccharomyces cerevisiae and found that simultaneous deletion of Coa6 and Sco2, a mitochondrial copper metallochaperone, or Coa6 and Cox12/COX6B, a structural subunit of CcO, completely abrogates Cox2 biogenesis. Unlike Coa6 deficient cells, copper supplementation fails to rescue Cox2 levels of these double mutants. Overexpression of Cox12 or Sco proteins partially rescues the coa6Δ phenotype, suggesting their overlapping but non-redundant roles in copper delivery to Cox2. These genetic data are strongly corroborated by biochemical studies demonstrating physical interactions between Coa6, Cox2, Cox12 and Sco proteins. Furthermore, we show that patient mutations in Coa6 disrupt Coa6–Cox2 interaction, providing the biochemical basis for disease pathogenesis. Taken together, these results place COA6 in the copper delivery pathway to CcO and, surprisingly, link it to a previously unidentified function of CcO subunit Cox12 in Cox2 biogenesis. PMID:26669719

  3. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  4. A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity.

    PubMed

    McFie, Pamela J; Stone, Scot J

    2011-09-01

    Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.

  5. Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases.

    PubMed

    Zhang, Wei; Shrestha, Ruben; Buckley, Rachael M; Jewell, Jamie; Bossmann, Stefan H; Stubbe, JoAnne; Li, Ping

    2014-08-15

    Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH2-CoA), saturated dimer-([(3)H]-sD-CO2H), and trimer-acid ([(3)H]-sT-CO2H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH2-CoA). Detection of [(3)H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.

  6. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

    PubMed

    Peter, Dominik M; Vögeli, Bastian; Cortina, Niña Socorro; Erb, Tobias J

    2016-04-20

    Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.

  7. Specific Inhibition of Acyl-CoA Oxidase-1 by an Acetylenic Acid Improves Hepatic Lipid and Reactive Oxygen Species (ROS) Metabolism in Rats Fed a High Fat Diet.

    PubMed

    Zeng, Jia; Deng, Senwen; Wang, Yiping; Li, Ping; Tang, Lian; Pang, Yefeng

    2017-03-03

    A chronic high fat diet results in hepatic mitochondrial dysfunction and induction of peroxisomal fatty acid oxidation (FAO); whether specific inhibition of peroxisomal FAO benefits mitochondrial FAO and reactive oxygen species (ROS) metabolism remains unclear. In this study a specific inhibitor for the rate-limiting enzyme involved in peroxisomal FAO, acyl-CoA oxidase-1 (ACOX1) was developed and used for the investigation of peroxisomal FAO inhibition upon mitochondrial FAO and ROS metabolism. Specific inhibition of ACOX1 by 10,12-tricosadiynoic acid increased hepatic mitochondrial FAO via activation of the SIRT1-AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway and proliferator activator receptor α and reduced hydrogen peroxide accumulation in high fat diet-fed rats, which significantly decreased hepatic lipid and ROS contents, reduced body weight gain, and decreased serum triglyceride and insulin levels. Inhibition of ACOX1 is a novel and effective approach for the treatment of high fat diet- or obesity-induced metabolic diseases by improving mitochondrial lipid and ROS metabolism.

  8. Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

    PubMed Central

    Garland, P. B.; Yates, D. W.; Haddock, B. A.

    1970-01-01

    1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do

  9. Metabolism of ricinoleic acid into gamma-decalactone: beta-oxidation and long chain acyl intermediates of ricinoleic acid in the genus Sporidiobolus sp.

    PubMed

    Blin-Perrin, C; Molle, D; Dufosse, L; Le-Quere, J L; Viel, C; Mauvais, G; Feron, G

    2000-07-01

    In order to study differences in gamma-decalactone production in yeast, four species of Sporidiobolus were cultivated with 5% of methyl ricinoleate as the lactone substrate. In vivo studies showed different time courses of intermediates of ricinoleic acid breakdown between the four species. In vitro studies of the beta-oxidation system were conducted with crude cell extracts of Sporidiobolus spp. and with ricinoleyl-CoA (RCoA) as substrate. The beta-oxidation was detected by measuring acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase activities, and acetyl-CoA production. The time courses of the CoA esters resulting from RCoA breakdown by crude extract of Sporidiobolus spp. permit the proposal of different metabolic models in the yeast. These models explained the differences observed during in vivo studies.

  10. Fatty acid acylation of rat brain myelin proteolipid protein in vitro: identification of the lipid donor.

    PubMed

    Bizzozero, O A; Lees, M B

    1986-02-01

    The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.

  11. The Role of Coa2 in Hemylation of Yeast Cox1 Revealed by Its Genetic Interaction with Cox10 ▿

    PubMed Central

    Bestwick, Megan; Khalimonchuk, Oleh; Pierrel, Fabien; Winge, Dennis R.

    2010-01-01

    Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2Δ cells is suppressed either by the presence of a mutant allele of the Cox10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. Cox10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2Δ cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1Δ and shy1Δ mutants. The suppressor activity of N196K mutant Cox10 is dependent on its catalytic function and the presence of Cox15, the second enzyme involved in heme a biosynthesis. Varying the substitution at Asn196 reveals a correlation between the suppressor activity and the stabilization of the high-mass homo-oligomeric Cox10 complex. We postulate that the mutant Cox10 complex has enhanced efficiency in the addition of heme a to Cox1. Coa2 appears to impart stability to the oligomeric wild-type Cox10 complex involved in Cox1 hemylation. PMID:19841065

  12. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  13. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  14. Fatty acyl-CoA elongation in Blatella germanica integumental microsomes.

    PubMed

    Juárez, M Patricia

    2004-08-01

    Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 microg/microl. The effect of the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 microM for C16-CoA, between 8 and 12 microM for C18-CoA, and close to 3 microM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.

  15. Insulin Signaling Regulates Fatty Acid Catabolism at the Level of CoA Activation

    PubMed Central

    Xu, Xiaojun; Gopalacharyulu, Peddinti; Seppänen-Laakso, Tuulikki; Ruskeepää, Anna-Liisa; Aye, Cho Cho; Carson, Brian P.; Mora, Silvia; Orešič, Matej; Teleman, Aurelio A.

    2012-01-01

    The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG) catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS). We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis. PMID:22275878

  16. Lutein prevents high fat diet-induced atherosclerosis in ApoE-deficient mice by inhibiting NADPH oxidase and increasing PPAR expression.

    PubMed

    Han, Hao; Cui, Wei; Wang, Linzhi; Xiong, Yufang; Liu, Liegang; Sun, Xiufa; Hao, Liping

    2015-03-01

    Epidemiological and experimental studies provide supportive evidence that lutein, a major carotenoid, may act as a chemopreventive agent against atherosclerosis, although the underlying molecular mechanisms are not well understood. The main aim of this study was to investigate the effects of lutein on the alleviation of atherosclerosis and its molecular mechanisms involved in oxidative stress and lipid metabolism. Male apolipoprotein E knockout mice (n = 55) were fed either a normal chow diet or a high fat diet (HFD) supplemented with or without lutein for 24 weeks. The results showed that a HFD induced atherosclerosis formation, lipid metabolism disorders and oxidative stress, but noticeable improvements were observed in the lutein treated group. Additionally, lutein supplementation reversed the decreased protein expression of aortic heme oxygenase-1 and increased the mRNA and protein expressions of aortic nicotinamide-adenine dinucleotide phosphate oxidase stimulated by a HFD. Furthermore, the decreased mRNA and protein expression levels of hepatic peroxisome proliferator-activated receptor-α, carnitine palmitoyltransferase 1A, acyl CoA oxidase 1, low density lipoprotein receptors and scavenger receptor class B type I observed in mice with atherosclerosis were markedly enhanced after treatment with lutein. Taken together, these data add new evidence supporting the anti-atherogenic properties of lutein and describing its mechanisms of action in atherosclerosis prevention, including oxidative stress and lipid metabolism improvements.

  17. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  18. Oxidative acylation using thioacids

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.

    1997-01-01

    Several important prebiotic reactions, including the coupling of amino acids into polypeptides by the formation of amide linkages, involve acylation. Theae reactions present a challenge to the understanding of prebiotic synthesis. Condensation reactions relying on dehydrating agents are either inefficient in aqueous solution or require strongly acidic conditions and high temperatures. Activated amino acids such as thioester derivatives have therefore been suggested as likely substrates for prebiotic peptide synthesis. Here we propose a closely related route to amide bond formation involving oxidative acylation by thioacids. We find that phenylalanine, leucine and phenylphosphate are acylated efficiently in aqueous solution by thioacetic acid and an oxidizing agent. From a prebiotic point of view, oxidative acylation has the advantage of proceeding efficiently in solution and under mild conditions. We anticipate that oxidative acylation should prove to be a general method for activating carboxylic acids, including amino acids.

  19. Measuring long-chain acyl-coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring.

    PubMed

    Blachnio-Zabielska, Agnieszka U; Koutsari, Christina; Jensen, Michael D

    2011-08-15

    Long-chain acyl-coenzymes A (acyl-CoAs) (LCACoA) are the activated forms of long-chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-(13) C]16-CoA) and oleoyl-CoA ([U-(13) C]18:1-CoA) using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl-CoA ([U(-13)C]16-CoA) and oleoyl-CoA ([U(-13)C]18:1-CoA) using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H](+) and [U(13)C]16-CoA and [U(13)C]18:1-CoA were monitored as [M+16+H](+) and [M+18+H](+), respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U(13) C]16-CoA and [U(13) C]18:1-CoA during a low dose intravenous infusion of [U(13

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

    PubMed Central

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

    2016-01-01

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

  1. Structural characterization of CalO2: A putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic

    SciTech Connect

    McCoy, Jason G.; Johnson, Heather D.; Singh, Shanteri; Bingman, Craig A.; Lei, In-Kyoung; Thorson, Jon S.; Phillips, Jr., George N.

    2009-08-13

    Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.

  2. Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

    SciTech Connect

    Willis, Mark A.; Zhuang, Zhihao; Song, Feng; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat

    2008-04-02

    The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.

  3. Fatty acyl-CoA inhibition of beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

    PubMed

    Faas, F H; Carter, W J; Wynn, J O

    1978-11-22

    The influence of the fatty acyl-CoA thioesters on rat liver microsomal hydroxymethylglutaryl-CoA reductase activity was tested in vitro to determine if the previously demonstrated inhibition of [14C]acetate incorporation into cholesterol is due to inhibition of this rate limiting step in cholesterol synthesis. The polyunsaturated fatty acyl-CoA thioesters caused the greatest inhibition of enzyme activity, 50 micron arachidonoyl-CoA inhibiting 67% and 5 micron inhibiting 22%. 50 micron linoleoyl-CoA inhibited 56% with the more saturated thioesters causing less inhibition. 50--100 micron free fatty acids, free CoA, cholesterol esters, phospholipids, carnitine derivatives, prostaglandins and non-specific detergents caused little or no inhibition of enzyme activity. Kinetic studies revealed the inhibition to be noncompetitive with respect to hydroxymethylglutaryl-CoA with a Ki for arachidonoyl CoA of 3.10 micron. Fatty acyl-CoA inhibition of in vitro cholesterol synthesis is due to inhibition of hydroxymethylglutaryl-CoA reductase activity. Variation in intracellular concentrations of fatty acyl-CoA thioesters may signficantly alter cholesterol synthesis.

  4. Ralstonia solanacearum RSp0194 Encodes a Novel 3-Keto-Acyl Carrier Protein Synthase III.

    PubMed

    Mao, Ya-Hui; Ma, Jin-Cheng; Li, Feng; Hu, Zhe; Wang, Hai-Hong

    2015-01-01

    Fatty acid synthesis (FAS), a primary metabolic pathway, is essential for survival of bacteria. Ralstonia solanacearum, a β-proteobacteria member, causes a bacterial wilt affecting more than 200 plant species, including many economically important plants. However, thus far, the fatty acid biosynthesis pathway of R. solanacearum has not been well studied. In this study, we characterized two forms of 3-keto-ACP synthase III, RsFabH and RsFabW, in R. solanacearum. RsFabH, the homologue of Escherichia coli FabH, encoded by the chromosomal RSc1050 gene, catalyzes the condensation of acetyl-CoA with malonyl-ACP in the initiation steps of fatty acid biosynthesis in vitro. The RsfabH mutant lost de novo fatty acid synthetic ability, and grows in medium containing free fatty acids. RsFabW, a homologue of Pseudomonas aeruginosa PA3286, encoded by a megaplasmid gene, RSp0194, condenses acyl-CoA (C2-CoA to C10-CoA) with malonyl-ACP to produce 3-keto-acyl-ACP in vitro. Although the RsfabW mutant was viable, RsfabW was responsible for RsfabH mutant growth on medium containing free fatty acids. Our results also showed that RsFabW could condense acyl-ACP (C4-ACP to C8-ACP) with malonyl-ACP, to produce 3-keto-acyl-ACP in vitro, which implies that RsFabW plays a special role in fatty acid synthesis of R. solanacearum. All of these data confirm that R. solanacearum not only utilizes acetyl-CoA, but also, utilizes medium-chain acyl-CoAs or acyl-ACPs as primers to initiate fatty acid synthesis.

  5. Global Hawk Pacific (GloPac) COA and Mission Coordination

    NASA Technical Reports Server (NTRS)

    Dillon, Mark; Hall, Philip

    2010-01-01

    This slide presentation reviews the science objectives of the Global Hawk unmanned aircraft system (UAS) in the Pacific region, shows examp le flight tracks, the satellite under-flight requirement, the flight planning, and the agencies coordination of the airspace required for the Certificate of Authorization (COA).

  6. Structural Basis for Substrate Fatty Acyl Chain Specificity: Crystal Structure of Human Very-Long-Chain Acyl-CoA Dehydrogenase

    SciTech Connect

    McAndrew, Ryan P.; Wang, Yudong; Mohsen, Al-Walid; He, Miao; Vockley, Jerry; Kim, Jung-Ja P.

    2008-08-26

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-{angstrom} resolution. The overall fold of the N-terminal {approx}400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an {alpha}-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12{angstrom} and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial {beta}-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.

  7. Acyl-Lipid Metabolism

    PubMed Central

    Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

    2010-01-01

    Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:22303259

  8. Acyl-Lipid Metabolism

    PubMed Central

    Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

    2013-01-01

    Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:23505340

  9. Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds.

    PubMed

    Banaś, Walentyna; Sanchez Garcia, Alicia; Banaś, Antoni; Stymne, Sten

    2013-06-01

    The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.

  10. Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation

    PubMed Central

    Xue, Yan; Xiao, Shi; Kim, Juyoung; Lung, Shiu-Cheung; Chen, Liang; Tanner, Julian A.; Suh, Mi Chung; Chye, Mee-Len

    2014-01-01

    The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)–flame ionization detector (FID) and GC–mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs. PMID:25053648

  11. Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation.

    PubMed

    Xue, Yan; Xiao, Shi; Kim, Juyoung; Lung, Shiu-Cheung; Chen, Liang; Tanner, Julian A; Suh, Mi Chung; Chye, Mee-Len

    2014-10-01

    The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)-flame ionization detector (FID) and GC-mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs.

  12. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    NASA Astrophysics Data System (ADS)

    Desai, P.; Ashokaan, N.; Masud, J.; Pariti, A.; Nath, M.

    2015-03-01

    This article provides a comprehensive guide on the synthesis and characterization of superparamagnetic CoAs nanoparticles and elongated nanostructures with high blocking temperature, (TB), via hot-injection precipitation and solvothermal methods. Cobalt arsenides constitute an important family of magnetically active solids that find a variety of applications ranging from magnetic semiconductors to biomedical imaging. While the higher temperature hot-injection precipitation technique (300 °C) yields pure CoAs nanostructures, the lower temperature solvothermal method (200 °C) yields a mixture of CoAs nanoparticles along with other Co-based impurity phases. The synthesis in all these cases involved usage of triphenylarsine ((C6H5)3As) as the As precursor which reacts with solid Co2(CO)8 by ligand displacement to yield a single source precursor. The surfactant, hexadecylamine (HDA) further assists in controlling the morphology of the nanostructures. HDA also provides a basic medium and molten flux-like conditions for the redox chemistry to occur between Co and As at elevated temperatures. The influence of the length of reaction time was investigated by studying the evolution of product morphology over time. It was observed that while spontaneous nucleation at higher temperature followed by controlled growth led to the predominant formation of short nanorods, with longer reaction time, the nanorods were further converted to nanoparticles. The size of the nanoparticles obtained, was mostly in the range of 10-15 nm. The key finding of this work is exceptionally high coercivity in CoAs nanostructures for the first time. Coercivity observed was as high as 0.1 T (1000 Oe) at 2 K. These kinds of magnetic nanostructures find multiple applications in spintronics, whereas the superparamagnetic nanoparticles are viable for use in magnetic storage, ferrofluids and as contrast enhancing agents in MRI.

  13. Crystal structure of human mitochondrial acyl-CoA thioesterase (ACOT2)

    PubMed Central

    Mandel, Corey R.; Tweel, Benjamin; Tong, Liang

    2009-01-01

    Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of CoA esters to free CoA and carboxylic acids and have important functions in lipid metabolism and other cellular processes. Type I ACOTs are found only in animals and contain an α/β hydrolase domain, through currently no structural information is available on any of these enzymes. We report here the crystal structure at 2.1 Å resolution of human mitochondrial ACOT2, a type I enzyme. The structure contains two domains, N and C domains. The C domain has the α/β hydrolase fold, with the catalytic triad Ser294-His422-Asp388. The N domain contains a seven-stranded β-sandwich, which has some distant structural homologs in other proteins. The active site is located in a large pocket at the interface between the two domains. The structural information has significant relevance for other type I ACOTs and related enzymes. PMID:19497300

  14. Crystal structure of human mitochondrial acyl-CoA thioesterase (ACOT2).

    PubMed

    Mandel, Corey R; Tweel, Benjamin; Tong, Liang

    2009-08-07

    Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of CoA esters to free CoA and carboxylic acids and have important functions in lipid metabolism and other cellular processes. Type I ACOTs are found only in animals and contain an alpha/beta hydrolase domain, through currently no structural information is available on any of these enzymes. We report here the crystal structure at 2.1A resolution of human mitochondrial ACOT2, a type I enzyme. The structure contains two domains, N and C domains. The C domain has the alpha/beta hydrolase fold, with the catalytic triad Ser294-His422-Asp388. The N domain contains a seven-stranded beta-sandwich, which has some distant structural homologs in other proteins. The active site is located in a large pocket at the interface between the two domains. The structural information has significant relevance for other type I ACOTs and related enzymes.

  15. Genetics Home Reference: peroxisomal acyl-CoA oxidase deficiency

    MedlinePlus

    ... including widely spaced eyes (hypertelorism), a low nasal bridge, and low-set ears. Extra fingers or toes ( ... This enzyme is found in sac-like cell structures (organelles) called peroxisomes, which contain a variety of ...

  16. Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

    SciTech Connect

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie

    2012-10-15

    Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

  17. Interactions of acyl-coenzyme A with phosphatidylcholine bilayers and serum albumin

    SciTech Connect

    Boylan, J.G.; Hamilton, J.A. )

    1992-01-21

    Interactions of oleoyl- and octanoyl-coenzyme A (CoA) with phosphatidylcholine (PC) vesicles and bovine serum albumin (BSA) were investigated by NMR spectroscopy. Binding of acyl-CoA to small unilamellar PC vesicles and to BSA was detected by changes in {sup 13}C and {sup 31}P chemical shifts relative to the chemical shifts for aqueous acyl-CoA. PC vesicles remained intact with {le} 15 mol % oleoyl-CoA, while higher oleoyl-CoA proportions produced mixed micelles. In contrast, {sup 13}C spectra revealed rapid exchange (ms) of octanoyl-CoA between the aqueous phase and PC vesicles and a low affinity for the bilayer. Thus, the binding affinity of acyl-CoA for PC bilayers is dependent on the acyl chain length. Addition of ({sup 13}C)carboxyl-enriched oleic acid to oleoyl-CoA/BSA mixtures revealed simultaneous binding of oleic acid and oleoyl-CoA to BSA, with some perturbation of binding interactions. Thus, BSA contains multiple binding sites for oleoyl-CoA and can bind fatty acid and acyl-CoA simultaneously.

  18. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

    DOE PAGES

    Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; ...

    2015-09-18

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

  19. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

    SciTech Connect

    Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; Martí-Arbona, Ricardo

    2015-09-18

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

  20. Acylation of Ferrocene: A Greener Approach

    ERIC Educational Resources Information Center

    Birdwhistell, Kurt R.; Nguyen, Andy; Ramos, Eric J.; Kobelja, Robert

    2008-01-01

    The acylation of ferrocene is a common reaction used in organic laboratories to demonstrate Friedel-Crafts acylation and the purification of compounds using column chromatography. This article describes an acylation of ferrocene experiment that is more eco-friendly than the conventional acylation experiment. The traditional experiment was modified…

  1. Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase III (mtFabH) assay: principles and method.

    PubMed

    Sachdeva, Sarbjot; Reynolds, Kevin A

    2008-01-01

    Fatty acid biosynthesis is one of the relatively newer targets in antibacterial drug discovery. The presence of distinct fatty acid synthases (FAS) in mammals and bacteria and the fact that most bacterial FAS enzymes are essential for viability make this a very attractive antimicrobial drug target. The enzyme beta-ketoacyl ACP synthase (KASIII or FabH) is the key enzyme that initiates fatty acid biosynthesis in a type II dissociated FAS. This enzyme catalyzes the condensation of acyl CoA and malonyl ACP (acyl carrier protein) to form a beta-ketoacyl ACP product, which is further processed to form mature fatty acids that are involved in various essential cellular processes and structures like phospholipid biosynthesis, cell wall formation, etc. Herein we describe a new assay for the Mycobacterium tuberculosis FabH (mtFabH) enzyme involved in a key initiation step in the synthesis of mycolic acids, which are an integral component of the cell wall. The assay eliminates the need for the cumbersome washing steps or specialty scintillation proximity assay beads and the preparation of acyl carrier proteins required in other assay formats. This discontinuous assay involves the reduction of radiolabled long-chain beta-ketoacyl CoA product to its dihydroxy derivative, which partitions into a nonpolar phase for quantitation, while the reduced radiolabeled substrate derivative remains in the aqueous phase.

  2. Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

    PubMed Central

    Fernández-Valverde, M; Reglero, A; Martinez-Blanco, H; Luengo, J M

    1993-01-01

    Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins. Images PMID:8476289

  3. Analysis of Leigh syndrome mutations in the yeast SURF1 homolog reveals a new member of the cytochrome oxidase assembly factor family.

    PubMed

    Bestwick, Megan; Jeong, Mi-Young; Khalimonchuk, Oleh; Kim, Hyung; Winge, Dennis R

    2010-09-01

    Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F(249)T and Y(344)D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G(137)E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G(137)E Shy1 mutant phenocopied shy1Delta cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G(137)E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX(9)C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Delta cells was conducted. Respiratory function of coa4Delta cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.

  4. Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds1[C][W][OPEN

    PubMed Central

    Yurchenko, Olga; Singer, Stacy D.; Nykiforuk, Cory L.; Gidda, Satinder; Mullen, Robert T.; Moloney, Maurice M.; Weselake, Randall J.

    2014-01-01

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. PMID:24740000

  5. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    SciTech Connect

    Roman-Lopez, C.R.; Allred, J.B.

    1986-05-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind (/sup 14/C)methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase.

  6. Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry.

    PubMed Central

    Aplin, R T; Baldwin, J E; Roach, P L; Robinson, C V; Schofield, C J

    1993-01-01

    Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9. Images Figure 1 PMID:8396910

  7. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  8. The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers.

    PubMed

    Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K; Tomasi, Pernell; Dyer, John M; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Parsons, Eugene P; Jenks, Matthew A; Lü, Shiyou

    2017-02-01

    We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem.

  9. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

    PubMed

    Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  10. Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

    PubMed Central

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2014-01-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  11. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    SciTech Connect

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  12. Subcellular relocalization of a long-chain fatty acid CoA ligase by a suppressor mutation alleviates a respiration deficiency in Saccharomyces cerevisiae.

    PubMed Central

    Harington, A; Schwarz, E; Slonimski, P P; Herbert, C J

    1994-01-01

    We have isolated an extragenic suppressor, FAM1-1, which is able to restore respiratory growth to a deletion of the CEM1 gene (mitochondrial beta-keto-acyl synthase). The sequence of the suppressor strongly suggests that it encodes a long-chain fatty acid CoA ligase (fatty-acyl-CoA synthetase). We have also cloned and sequenced the wild-type FAM1 gene, which is devoid of suppressor activity. The comparison of the two sequences shows that the suppressor mutation is an A-->T transversion, which creates a new initiation codon and adds 18 amino acids to the N-terminus of the protein. This extension has all the characteristics of a mitochondrial targeting sequence, whilst the N-terminus of the wild-type protein has none of these characteristics. In vitro mitochondrial import experiments show that the N-terminal half of the suppressor protein, but not of the wild-type, is transported into mitochondria. Thus, we hypothesize that the suppressor acts by changing the subcellular localization of the protein and relocating at least some of the enzyme from the cytosol to the mitochondria. These results support the hypothesis that some form of fatty acid synthesis, specific for the mitochondria, is essential for the function of the organelle. Images PMID:7988550

  13. Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

    PubMed

    Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

    2017-02-21

    Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases (ACCase) whose subunit composition and physiological roles have not yet been clearly established. A rather controversial data in the literature refers to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs; one of the substrates involved in the last step of condensation mediated by the polyketide synthase Pks13 to synthesize mature mycolic acids. Here we have successfully reconstituted the so called long-chain acyl-CoA carboxylase complex (LCC) from its purified components: the α-subunit AccA3, the ε-subunit AccE5 and the two β-subunits AccD4 and AccD5, and demonstrated that the four subunits are essential for its LCC activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor of the AccD5 subunit, that its role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. This article is protected by copyright. All rights reserved.

  14. Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor

    SciTech Connect

    Chemler, Joseph A.; Buchholz, Tonia J.; Geders, Todd W.; Akey, David L.; Rath, Christopher M.; Chlipala, George E.; Smith, Janet L.; Sherman, David H.

    2012-08-10

    Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 {angstrom} germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.

  15. Ghrelin acylation and metabolic control.

    PubMed

    Al Massadi, O; Tschöp, M H; Tong, J

    2011-11-01

    Since its discovery, many physiologic functions have been ascribed to ghrelin, a gut derived hormone. The presence of a median fatty acid side chain on the ghrelin peptide is required for the binding and activation of the classical ghrelin receptor, the growth hormone secretagogue receptor (GHSR)-1a. Ghrelin O-acyl transferase (GOAT) was recently discovered as the enzyme responsible for this acylation process. GOAT is expressed in all tissues that have been found to express ghrelin and has demonstrated actions on several complex endocrine organ systems such as the hypothalamus-pituitary-gonadal, insular and adrenal axis as well as the gastrointestinal (GI) tract, bone and gustatory system. Ghrelin acylation is dependent on the function of GOAT and the availability of substrates such as proghrelin and short- to medium-chain fatty acids (MCFAs). This process is governed by GOAT activity and has been shown to be modified by dietary lipids. In this review, we provided evidence that support an important role of GOAT in the regulation of energy homeostasis and glucose metabolism by modulating acyl ghrelin (AG) production. The relevance of GOAT and AG during periods of starvation remains to be defined. In addition, we summarized the recent literature on the metabolic effects of GOAT specific inhibitors and shared our view on the potential of targeting GOAT for the treatment of metabolic disorders such as obesity and type 2 diabetes.

  16. In vitro acylation of okadaic acid in the presence of various bivalves' extracts.

    PubMed

    Konoki, Keiichi; Onoda, Tatsuya; Watanabe, Ryuichi; Cho, Yuko; Kaga, Shinnosuke; Suzuki, Toshiyuki; Yotsu-Yamashita, Mari

    2013-01-29

    The dinoflagellate Dinophysis spp. is responsible for diarrhetic shellfish poisoning (DSP). In the bivalves exposed to the toxic bloom of the dinoflagellate, dinophysistoxin 3 (DTX3), the 7-OH acylated form of either okadaic acid (OA) or DTX1, is produced. We demonstrated in vitro acylation of OA with palmitoyl CoA in the presence of protein extract from the digestive gland, but not other tissues of the bivalve Mizuhopecten yessoensis. The yield of 7-O-palmitoyl OA reached its maximum within 2 h, was the highest at 37 °C followed by 28 °C, 16 °C and 4 °C and was the highest at pH 8 in comparison with the yields at pH 6 and pH 4. The transformation also proceeded when the protein extract was prepared from the bivalves Corbicula japonica and Crassostrea gigas. The OA binding protein OABP2 identified in the sponge Halichondria okadai was not detected in the bivalve M. yessoensis, the bivalve Mytilus galloprovincialis and the ascidian Halocynthia roretzi, though they are known to accumulate diarrhetic shellfish poisoning toxins. Since DTX3 does not bind to protein phosphatases 1 and 2A, the physiological target for OA and DTXs in mammalian cells, the acylation of DSP toxins would be related to a detoxification mechanism for the bivalve species.

  17. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    SciTech Connect

    Halavaty, Andrei S.; Kim, Youngchang; Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James; Zhou, Min; Onopriyenko, Olena; Skarina, Tatiana; Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N.; Joachimiak, Andrzej; Savchenko, Alexei; Anderson, Wayne F.

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  18. Chemical Reporters for Exploring Protein Acylation

    PubMed Central

    Thinon, Emmanuelle; Hang, Howard C.

    2015-01-01

    Proteins are acylated by a variety of metabolites that regulates many important cellular pathways in all kingdoms of life. Acyl groups in cells can vary in structure from the smallest unit, acetate, to modified long chain fatty acids, all of which can be activated and covalently attached to diverse amino acid side chains and consequently modulate protein function. For example, acetylation of Lys residues can alter the charge state of proteins and generate new recognition elements for protein–protein interactions. Alternatively, long chain fatty-acylation targets proteins to membranes and enables spatial control of cell signalling. To facilitate the analysis of protein acylation in biology, acyl analogues bearing alkyne or azide tags have been developed that enable fluorescent imaging and proteomic profiling of modified proteins using bioorthogonal ligation methods. Herein, we summarize the currently available acylation chemical reporters and highlight their utility to discover and quantify the roles of protein acylation in biology. PMID:25849926

  19. Determination of individual long-chain fatty acyl-CoA esters in heart and skeletal muscle.

    PubMed

    Molaparast-Saless, F; Shrago, E; Spennetta, T L; Donatello, S; Kneeland, L M; Nellis, S H; Liedtke, A J

    1988-05-01

    A method has been developed for determination of individual long-chain fatty acyl-CoA esters from heart and skeletal muscle using high performance liquid chromatography (HPLC). The esters were extracted from freeze-clamped tissue of pig and rat hearts and rat skeletal muscle for analysis on a radially compressed C18 5mu reverse-phase column. Nine peaks in the extract with carbon chain lengths from C12 to C20 that subsequently disappeared on alkaline hydrolysis were identified. The major acyl-CoA peaks were 14:1, 18:2, 16:0 and 18:1 and additionally in rat heart 18:0. Total long-chain acyl-CoA esters obtained by summation of the individual molecular species was 11.34 +/- 1.48 nmol/g wet wt. pig heart; 14.51 +/- 2.11 nmol/g wet wt. in rat heart, and 4.35 +/- 0.71 nmol/g wet wt. in rat skeletal muscle. These values were approximately 132% of those obtained using a separate procedure that measured total CoA by HPLC after alkaline hydrolysis of the esters. The described method demonstrates the quantitation of individual acyl-CoA species in muscle tissue. Therefore, it has a number of advantages in that it permits information to be obtained on the individual molecular species under various nutritional and metabolic conditions.

  20. Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

    PubMed Central

    Maloney, Finn P.; Gerwick, Lena; Gerwick, William H.; Sherman, David H.; Smith, Janet L.

    2016-01-01

    Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA. PMID:27573844

  1. Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

    PubMed

    Valsangkar, Deepa S; Downs, Stephen M

    2015-09-01

    In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

  2. Acyl-CoA binding protein expression is fiber type- specific and elevated in muscles from the obese insulin-resistant Zucker rat.

    PubMed

    Franch, Jesper; Knudsen, Jens; Ellis, Bronwyn A; Pedersen, Preben K; Cooney, Gregory J; Jensen, Jørgen

    2002-02-01

    Accumulation of acyl-CoA is hypothesized to be involved in development of insulin resistance. Acyl-CoA binds to acyl-CoA binding protein (ACBP) with high affinity, and therefore knowledge about ACBP concentration is important for interpreting acyl-CoA data. In the present study, we used a sandwich enzyme-linked immunosorbent assay to quantify ACBP concentration in different muscle fiber types. Furthermore, ACBP concentration was compared in muscles from lean and obese Zucker rats. Expression of ACBP was highest in the slow-twitch oxidative soleus muscle and lowest in the fast-twitch glycolytic white gastrocnemius (0.46 +/- 0.02 and 0.16 +/- 0.005 microg/mg protein, respectively). Expression of ACBP was soleus > red gastrocnemius > extensor digitorum longus > white gastrocnemius. Similar fiber type differences were found for carnitine palmitoyl transferase (CPT)-1, and a correlation was observed between ACBP and CPT-1. Muscles from obese Zucker rats had twice the triglyceride content, had approximately twice the long-chain acyl CoA content, and were severely insulin resistant. ACBP concentration was approximately 30% higher in all muscles from obese rats. Activities of CPT-1 and 3-hydroxy-acyl-CoA dehydrogenase were increased in muscles from obese rats, whereas citrate synthase activity was similar. In conclusion, ACBP expression is fiber type-specific with the highest concentration in oxidative muscles and the lowest in glycolytic muscles. The 90% increase in the concentration of acyl-CoA in obese Zucker muscle compared with only a 30% increase in the concentration of ACBP supports the hypothesis that an increased concentration of free acyl-CoA is involved in the development of insulin resistance.

  3. Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj

    2011-03-01

    Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.

  4. Acyl-CoA synthetase activity links wild-type but not mutant α-synuclein to brain arachidonate metabolism

    PubMed Central

    Golovko, Mikhail Y.; Rosenberger, Thad A.; Faergeman, Nils J.; Feddersen, Søren; Cole, Nelson B.; Pribill, Ingrid; Berger, Johannes; Nussbaum, Robert L.; Murphy, Eric J.

    2008-01-01

    Because α-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of α-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-14C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetases (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wt mouse or human α-synuclein, but not by A30P, E46K, and A53T forms of α-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools was markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that α-synuclein bound free 20:4n-6 (Kd of 3.7 μM), but did not bind 20:4n-6-CoA. These data suggest α-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that α-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum localized acyl-CoA synthetase activity, although mutants forms of α-synuclein fail to restore this activity. PMID:16734431

  5. The Physiology of Protein S-acylation

    PubMed Central

    Chamberlain, Luke H.; Shipston, Michael J.

    2015-01-01

    Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field. PMID:25834228

  6. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

    1998-01-06

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

  7. The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers1[OPEN

    PubMed Central

    Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K.; Dyer, John M.; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Jenks, Matthew A.

    2017-01-01

    We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4’s principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation’s effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem. PMID:28069670

  8. Selective Oma1 Protease-mediated Proteolysis of Cox1 Subunit of Cytochrome Oxidase in Assembly Mutants*

    PubMed Central

    Khalimonchuk, Oleh; Jeong, Mi-Young; Watts, Talina; Ferris, Elliott; Winge, Dennis R.

    2012-01-01

    Stalled biogenesis of the mitochondrial cytochrome c oxidase (CcO) complex results in degradation of subunits containing redox cofactors. The conserved Oma1 metalloproteinase mediates facile Cox1 degradation in cells lacking the Coa2 assembly factor, but not in a series of other mutants stalled in CcO maturation. Oma1 is activated in coa2Δ cells, but the selective Cox1 degradation does not arise merely from its activation. Oma1 is also active in cells with dysfunctional mitochondria and cox11Δ cells impaired in CcO maturation, but this activation does not result in Oma1-mediated Cox1 degradation. The facile and selective degradation of Cox1 in coa2Δ cells, relative to other CcO assembly mutants, is likely due to impaired hemylation and subsequent misfolding of the subunit. Specific Cox1 proteolysis in coa2Δ cells arises from a combination of Oma1 activation and a susceptible conformation of Cox1. PMID:22219186

  9. Spectroscopic Classification of SN 2017coa as a Type Ia Supernova

    NASA Astrophysics Data System (ADS)

    Xiang, Danfeng; Rui, Liming; Wang, Xiaofeng; Tan, Hanjie; Li, Wenxiong; Zhang, Tianmeng; Xu, Zhijian; Yang, Zesheng; Song, Hao; Mo, Jun; Wang, Yuanhao; Zhou, Ziheng; Meng, Xianmin; Qian, Shenban; Jia, Junjun; Zhou, Xu; Zhang, Jujia

    2017-04-01

    We obtained an optical spectrum (range 360-840 nm) of SN 2017coa,discovered by Tsinghua-NAOC Transient Survey (TNTS), on UT Mar.31.49 2017 with the 2.16-m telescope (+BFOSC) at Xinglong Station of National Astronomical Observatories of China (NAOC).

  10. Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene.

    PubMed Central

    Zou, J; Katavic, V; Giblin, E M; Barton, D L; MacKenzie, S L; Keller, W A; Hu, X; Taylor, D C

    1997-01-01

    A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs. PMID:9212466

  11. The utilization of the acyl-CoA and the involvement PDAT and DGAT in the biosynthesis of erucic acid-rich triacylglycerols in Crambe seed oil.

    PubMed

    Furmanek, Tomasz; Demski, Kamil; Banaś, Walentyna; Haslam, Richard; Napier, Jonathan; Stymne, Sten; Banaś, Antoni

    2014-04-01

    The triacylglycerol of Crambe abyssinica seeds consist of 95% very long chain (>18 carbon) fatty acids (86% erucic acid; 22:1∆13) in the sn-1 and sn-3 positions. This would suggest that C. abyssinica triacylglycerols are not formed by the action of the phospholipid:diacylglycerol acyltransferase (PDAT), but are rather the results of acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. However, measurements of PDAT and DGAT activities in microsomal membranes showed that C. abyssinica has significant PDAT activity, corresponding to about 10% of the DGAT activity during periods of rapid seed oil accumulation. The specific activity of DGAT for erucoyl-CoA had doubled at 19 days after flowering compared to earlier developmental stages, and was, at that stage, the preferred acyl donor, whereas the activities for 16:0-CoA and 18:1-CoA remained constant. This indicates that an expression of an isoform of DGAT with high specificity for erucoyl-CoA is induced at the onset of rapid erucic acid and oil accumulation in the C. abyssinica seeds. Analysis of the composition of the acyl-CoA pool during different stages of seed development showed that the percentage of erucoyl groups in acyl-CoA was much higher than in complex lipids at all stages of seed development except in the desiccation phase. These results are in accordance with published results showing that the rate limiting step in erucic acid accumulation in C. abyssinica oil is the utilization of erucoyl-CoA by the acyltransferases in the glycerol-3-phosphate pathway.

  12. Succinate oxidase in Neurospora.

    PubMed

    West, D J; Woodward, D O

    1973-02-01

    Two kinetically distinct states of succinate oxidase have been detected in the mitochondria of Neruospora crassa. One state has a K(m) for succinate of 4.1 x 10(-3)m, and the other has a K(m) for succinate of 3.5 x 10(-4)m. The high K(m) state was found in freshly extracted mitochondria from either 20- or 72-hr mycelium. However, the succinate oxidase activity in mitochondria from 20-hr mycelium rapidly deteriorated in vitro, leaving a stable residual activity with the lower K(m) for succinate. Adenosine triphosphate (ATP) plus Mg(2+) stabilized the high K(m) state in these preparations. The high K(m) state of succinate oxidase was further characterized by a two- to threefold increase in activity over the pH range 6.6 to 8.0 and by classical competitive inhibition by fumarate and malonate. By contrast, the low K(m) state of succinate oxidase showed a relatively flat response to pH over the range 6.6 to 8.0 and a nonclassical pattern of inhibition by fumarate and malonate, as shown by nonlinear plots of reciprocal velocity versus reciprocal substrate concentration in the presence of inhibitor or reciprocal velocity versus inhibitor concentration at fixed substrate concentrations. The relationship of mycelial age to the in vitro stability of succinate oxidase is considered with reference to probable changes in the relative pool sizes of extra- and intramitochondrial ATP in response to changes in the rate of glycolysis.

  13. Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

    PubMed Central

    Casabon, Israël; Swain, Kendra; Crowe, Adam M.

    2014-01-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

  14. Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins.

    PubMed

    Jenner, Matthew; Afonso, Jose P; Kohlhaas, Christoph; Karbaum, Petra; Frank, Sarah; Piel, Jörn; Oldham, Neil J

    2016-04-18

    Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

  15. Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases.

    PubMed

    Wältermann, Marc; Stöveken, Tim; Steinbüchel, Alexander

    2007-02-01

    Triacylglycerols (TAGs) and wax esters (WEs) are beside polyhydroxyalkanoates (PHAs) important storage lipids in some groups of prokaryotes. Accumulation of these lipids occurs in cells when they are cultivated under conditions of unbalanced growth in the presence of high concentrations of a suitable carbon source, which can be used for fatty acid and storage lipid biosyntheses. The key enzymes, which mediate both WE and TAG formations from long-chain acyl-coenzyme A (CoA) as acyl donor and long-chain fatty alcohols or diacylglycerols as respective acyl acceptors in bacteria, are WE synthases/acyl-CoA:diacylglycerol acyltransferases (WS/DGATs). The WS/DGATs identified so far represent rather unspecific enzymes with broad spectra of possible substrates; this makes them interesting for many biotechnological applications. This review traces the molecular structure and biochemical properties including the probable regions responsible for acyltransferase properties, enzymatic activity and substrate specifities. The phylogenetic relationships based on amino acid sequence similarities of this unique class of enzymes were revealed. Furthermore, recent advances in understanding the physiological functions of WS/DGATs in their natural hosts including pathogenic Mycobacterium tuberculosis were discussed.

  16. The Endoplasmic Reticulum-Associated Maize GL8 Protein Is a Component of the Acyl-Coenzyme A Elongase Involved in the Production of Cuticular Waxes1

    PubMed Central

    Xu, Xiaojie; Dietrich, Charles R.; Lessire, Rene; Nikolau, Basil J.; Schnable, Patrick S.

    2002-01-01

    The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a β-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes. PMID:11891248

  17. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  18. Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production.

    PubMed

    Noguchi, Shingo; Putri, Sastia P; Lan, Ethan I; Laviña, Walter A; Dempo, Yudai; Bamba, Takeshi; Liao, James C; Fukusaki, Eiichiro

    Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using (13)C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.

  19. Cox1 mutation abrogates need for Cox23 in cytochrome c oxidase biogenesis

    PubMed Central

    Dela Cruz, Richard; Jeong, Mi-Young; Winge, Dennis R.

    2016-01-01

    Cox23 is a known conserved assembly factor for cytochrome c oxidase, although its role in cytochrome c oxidase (CcO) biogenesis remains unresolved. To gain additional insights into its role, we isolated spontaneous suppressors of the respiratory growth defect in cox23∆ yeast cells. We recovered independent colonies that propagated on glycerol/lactate medium for cox23∆ cells at 37°C. We mapped these mutations to the mitochondrial genome and specifically to COX1 yielding an I101F substitution. The I101F Cox1 allele is a gain-of-function mutation enabling yeast to respire in the absence of Cox23. CcO subunit steady-state levels were restored with the I101F Cox1 suppressor mutation and oxygen consumption and CcO activity were likewise restored. Cells harboring the mitochondrial genome encoding I101F Cox1 were used to delete genes for other CcO assembly factors to test the specificity of the Cox1 mutation as a suppressor of cox23∆ cells. The Cox1 mutant allele fails to support respiratory growth in yeast lacking Cox17, Cox19, Coa1, Coa2, Cox14 or Shy1, demonstrating its specific suppressor activity for cox23∆ cells.

  20. Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum.

    PubMed

    Koetsier, Martijn J; Jekel, Peter A; Wijma, Hein J; Bovenberg, Roel A L; Janssen, Dick B

    2011-03-23

    Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)- and (S)-β-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-β-phenylalanine, forming (R)-β-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with β-phenylalanines and D-α-phenylalanine.

  1. Mosaic Conservation Opportunity Areas - Liberal Model (ECO_RES.COA_MOSAIC33)

    EPA Pesticide Factsheets

    The COA_Mosaic33 layer designates areas with potential for forest/grassland mosaic conservation. These are areas of natural or semi-natural forest/grassland mosaic land cover patches that are at least 75 meters away from roads and away from patch edges. OAs were modeled by creating distance grids using the National Land Cover Database and the Census Bureau's TIGER roads files

  2. Mosaic Conservation Opportunity Areas - Conservativel Model (ECO_RES.COA_MOSAIC66)

    EPA Pesticide Factsheets

    The COA_Mosaic66 layer designates areas with potential for forest/grassland mosaic conservation. These are areas of natural or semi-natural forest/grassland land cover patches that area at least 395 meters away from roads and away from patch edges. OAs were modeled by creating distance grids using the National Land Cover Database and the Census Bureau's TIGER road files.

  3. Regulation of schistosome egg production by HMG CoA reductase

    SciTech Connect

    VandeWaa, E.A.; Bennett, J.L.

    1986-03-05

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of /sup 14/C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production.

  4. Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

    PubMed

    Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

    2015-03-01

    The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

  5. Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism.

    PubMed

    Garcia, Matthew; Leonardi, Roberta; Zhang, Yong-Mei; Rehg, Jerold E; Jackowski, Suzanne

    2012-01-01

    Pantothenate kinase (PanK) phosphorylates pantothenic acid (vitamin B(5)) and controls the overall rate of coenzyme A (CoA) biosynthesis. Pank1 gene deletion in mice results in a metabolic phenotype where fatty acid oxidation and gluconeogenesis are impaired in the fasted state, leading to mild hypoglycemia. Inactivating mutations in the human PANK2 gene lead to childhood neurodegeneration, but Pank2 gene inactivation in mice does not elicit a phenotype indicative of the neuromuscular symptoms or brain iron accumulation that accompany the human disease. Pank1/Pank2 double knockout (dKO) mice were derived to determine if the mild phenotypes of the single knockout mice are due to the ability of the two isoforms to compensate for each other in CoA biosynthesis. Postnatal development was severely affected in the dKO mice. The dKO pups developed progressively severe hypoglycemia and hyperketonemia by postnatal day 10 leading to death by day 17. Hyperketonemia arose from impaired whole-body ketone utilization illustrating the requirement for CoA in energy generation from ketones. dKO pups had reduced CoA and decreased fatty acid oxidation coupled with triglyceride accumulation in liver. dKO hepatocytes could not maintain the NADH levels compared to wild-type hepatocytes. These results revealed an important link between CoA and NADH levels, which was reflected by deficiencies in hepatic oleate synthesis and gluconeogenesis. The data indicate that PanK1 and PanK2 can compensate for each other to supply tissue CoA, but PanK1 is more important to CoA levels in liver whereas PanK2 contributes more to CoA synthesis in the brain.

  6. Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target. PMID:27676316

  7. SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase

    PubMed Central

    Laurent, Gaëlle; German, Natalie J.; Saha, Asish K.; de Boer, Vincent C. J.; Davies, Michael; Koves, Timothy R.; Dephoure, Noah; Fischer, Frank; Boanca, Gina; Vaitheesvaran, Bhavapriya; Lovitch, Scott B.; Sharpe, Arlene H.; Kurland, Irwin J.; Steegborn, Clemens; Gygi, Steven P.; Muoio, Deborah M.; Ruderman, Neil B.; Haigis, Marcia C.

    2013-01-01

    Summary Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a novel regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a novel SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis. PMID:23746352

  8. ROMPgel beads in IRORI format: acylations revisited.

    PubMed

    Roberts, Richard S

    2005-01-01

    Functionalized "designer" polymers derived from ring-opening metathesis polymerization (ROMPgels) are attractive for their high loading, high purity, and ease of synthesis. Their physical state may vary from liquid to gel to granular solid, making a general method of handling these polymers difficult. By incorporating a suitable norbornene-substituted linker on standard Wang beads, ROMPgels can be easily grafted onto the resin, adding the convenience of a bead format while still maintaining the high loading and excellent site accessibility. This advantage is demonstrated by the use of an N-hydroxysuccinimide ROMPgel (3.3 mmol g(-1), a 3-fold increase from the parent linker resin) in IRORI Kan format. Conditions for the acylation of these IRORI-formatted ROMPgels are reported, along with the scope and limitations of the choice of acylating reagents. Yields are greatly improved by the use of perfluorinated solvents as a nonparticipating cosolvent in the acylation process. A simple titration method for the quantification of the acylated ROMPgels is also reported. Spent Kans are regenerated after each use without apparent loss of activity or purity after several cycles. Due to the high loading and reduced swelling of the ROMPgel resin, up to 0.39 mmol acyl group has successfully been recovered from a single IRORI miniKan, demonstrating the high capacity of the resin and applicability to both lead discovery and optimization programs.

  9. In silico prediction of acyl glucuronide reactivity

    NASA Astrophysics Data System (ADS)

    Potter, Tim; Lewis, Richard; Luker, Tim; Bonnert, Roger; Bernstein, Michael A.; Birkinshaw, Timothy N.; Thom, Stephen; Wenlock, Mark; Paine, Stuart

    2011-11-01

    Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted 13C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

  10. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C. Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease. PMID:19141679

  11. Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

    NASA Technical Reports Server (NTRS)

    White, D. H.; Kennedy, R. M.; Macklin, J.

    1984-01-01

    Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. The proposed mechanism has been confirmed by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespead, geologically realistic setting for prebiotic peptide formation via in situ activation.

  12. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction†

    PubMed Central

    Héroux, Annie; Bozinovski, Dragana M.; Valley, Michael P.; Fitzpatrick, Paul F.; Orville, Allen M.

    2009-01-01

    The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 Å resolution or better are described of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct. The D402N enzyme has no detectable activity with neutral nitroalkanes (Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 23, 8738–8739). The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2’-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062–2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2’-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle. PMID:19265437

  13. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

    SciTech Connect

    Heroux, A.; Bozinovski, D; Valley, M; Fitzpatrick, P; Orville, A

    2009-01-01

    The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.

  14. The multiple acyl-coenzyme A dehydrogenation disorders, glutaric aciduria type II and ethylmalonic-adipic aciduria. Mitochondrial fatty acid oxidation, acyl-coenzyme A dehydrogenase, and electron transfer flavoprotein activities in fibroblasts.

    PubMed Central

    Amendt, B A; Rhead, W J

    1986-01-01

    The multiple acyl-coenzyme A (CoA) dehydrogenation disorders (MAD) include severe (S) and mild (M) variants, glutaric aciduria type II (MAD:S) and ethylmalonic-adipic aciduria (MAD:M). Intact MAD:M mitochondria oxidized [1-14C]octanoate, [1-14C]palmityl-CoA, and [1,5-14C]glutarate at 20-46% of control levels; MAD:S mitochondria oxidized these three substrates at 0.4-18% of control levels. In MAD:M mitochondria, acyl-CoA dehydrogenase (ADH) activities were similar to control, whereas MAD:S ADH activities ranged from 38% to 73% of control. Electron transfer flavoprotein (ETF) activities in five MAD:M cell lines ranged from 29 to 51% of control (P less than 0.01); ETF deficiency was the primary enzymatic defect in two MAD:M lines. In four MAD:S patients, ETF activities ranged from 3% to 6% of control (P less than 0.001); flavin adenine dinucleotide addition increased residual ETF activity from 4% to 21% of control in a single MAD:S line (P less than 0.01). Three MAD:S patients had ETF activities ranging from 33 to 53% of control; other investigators found deficient ETF-dehydrogenase activity in these MAD:S and three of our MAD:M cell lines. PMID:3722376

  15. Acyl anion free N-heterocyclic carbene organocatalysis.

    PubMed

    Ryan, Sarah J; Candish, Lisa; Lupton, David W

    2013-06-21

    Reaction discovery using N-heterocyclic carbene organocatalysis has been dominated by the chemistry of acyl anion equivalents. Recent studies demonstrate that NHCs are far more diverse catalysts, with a variety of reactions discovered that proceed without acyl anion equivalent formation. In this tutorial review selected examples of acyl anion free NHC catalysis using carbonyl compounds are presented.

  16. Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis.

    PubMed

    Padanad, Mahesh S; Konstantinidou, Georgia; Venkateswaran, Niranjan; Melegari, Margherita; Rindhe, Smita; Mitsche, Matthew; Yang, Chendong; Batten, Kimberly; Huffman, Kenneth E; Liu, Jingwen; Tang, Ximing; Rodriguez-Canales, Jaime; Kalhor, Neda; Shay, Jerry W; Minna, John D; McDonald, Jeffrey; Wistuba, Ignacio I; DeBerardinis, Ralph J; Scaglioni, Pier Paolo

    2016-08-09

    KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

  17. Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus

    DOE PAGES

    Scott, Joseph W.; Poole, Farris L.; Adams, Michael W. W.

    2014-01-01

    Tmore » he hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α 2 β 2 structures but the genome contains genes encoding three additional α -subunits.he ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α 2 β 2 isoenzymes.he α -subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids.he β -subunit determined preference for adenine or guanine nucleotides.he GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes.ranscriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of thehermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.« less

  18. High acyl gellan as an emulsion stabilizer.

    PubMed

    Vilela, Joice Aline Pires; da Cunha, Rosiane Lopes

    2016-03-30

    High acyl gellan (0.01-0.2% w/w) was used as stabilizer in oil in water emulsions containing 30% (w/w) of sunflower oil and prepared under different process conditions. Stable emulsions to phase separation could be obtained using high acyl gellan (HA) content above 0.05% (w/w), while low acyl gellan (LA) prepared at the same conditions could not stabilize emulsions. Emulsions properties depended on the process used to mix the oil and gellan dispersion since high pressure homogenization favored stabilization while very high energy density applied by ultrasound led to systems destabilization. Emulsions prepared using high pressure homogenization showed zeta potential values ranging from -50 up to -59 mV, suggesting that electrostatic repulsion could be contributing to the systems stability. Rheological properties of continuous phase were also responsible for emulsions stabilization, since HA gellan dispersions showed high viscosity and gel-like behavior. The high viscosity of the continuous phase could be associated to the presence of high acyl gellan microgels/aggregates. Disentanglement of these aggregates performed by ultrasound strongly decreased the viscosity and consequently affected the emulsions behavior, reducing the stability to phase separation.

  19. Acylated pregnane glycosides from Caralluma russeliana.

    PubMed

    Abdel-Sattar, Essam; Ahmed, Ahmed A; Hegazy, Mohamed-Elamir F; Farag, Mohamed A; Al-Yahya, Mohammad Abdul-Aziz

    2007-05-01

    The chloroform extract of the aerial parts of Caralluma russeliana yielded four acylated pregnane glycosides, namely russeliosides E-H, three were found now. The structures of the compounds were elucidated using MS, 1H NMR, 13C NMR, 1H-1H COSY, HMQC, NOESY and HMBC experiments.

  20. Acylation of the Type 3 Secretion System Translocon Using a Dedicated Acyl Carrier Protein

    PubMed Central

    Agrebi, Rym; Canestrari, Mickaël J.; Mignot, Tâm; Lebrun, Régine; Bouveret, Emmanuelle

    2017-01-01

    Bacterial pathogens often deliver effectors into host cells using type 3 secretion systems (T3SS), the extremity of which forms a translocon that perforates the host plasma membrane. The T3SS encoded by Salmonella pathogenicity island 1 (SPI-1) is genetically associated with an acyl carrier protein, IacP, whose role has remained enigmatic. In this study, using tandem affinity purification, we identify a direct protein-protein interaction between IacP and the translocon protein SipB. We show, by mass spectrometry and radiolabelling, that SipB is acylated, which provides evidence for a modification of the translocon that has not been described before. A unique and conserved cysteine residue of SipB is identified as crucial for this modification. Although acylation of SipB was not essential to virulence, we show that this posttranslational modification promoted SipB insertion into host-cell membranes and pore-forming activity linked to the SPI-1 T3SS. Cooccurrence of acyl carrier and translocon proteins in several γ- and β-proteobacteria suggests that acylation of the translocon is conserved in these other pathogenic bacteria. These results also indicate that acyl carrier proteins, known for their involvement in metabolic pathways, have also evolved as cofactors of new bacterial protein lipidation pathways. PMID:28085879

  1. Molar absorptivity and color characteristics of acylated and non-acylated pelargonidin-based anthocyanins.

    PubMed

    Giusti, M M; Rodríguez-Saona, L E; Wrolstad, R E

    1999-11-01

    The effects of glycosylation and acylation on the spectral characteristics, molar absorptivity, and color attributes of purified acylated and non-acylated pelargonidin derivatives were compared. Pigments were obtained from strawberries, radishes, red-fleshed potatoes, and partially hydrolyzed radish pigments. Individual pigments were isolated by using semipreparative HPLC. Spectral and color (CIELch) attributes of purified pigments were measured. Molar absorptivity ranged from 15 600 to 39 590 for pelargonidin-3-glucoside (pg-3-glu) and pg-3-rutinoside-5-glucoside acylated with p-coumaric acid, respectively. The presence of cinnamic acid acylation had a considerable impact on spectral and color characteristics, causing a bathochromic shift of lambda(max). Sugar substitution also played an important role, with a hypsochromic shift caused by the presence of glycosylation. Pg-3, 5-diglu and pg-3,5-triglu possessed a higher hue angle (>40 degrees ) than the other pg derivatives at pH 1.0, corresponding to the yellow-orange region of the color solid. Acylation with malonic acid did not affect lambda(max) and showed little effect on color characteristics. The solvent system had an effect not only on the molar absorptivity, but also on the visual color characteristic of the pigments.

  2. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Schnable, Patrick S.; Wen, Tsui-Jung

    2009-04-28

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  3. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2005-09-13

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  4. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2004-07-20

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.

  5. {alpha}-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

    SciTech Connect

    Lee, Young; Naseem, R. Haris; Park, Byung-Hyun; Garry, Daniel J.; Richardson, James A.; Schaffer, Jean E.; Unger, Roger H. . E-mail: roger.unger@utsouthwestern.edu

    2006-05-26

    {alpha}-Lipoic acid ({alpha}-LA) mimics the hypothalamic actions of leptin on food intake, energy expenditure, and activation of AMP-activated protein kinase (AMPK). To determine if, like leptin, {alpha}-LA protects against cardiac lipotoxicity, {alpha}-LA was fed to transgenic mice with cardiomyocyte-specific overexpression of the acyl CoA synthase (ACS) gene. Untreated ACS-transgenic mice died prematurely with increased triacylglycerol content and dilated cardiomyopathy, impaired systolic function and myofiber disorganization, apoptosis, and interstitial fibrosis on microscopy. In {alpha}-LA-treated ACS-transgenic mice heart size, echocardiogram and TG content were normal. Plasma TG fell 50%, hepatic-activated phospho-AMPK rose 6-fold, sterol regulatory element-binding protein-1c declined 50%, and peroxisome proliferator-activated receptor-{gamma} cofactor-1{alpha} mRNA rose 4-fold. Since food restriction did not prevent lipotoxicity, we conclude that {alpha}-LA treatment, like hyperleptinemia, protects the heart of ACS-transgenic mice from lipotoxicity.

  6. Adaptive changes in individual acyl-CoA esters from hamster BAT during cold acclimation.

    PubMed

    Donatello, S; Spennetta, T; Strieleman, P; Woldegiorgis, G; Shrago, E

    1988-02-01

    Long-chain fatty acyl-CoA esters (LCFACoAE) were extracted from freeze-clamped powdered brown adipose tissue (BAT) obtained from thermoneutral control and cold-acclimated hamsters and the CoA esters individually separated by high-performance liquid chromatography. LCFACoAE of carbon chain length C12 to C20 were identified by increasing column retention time in the following order: C12:0, C14:1, C14:0, C16:1, C18:2, C16:0, C18:1, C18:0, and C20:4. The mean total LCFACoAE concentrations were 235 +/- 40 nmol/g protein for the control hamsters and 648 +/- 105 nmol/g protein for the 22-day cold-acclimated hamsters. A rapid fourfold increase in the levels of C16:0, C18:0, and C18:1 occurred within hours after initiation of the cold temperature, whereas the concentrations of the other six LCFACoAE either increased only slightly or remained unchanged. Almost 50% of the total LCFACoAE in the BAT of cold-acclimated hamsters was made up of C16:0, C18:0, and C18:1. These results, which demonstrate some dynamic changes in adipose tissue LCFACoAE, are consistent with their proposed role in the initiation and maintenance of BAT thermogenesis.

  7. The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

    SciTech Connect

    Hayashi, H.; Miwa, A. )

    1989-11-01

    The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using (1-{sup 14}C)butyric acid and (1-{sup 14}C)lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of ({sup 14}C)lignoceric acid into primary bile acids was approximately four times higher than that of ({sup 14}C)butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both ({sup 14}C)lignoceric acid and ({sup 14}C)butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.

  8. Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation.

    PubMed

    Forwood, Jade K; Thakur, Anil S; Guncar, Gregor; Marfori, Mary; Mouradov, Dmitri; Meng, Weining; Robinson, Jodie; Huber, Thomas; Kellie, Stuart; Martin, Jennifer L; Hume, David A; Kobe, Bostjan

    2007-06-19

    Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.

  9. Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation

    PubMed Central

    Forwood, Jade K.; Thakur, Anil S.; Guncar, Gregor; Marfori, Mary; Mouradov, Dmitri; Meng, Weining; Robinson, Jodie; Huber, Thomas; Kellie, Stuart; Martin, Jennifer L.; Hume, David A.; Kobe, Bostjan

    2007-01-01

    Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease. PMID:17563367

  10. Lysyl oxidase in colorectal cancer.

    PubMed

    Cox, Thomas R; Erler, Janine T

    2013-11-15

    Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to ~600,000 deaths annually, predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion, and angiogenesis. This emerging research has stimulated significant interest in lysyl oxidase as a strong candidate for developing and deploying inhibitors as functional efficacious cancer therapeutics. In this review, we discuss the rapidly expanding body of knowledge concerning lysyl oxidase in solid tumor progression, highlighting recent advancements in the field of colorectal cancer.

  11. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress.

    PubMed

    Vu, Hieu Sy; Roth, Mary R; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A; Williams, Todd D; Welti, Ruth

    2014-04-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.

  12. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  13. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A/sub 2/

    SciTech Connect

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

    1986-05-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1-/sup 14/C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A/sub 2/ (PLA/sub 2/) activity (64.3-545.6 nmols/min/mg). The PLA/sub 2/ was maximally active in the neutral-alkaline pH range, was Ca/sup 2 +/-dependent, and was unaffected by the addition of xanthine. PLA/sub 2/ activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA/sub 2/ activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca/sup 2 +/-dependent PLA/sub 2/ measured in various tissue homogenates (less than or equal to 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA/sub 2/ may have influenced previously published reports, and such studies should be interpreted cautiously.

  14. Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

    PubMed

    Bareth, Bettina; Nikolov, Miroslav; Lorenzi, Isotta; Hildenbeutel, Markus; Mick, David U; Helbig, Christin; Urlaub, Henning; Ott, Martin; Rehling, Peter; Dennerlein, Sven

    2016-05-15

    The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

  15. A key role of PGC-1α transcriptional coactivator in production of VEGF by a novel angiogenic agent COA-Cl in cultured human fibroblasts.

    PubMed

    Igarashi, Junsuke; Okamoto, Ryuji; Yamashita, Tetsuo; Hashimoto, Takeshi; Karita, Sakiko; Nakai, Kozo; Kubota, Yasuo; Takata, Maki; Yamaguchi, Fuminori; Tokuda, Masaaki; Sakakibara, Norikazu; Tsukamoto, Ikuko; Konishi, Ryoji; Hirano, Katsuya

    2016-03-01

    We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.

  16. Respiratory burst oxidase of fertilization.

    PubMed Central

    Heinecke, J W; Shapiro, B M

    1989-01-01

    Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product. PMID:2537493

  17. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

    PubMed

    Liu, Qingqing; Gauthier, Marie-Soleil; Sun, Lei; Ruderman, Neil; Lodish, Harvey

    2010-11-01

    Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.

  18. Acyl glucuronides: the good, the bad and the ugly.

    PubMed

    Regan, Sophie L; Maggs, James L; Hammond, Thomas G; Lambert, Craig; Williams, Dominic P; Park, B Kevin

    2010-10-01

    Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response.

  19. Multiple acyl-CoA dehydrogenation deficiency as decreased acyl-carnitine profile in serum.

    PubMed

    Wen, Bing; Li, Duoling; Li, Wei; Zhao, Yuying; Yan, Chuanzhu

    2015-06-01

    We report a case with late onset riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency (MADD) characterized by decreased acyl-carnitine profile in serum which is consistent with primary systemic carnitine deficiency (CDSP) while just the contrary to a typical MADD. This patient complained with muscle weakness, muscle pain and intermittent vomiting, and was diagnosed as polymyositis, received prednisone therapy before consulted with us. Muscle biopsy revealed mild lipid storage. The findings of serum acyl-carnitines were consistent with CDSP manifesting as decreased free and total carnitines in serum. But oral L-carnitine supplementation was not very effective to this patient and mutation analysis of the SLC22A5 gene for CDSP was normal. Later, another acyl-carnitine analysis revealed a typical MADD profile in serum, which was characterized by increased multiple acyl-carnitines. Compound heterozygous mutations were identified in electron transferring-flavoprotein dehydrogenase (ETFDH) gene which confirmed the diagnosis of MADD. After administration of riboflavin, he improved dramatically, both clinically and biochemically. Thus, late onset riboflavin-responsive MADD should be included in the differential diagnosis for adult carnitine deficiency.

  20. Fatty Acyl Chains of Mycobacterium marinum Lipooligosaccharides

    PubMed Central

    Rombouts, Yoann; Alibaud, Laeticia; Carrère-Kremer, Séverine; Maes, Emmanuel; Tokarski, Caroline; Elass, Elisabeth; Kremer, Laurent; Guérardel, Yann

    2011-01-01

    We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly. PMID:21803773

  1. Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.

    PubMed Central

    Kassovska-Bratinova, S.; Fukao, T.; Song, X. Q.; Duncan, A. M.; Chen, H. S.; Robert, M. F.; Pérez-Cerdá, C.; Ugarte, M.; Chartrand, C.; Vobecky, S.; Kondo, N.; Mitchell, G. A.

    1996-01-01

    Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes >> fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency. Images Figure 2 Figure 6 PMID:8751852

  2. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  3. Discovery of Tumor-Specific Irreversible Inhibitors of Stearoyl CoA Desaturase

    PubMed Central

    Theodoropoulos, Panayotis C.; Gonzales, Stephen S.; Winterton, Sarah E.; Rodriguez-Navas, Carlos; McKnight, John S.; Morlock, Lorraine K.; Hanson, Jordan M.; Cross, Bethany; Owen, Amy E.; Duan, Yingli; Moreno, Jose R.; Lemoff, Andrew; Mirzaei, Hamid; Posner, Bruce A.; Williams, Noelle S.

    2016-01-01

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic to the same four of 12 human lung cancer cell lines at low nanomolar concentrations. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible stearoyl CoA desaturase (SCD) inhibitors. SCD is recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes were unable to activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors. PMID:26829472

  4. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  5. Spectroscopic Classification of ASASSN-16fn/AT2016coa and MASTER J202606.27-200732.6

    NASA Astrophysics Data System (ADS)

    Falco, E.; Calkins, M.; Challis, P.; Kirshner, R.; Prieto, J. L.; Stanek, K. Z.

    2016-06-01

    Optical spectra (range 350-760nm) of the supernova candidates ASASSN-16fn/AT2016coa (ATel #9081) and MASTER J202606.27-200732.6 (ATel #9056) were obtained on UT 2016 June 3 with the F. L. Whipple Observatory 1.5-m telescope (+ FAST).

  6. Geranylgeraniol suppresses the viability of human DU145 prostate carcinoma cells and the level of HMG CoA reductase

    PubMed Central

    Fernandes, Nicolle V.; Yeganehjoo, Hoda; Katuru, Rajasekhar; DeBose-Boyd, Russell A.; Morris, Lindsey L.; Michon, Renee; Yu, Zhi-Ling; Mo, Huanbiao

    2014-01-01

    The rate-limiting enzyme of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation of nuclear lamins and Ras and dolichol-mediated glycosylation of growth factor receptors. The diterpene geranylgeraniol downregulates the level of HMG CoA reductase and suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach, and blood tumors. We evaluated the growth-suppressive activity of geranylgeraniol in human prostate carcinoma cells. Geranylgeraniol induced dose-dependent suppression of the viability of human DU145 prostate carcinoma cells (IC50 = 80 ±18 μmol/L, n =5) following 72-h incubations in 96-well plates. Cell cycle was arrested at the G1 phase with a concomitant decrease in cyclin D1 protein. Geranylgeraniol-induced apoptosis was detected by flow cytometric analysis, fluorescence microscopy following acridine orange and ethidium bromide dual staining, and caspase-3 activation. Geranylgeraniol-induced viability suppression was accompanied by concentration-dependent decrease in the level of HMG CoA reductase protein. As a nonsterol molecule that downregulates HMG CoA reductase in the presence of sterols, geranylgeraniol may have potential in the chemoprevention and/or therapy of human prostate cancer. PMID:24006306

  7. Carnitine palmitoyl transferase activity in Morris Hepatoma 7777 mitochondria and its sensitivity to malonyl CoA inhibition

    SciTech Connect

    Woldegiorgis, G.; Shrago, E.

    1986-05-01

    Earlier reports in the literature have indicated no detectable Carnitine Palymitoyl Transferase (CPT) activity in homogenates prepared from Morris Hepatoma 7777. In its study CPT activity in isolated mitochondria (mito) was measured by butanol extraction of the (/sup 3/H)palmitoyl carnitine formed as outlined by Bremer et al. Contrary to the earlier work where no appreciable activity of CPT was observed the authors find significant levels of CPT (2.6 nMol/min/mg protein) in isolated mito from Morris Hepatoma 7777 (MH 7777). The level of CPT activity observed in MH 7777 mito was, however, 36% lower compared to the host liver CPT activity (4.1 nMol/min/mg protein). The enzyme in MH 7777 mito showed 83% inhibition in the presence of 10 ..mu..M malonyl CoA, in agreement with the degree of sensitivity observed with the host liver isolated mito. On freeze thawing host mito, total CPT activity increased and the sensitivity of the enzyme to malonyl CoA decreased. Frozen thawed MH 7777 mito showed a similar response to malonyl CoA but no change in the total CPT level was observed. The authors results establish for the first time the presence of a malonyl CoA sensitive CPT in MH 7777 mito, which may have slightly different properties from normal due to the membrane environment of the enzyme.

  8. Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase.

    PubMed

    McDonald, Allison E; Amirsadeghi, Sasan; Vanlerberghe, Greg C

    2003-12-01

    The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.

  9. Human acyl-CoA dehydrogenase-9 plays a novel role in the mitochondrial beta-oxidation of unsaturated fatty acids.

    PubMed

    Ensenauer, Regina; He, Miao; Willard, Jan-Marie; Goetzman, Eric S; Corydon, Thomas J; Vandahl, Brian B; Mohsen, Al-Walid; Isaya, Grazia; Vockley, Jerry

    2005-09-16

    Unsaturated fatty acids play an important role in the prevention of human diseases such as diabetes, obesity, cancer, and neurodegeneration. However, their oxidation in vivo by acyl-CoA dehydrogenases (ACADs) that catalyze the first step of each cycle of mitochondrial fatty acid beta-oxidation is not entirely understood. Recently, a novel ACAD (ACAD-9) of unknown function that is highly homologous to human very-long-chain acyl-CoA dehydrogenase was identified by large-scale random sequencing. To characterize its enzymatic role, we have expressed ACAD-9 in Escherichia coli, purified it, and determined its pattern of substrate utilization. The N terminus of the mature form of the enzyme was identified by in vitro mitochondrial import studies of precursor protein. A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit. Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane. Gel filtration analysis indicated that, like very-long-chain acyl-CoA dehydrogenase, ACAD-9 is a dimer, in contrast to the other known ACADs, which are tetramers. Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA). These results suggest a previously unrecognized role for ACAD-9 in the mitochondrial beta-oxidation of long-chain unsaturated fatty acids. Because of the substrate specificity and abundance of ACAD-9 in brain, we speculate that it may play a role in the turnover of lipid membrane unsaturated fatty acids that are essential for membrane integrity and structure.

  10. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

  11. Physiological Consequences of Compartmentalized Acyl-CoA Metabolism*

    PubMed Central

    Cooper, Daniel E.; Young, Pamela A.; Klett, Eric L.; Coleman, Rosalind A.

    2015-01-01

    Meeting the complex physiological demands of mammalian life requires strict control of the metabolism of long-chain fatty acyl-CoAs because of the multiplicity of their cellular functions. Acyl-CoAs are substrates for energy production; stored within lipid droplets as triacylglycerol, cholesterol esters, and retinol esters; esterified to form membrane phospholipids; or used to activate transcriptional and signaling pathways. Indirect evidence suggests that acyl-CoAs do not wander freely within cells, but instead, are channeled into specific pathways. In this review, we will discuss the evidence for acyl-CoA compartmentalization, highlight the key modes of acyl-CoA regulation, and diagram potential mechanisms for controlling acyl-CoA partitioning. PMID:26124277

  12. OUTCROP-BASED HIGH RESOLUTION GAMMA-RAY CHARACTERIZATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA). CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The COA supplies drinking water to a number of municipalities in central Oklahoma. Two major stratigraphic units in the COA, the Garber Sandstone and Wellington Formation, contain naturally occurring arsenic that exceeds government mandated drinking-water standards (EPA, 2001). ...

  13. Effects of hypo- and hyperthyroidism on rat liver microsomal long-chain fatty acyl-CoA synthetase and hydrolase

    SciTech Connect

    Dang, A.Q.; Faas, F.H.; Carter, W.J.

    1986-05-01

    The effects of hyperthyroidism (hyperT/sub 3/), (tri-iodothryonine (T/sub 3/) injected rats), and hypothyroidism (hypoT/sub 3/) (thyroidectomized rats) on the activation of fatty acids by a microsomal long-chain fatty acyl-CoA (LCA-CoA) synthetase and the degradation of LCA-CoA by a microsomal LCA-CoA hydrolase was determined. MAS was assayed by measuring the (1-/sup 14/C)-palmitate or -1-/sup 14/C) oleate incorporated into its water soluble CoA ester. MAH was assayed spectrophotomerically by following the reduction of 5',5'-dithiobis-(2-nitrobenzoic acid) by the CoA released from palmitoyl-CoA or oleoyl-CoA. Enzyme activities are given as mean (nmoles/mg/min) +/- SEM. MAS activities were decreased 36-44% (p < 0.01) in both hypoT/sub 3/ and hyperT/sub 3/ (controls = 101 +/- 4 (n = 11, (1-/sup 14/C)-palmitate) of 72 +/- 2 (n = 5,(1-/sup 14/C)oleate)). These decreases may contribute to the decreased triacelyglycerol (TG) and phospholipid contents in the hyperT/sub 3/ liver and the decreased clearance rate of plasma TG in the hypoT/sub 3/. MAH was decreased 27-42% (p<0.01) only in hypoT/sub 3/ (controls = 77 +/- 3 (n = 11, palmitoyl-CoA) or 45 +/- 1 (n = 5, oleoyl-CoA)). This decrease was corrected by T/sub 3/ treatment. Since the decreased MAH would increase the availability of LCA-CoA, it may contribute to the increased TG synthesis in hypoT/sub 3/.

  14. Expression of alternative oxidase in tomato

    SciTech Connect

    Kakefuda, M.; McIntosh, L. )

    1990-05-01

    Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

  15. Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates†

    PubMed Central

    Fontes, Rui; Günther Sillero, Maria A.; Sillero, Antonio

    1998-01-01

    Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5′-tetraphosphate (p4A) and adenosine 5′-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-O-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), and adenosine(5′)pentaphospho(5′)adenosine (Ap5A) are also substrates of the reaction yielding p4(d)A in the presence of tripolyphosphate (P3). UTP, CTP, and AMP are not substrates of the reaction. The Km values for ATP and P3 are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl2 or CoCl2 equimolecular with the sum of ATP and P3. The relative rates of synthesis of p4A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p4A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p4A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap4A (from ATP), Ap5A (from p4A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap4N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates). PMID:9620965

  16. Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed.

    PubMed

    Cahoon, E B; Coughlan, S J; Shanklin, J

    1997-04-01

    A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1delta9)* and cis-vaccenic (18:1delta11) acids. Extracts of Escherichia coli that express the milkweed cDNA catalyzed delta9 desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known delta9-stearoyl (18:0)-ACP desaturases. Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized delta9-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a delta9-18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.

  17. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

    SciTech Connect

    Byers, D.M.; Meighen, E.A.

    1985-09-01

    Pulse-chase experiments with (/sup 3/H)tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a /sup 3/H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi (/sup 3/H)acylprotein and (/sup 3/H)palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (34K) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.

  18. Model simulations of cooking organic aerosol (COA) over the UK using estimates of emissions based on measurements at two sites in London

    NASA Astrophysics Data System (ADS)

    Ots, Riinu; Vieno, Massimo; Allan, James D.; Reis, Stefan; Nemitz, Eiko; Young, Dominique E.; Coe, Hugh; Di Marco, Chiara; Detournay, Anais; Mackenzie, Ian A.; Green, David C.; Heal, Mathew R.

    2016-11-01

    Cooking organic aerosol (COA) is currently not included in European emission inventories. However, recent positive matrix factorization (PMF) analyses of aerosol mass spectrometer (AMS) measurements have suggested important contributions of COA in several European cities. In this study, emissions of COA were estimated for the UK, based on hourly AMS measurements of COA made at two sites in London (a kerbside site in central London and an urban background site in a residential area close to central London) for the full calendar year of 2012 during the Clean Air for London (ClearfLo) campaign. Iteration of COA emissions estimates and subsequent evaluation and sensitivity experiments were conducted with the EMEP4UK atmospheric chemistry transport modelling system with a horizontal resolution of 5 km × 5 km. The spatial distribution of these emissions was based on workday population density derived from the 2011 census data. The estimated UK annual COA emission was 7.4 Gg per year, which is an almost 10 % addition to the officially reported UK national total anthropogenic emissions of PM2.5 (82 Gg in 2012), corresponding to 320 mg person-1 day-1 on average. Weekday and weekend diurnal variation in COA emissions were also based on the AMS measurements. Modelled concentrations of COA were then independently evaluated against AMS-derived COA measurements from another city and time period (Manchester, January-February 2007), as well as with COA estimated by a chemical mass balance model of measurements for a 2-week period at the Harwell rural site (˜ 80 km west of central London). The modelled annual average contribution of COA to ambient particulate matter (PM) in central London was between 1 and 2 µg m-3 (˜ 20 % of total measured OA1) and between 0.5 and 0.7 µg m-3 in other major cities in England (Manchester, Birmingham, Leeds). It was also shown that cities smaller than London can have a central hotspot of population density of smaller

  19. Acylated flavonol glycoside from Platanus orientalis.

    PubMed

    Tantry, Mudasir A; Akbar, Seema; Dar, Javid A; Irtiza, Syed; Galal, Ahmed; Khuroo, Mohammad A; Ghazanfar, Khalid

    2012-03-01

    The ethylacetate and n-butanol fractions of ethanolic extract of Platanus orientalis leaves led to the isolation of new acylated flavonol glycoside as 3',5,7-trihydroxy-4'-methoxyflavonol 3-[O-2-O-(2,4-Dihydroxy)-E-cinnamoyl-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl (1→2)]-β-D-glucopyranoside, along with seven known compounds. All the compounds were characterized by NMR including 2D NMR techniques. The isolates were evaluated for NF-κB, nitric oxide (NO), aromatase and QR2 chemoprevention activities and some of them appeared to be modestly active.

  20. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  1. Pre-heating and polyphenol oxidase inhibition impact on extraction of purple sweet potato anthocyanins.

    PubMed

    de Aguiar Cipriano, Paula; Ekici, Lutfiye; Barnes, Ryan C; Gomes, Carmen; Talcott, Stephen T

    2015-08-01

    Purple sweet potatoes (PSP) have been used as a natural food colorant with high acylated anthocyanins concentrations. Commercially extracting pigments from PSP can be challenging due to firm texture and high polyphenol oxidase (PPO) content. These studies evaluated hot water immersions (30, 50, 70, and 90°C for 10 min) as pre-heating treatments and addition of PPO inhibitors (citric acid, oxalic acid, and sodium borate) to aqueous extraction solutions to aid pigment recovery. Predominant PSP anthocyanins included acylated cyanidin or peonidin derivatives. Non-pigmented cinnamates acted as oxidase substrates and induced co-oxidation reactions with anthocyanins. Pre-heating PSP significantly increased polyphenolic yields in a temperature-dependent manner, consistent with tissue softening and PPO inactivation. The use of solvent modifiers in the extraction solution associated with heat helped minimize enzyme action and increased polyphenolic recovery. Minimizing the impact of PPO with heat was critical to the extraction and recovery of PSP anthocyanins, suitable for food use.

  2. Progress toward Understanding Protein S-acylation: Prospective in Plants.

    PubMed

    Li, Yaxiao; Qi, Baoxiu

    2017-01-01

    S-acylation, also known as S-palmitoylation or palmitoylation, is a reversible post-translational lipid modification in which long chain fatty acid, usually the 16-carbon palmitate, covalently attaches to a cysteine residue(s) throughout the protein via a thioester bond. It is involved in an array of important biological processes during growth and development, reproduction and stress responses in plant. S-acylation is a ubiquitous mechanism in eukaryotes catalyzed by a family of enzymes called Protein S-Acyl Transferases (PATs). Since the discovery of the first PAT in yeast in 2002 research in S-acylation has accelerated in the mammalian system and followed by in plant. However, it is still a difficult field to study due to the large number of PATs and even larger number of putative S-acylated substrate proteins they modify in each genome. This is coupled with drawbacks in the techniques used to study S-acylation, leading to the slower progress in this field compared to protein phosphorylation, for example. In this review we will summarize the discoveries made so far based on knowledge learnt from the characterization of protein S-acyltransferases and the S-acylated proteins, the interaction mechanisms between PAT and its specific substrate protein(s) in yeast and mammals. Research in protein S-acylation and PATs in plants will also be covered although this area is currently less well studied in yeast and mammalian systems.

  3. Progress toward Understanding Protein S-acylation: Prospective in Plants

    PubMed Central

    Li, Yaxiao; Qi, Baoxiu

    2017-01-01

    S-acylation, also known as S-palmitoylation or palmitoylation, is a reversible post-translational lipid modification in which long chain fatty acid, usually the 16-carbon palmitate, covalently attaches to a cysteine residue(s) throughout the protein via a thioester bond. It is involved in an array of important biological processes during growth and development, reproduction and stress responses in plant. S-acylation is a ubiquitous mechanism in eukaryotes catalyzed by a family of enzymes called Protein S-Acyl Transferases (PATs). Since the discovery of the first PAT in yeast in 2002 research in S-acylation has accelerated in the mammalian system and followed by in plant. However, it is still a difficult field to study due to the large number of PATs and even larger number of putative S-acylated substrate proteins they modify in each genome. This is coupled with drawbacks in the techniques used to study S-acylation, leading to the slower progress in this field compared to protein phosphorylation, for example. In this review we will summarize the discoveries made so far based on knowledge learnt from the characterization of protein S-acyltransferases and the S-acylated proteins, the interaction mechanisms between PAT and its specific substrate protein(s) in yeast and mammals. Research in protein S-acylation and PATs in plants will also be covered although this area is currently less well studied in yeast and mammalian systems. PMID:28392791

  4. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    SciTech Connect

    Ohlrogge, John B.

    2013-11-05

    Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

  5. Acyl peptidic siderophores: structures, biosyntheses and post-assembly modifications.

    PubMed

    Kem, Michelle P; Butler, Alison

    2015-06-01

    Acyl peptidic siderophores are produced by a variety of bacteria and possess unique amphiphilic properties. Amphiphilic siderophores are generally produced in a suite where the iron(III)-binding headgroup remains constant while the fatty acid appendage varies by length and functionality. Acyl peptidic siderophores are commonly synthesized by non-ribosomal peptide synthetases; however, the method of peptide acylation during biosynthesis can vary between siderophores. Following biosynthesis, acyl siderophores can be further modified enzymatically to produce a more hydrophilic compound, which retains its ferric chelating abilities as demonstrated by pyoverdine from Pseudomonas aeruginosa and the marinobactins from certain Marinobacter species. Siderophore hydrophobicity can also be altered through photolysis of the ferric complex of certain β-hydroxyaspartic acid-containing acyl peptidic siderophores.

  6. Xanthomonas campestris RpfB is a fatty Acyl-CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase.

    PubMed

    Bi, Hongkai; Yu, Yonghong; Dong, Huijuan; Wang, Haihong; Cronan, John E

    2014-07-01

    In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high

  7. Conformational transitions of cinnamoyl CoA reductase 1 from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant D; Khan, Bashir M; Gaikwad, Sushama M

    2014-03-01

    Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2M GdnHCl (as compared to drastic loss of native structure in 2M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein.

  8. Acyl-CoA binding proteins: multiplicity and function.

    PubMed

    Gossett, R E; Frolov, A A; Roths, J B; Behnke, W D; Kier, A B; Schroeder, F

    1996-09-01

    The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

  9. Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.

    PubMed

    Lee, C Y; Meighen, E A

    1997-04-04

    The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.

  10. Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition.

    PubMed

    Ye, Zi-Wei; Lung, Shiu-Cheung; Hu, Tai-Hua; Chen, Qin-Fang; Suen, Yung-Lee; Wang, Mingfu; Hoffmann-Benning, Susanne; Yeung, Edward; Chye, Mee-Len

    2016-12-01

    Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven β-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.

  11. Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

    PubMed

    Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

    2015-10-27

    MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

  12. Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

    PubMed Central

    Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

    2015-01-01

    MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

  13. The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake.

    PubMed

    Poppelreuther, Margarete; Rudolph, Berenice; Du, Chen; Großmann, Regina; Becker, Melanie; Thiele, Christoph; Ehehalt, Robert; Füllekrug, Joachim

    2012-05-01

    Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leaflet of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced by RNA interference of ACSL3. In conclusion, the structural organization of ACSL3 allows the fast and efficient movement from the ER to emerging LDs. ACSL3 not only esterifies fatty acids with CoA but is also involved in the cellular uptake of fatty acids, presumably indirectly by metabolic trapping. The unique localization of the acyl-CoA synthetase ACSL3 on LDs suggests a function in the local synthesis of lipids.

  14. Real-Time Decision Support for Course of Action/Enemy Course of Action (COA/ECOA) Analysis

    DTIC Science & Technology

    2005-10-01

    used. 3. A domain ontology must be given. Based on these assumptions, we propose the CAFSIN solution, standing for COA Analysis based on Fuzzified...the Pentagon” and “DoD” are wired together as the same word and are represented as a single node). 2. Polysemy : words with different meanings in the...Given an ontology with these requirements satisfied, a standard hashing function may be used to directly identify a specific node in the ontology

  15. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Khan, Bashir M

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.

  16. Correlation of ATP citrate lyase and acetyl CoA levels with trichothecene production in Fusarium graminearum.

    PubMed

    Sakamoto, Naoko; Tsuyuki, Rie; Yoshinari, Tomoya; Usuma, Jermnak; Furukawa, Tomohiro; Nagasawa, Hiromichi; Sakuda, Shohei

    2013-11-21

    The correlation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride.

  17. SUBSURFACE WELL-LOG CORRELATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA), CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The fluvial Garber Sandstone and the underlying Wellington Formation are important sources of drinking water in central Oklahoma. These formations, which make up much of the COA, consist of amalgamated sandstones with some interbedded mudstones, siltstones, and local mudstone- a...

  18. [Alternative oxidase - never ending story].

    PubMed

    Szal, Bożena; Rychter, Anna M

    2016-01-01

    Investigations of plant cyanide resistant respiration lead to the discovery in mitochondrial respiratory chain of the second terminal oxidase, alternative oxidase (AOX). AOX transfers electrons from reduced ubiquinone to oxygen omitting two coupling places thus lowering energetic efficiency of respiration. The presence of AOX was shown in all plants and also in some fungi, mollusca and protista. In termogenic plants the activity of AOX is connected with heat production. In other organisms AOX activity is important for maintaining metabolic homeostasis (carbon metabolism, cell redox state and energy demand) and ROS homeostasis. In this article structure of plant AOX protein and the regulation on molecular levels was described. Possible role of AOX as stress marker was pointed and the possibility of using AOX in human gene therapy was discussed.

  19. Lysyl oxidase in cancer research.

    PubMed

    Perryman, Lara; Erler, Janine T

    2014-01-01

    Metastasis is the main reason for cancer-associated deaths and therapies are desperately needed to target the progression of cancer. Lysyl oxidase (LOX) plays a pivotal role in cancer progression, including metastasis, and is therefore is an attractive therapeutic target. In this review we will breakdown the process of cancer progression and the various roles that LOX plays has in the advancement of cancer. We will highlight why LOX is an exciting therapeutic target for the future.

  20. The terminal oxidases of Paracoccus denitrificans.

    PubMed

    de Gier, J W; Lübben, M; Reijnders, W N; Tipker, C A; Slotboom, D J; van Spanning, R J; Stouthamer, A H; van der Oost, J

    1994-07-01

    Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (delta ctaDI, delta ctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. This protohaem-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used. In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

  1. Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes.

    PubMed

    Goepfert, Simon; Vidoudez, Charles; Tellgren-Roth, Christian; Delessert, Syndie; Hiltunen, J Kalervo; Poirier, Yves

    2008-12-01

    Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.

  2. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  3. Differential turnover of phospholipid acyl groups in mouse peritoneal macrophages

    SciTech Connect

    Kuwae, T.; Schmid, P.C.; Johnson, S.B.; Schmid, H.H. )

    1990-03-25

    Phospholipid acyl turnover was assessed in mouse peritoneal exudate cells which consisted primarily of macrophages. The cells were incubated for up to 5 h in media containing 40% H218O, and uptake of 18O into ester carbonyls of phospholipids was determined by gas chromatography-mass spectrometry of hydrogenated methyl esters. The uptake was highest in choline phospholipids and phosphatidylinositol, less in ethanolamine phospholipids, and much less in phosphatidylserine. Acyl groups at the sn-1 and sn-2 positions of diacyl glycerophospholipids, including arachidonic and other long-chain polyunsaturated fatty acids, acquired 18O at about the same rate. Acyl groups of alkylacyl glycerophosphocholine exhibited lower rates of 18O uptake, and acyl groups of ethanolamine plasmalogens (alkenylacyl glycerophosphoethanolamines) acquired only minimal amounts of 18O within 5 h, indicating a low average acyl turnover via free fatty acids. Pulse experiments with exogenous 3H-labeled arachidonic acid supported the concept that acylation of alkenyl glycerophosphoethanolamine occurs by acyl transfer from other phospholipids rather than via free fatty acids and acyl-CoA. The 18O content of intracellular free fatty acids increased gradually over a 5-h period, whereas in extracellular free fatty acids it reached maximal 18O levels within the first hour. Arachidonate and other long-chain polyunsaturated fatty acids were found to participate readily in deacylation-reacylation reactions but were present only in trace amounts in the free fatty acid pools inside and outside the cells. We conclude that acyl turnover of macrophage phospholipids through hydrolysis and reacylation is rapid but tightly controlled so that appreciable concentrations of free arachidonic acid do not occur.

  4. Phosphatidylglycerol of rat lung. Intracellular sites of formation de novo and acyl species pattern in mitochondria, microsomes and surfactant.

    PubMed Central

    Schlame, M; Rüstow, B; Kunze, D; Rabe, H; Reichmann, G

    1986-01-01

    The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant. PMID:3827844

  5. Defective Pollen Wall is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase

    SciTech Connect

    Shi, J.; Shanklin, J.; Tan, H.; Yu, X.-H.; Liu, Y.; Liang, W.; Ranathunge, K.; Franke, R. B.; Schreiber, L.; Wang, Y.; Kai, G.; Ma, H.; Zhang, D.

    2011-06-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

  6. Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase.

    PubMed

    Shi, Jing; Tan, Hexin; Yu, Xiao-Hong; Liu, Yuanyun; Liang, Wanqi; Ranathunge, Kosala; Franke, Rochus Benni; Schreiber, Lukas; Wang, Yujiong; Kai, Guoying; Shanklin, John; Ma, Hong; Zhang, Dabing

    2011-06-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

  7. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

  8. Role of acyl carrier protein isoforms in plant lipid metabolism

    SciTech Connect

    Not Available

    1990-01-01

    Although acyl carrier protein (ACP) is the best studied protein in plant fatty acid biosynthesis, the in vivo forms of ACPs and their steady state pools have not been examined previously in either seed or leaf. Information about the relative pool sizes of free ACP and its acyl-ACP intermediates is essential for understanding regulation of de novo fatty acid biosynthesis in plants. In this study we utilized antibodies directed against spinach ACP as a sensitive assay to analyze the acyl groups while they were still covalently attached to ACPs. 4 refs., 4 figs.

  9. The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

    NASA Astrophysics Data System (ADS)

    Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

    2017-03-01

    We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

  10. Acylated cyanidin 3-sambubioside-5-glucosides in Matthiola incana.

    PubMed

    Saito, N; Tatsuzawa, F; Nishiyama, A; Yokoi, M; Shigihara, A; Honda, T

    1995-03-01

    Four acylated cyanidin 3-sambubioside-5-glucosides were isolated from purple-violet flowers of Matthiola incana and their structures were determined by chemical and spectroscopic methods. Three acylated anthocyanins were cyanidin 3-O-(6-O-acyl-2-O-(2-O-sinapyl-beta-D-xylopyranosyl)-beta-D- glucopyranosides)-5-O-(6-O-malonyl-beta-D-glucopyranosides), in which the acyl group is p-coumaryl, caffeyl or ferulyl, respectively. The remaining pigment is free from malonic acid and was identified as cyanidin 3-O-(6-O-trans-ferulyl-2-O-(2- O-trans-sinapyl-beta-D-xylopyranosyl)-beta-D-glucopyranoside)-5-O- (beta-D-glucopyranoside). Analysis of the anthocyanin constituents in 16 purple-violet cultivars revealed that they contained the above triacylated anthocyanins in variable amounts as main pigments. An aromatic pair of pigments containing sinapic and ferulic acids are considered to produce an important intramolecular effect, making bluish colours in these flowers.

  11. Acyl Meldrum's acid derivatives: application in organic synthesis

    NASA Astrophysics Data System (ADS)

    Janikowska, K.; Rachoń, J.; Makowiec, S.

    2014-07-01

    This review is focused on an important class of Meldrum's acid derivatives commonly known as acyl Meldrum's acids. The preparation methods of these compounds are considered including the recently proposed and rather rarely used ones. The chemical properties of acyl Meldrum's acids are described in detail, including thermal stability and reactions with various nucleophiles. The possible mechanisms of these transformations are analyzed. The bibliography includes 134 references.

  12. Oxidative activation of dihydropyridine amides to reactive acyl donors.

    PubMed

    Funder, Erik Daa; Trads, Julie B; Gothelf, Kurt V

    2015-01-07

    Amides of 1,4-dihydropyridine (DHP) are activated by oxidation for acyl transfer to amines, alcohols and thiols. In the reduced form the DHP amide is stable towards reaction with amines at room temperature. However, upon oxidation with DDQ the acyl donor is activated via a proposed pyridinium intermediate. The activated intermediate reacts with various nucleophiles to give amides, esters, and thio-esters in moderate to high yields.

  13. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  14. Acylated pregnane glycosides from Caralluma quadrangula.

    PubMed

    Abdallah, Hossam M; Osman, Abdel-Moneim M; Almehdar, Hussein; Abdel-Sattar, Essam

    2013-04-01

    In a previous study, the methanolic extract as well as the chloroform fraction of the aerial parts of Caralluma quadrangula (Forssk.) N.E.Br. indigenous to Saudi Arabia showed significant in vitro cytotoxic activity against breast cancer (MCF7) cell line. In a biologically-guided fractionation approach, four acylated pregnane glycosides were isolated from the chloroform fraction of C. quadrangula. The structures of the isolated compounds were elucidated by the analysis of their MS and NMR data. The compounds were identified as 12,20-di-O-benzoylboucerin 3-O-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (1), 12,20-di-O-benzoylboucerin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (2), 12,20-di-O-benzoylboucerin 3-O-β-D-glucopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (3) and 12,20-di-O-benzoyl-3β,5α,12β,14β,20-pentahydroxy-(20R)-pregn-6-ene 3-O-β-D-glucopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (4). The isolated compounds were tested for their cytotoxic activity against breast cancer (MCF7) cell line.

  15. Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase | Office of Cancer Genomics

    Cancer.gov

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease.

  16. Mitochondrial cytochrome c oxidase deficiency.

    PubMed

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-03-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance of studying different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy.

  17. Immunological comparison of sulfite oxidase

    SciTech Connect

    Pollock, V.; Barber, M.J. )

    1991-03-11

    Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibited S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.

  18. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  19. 3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis DPN7T: crystal structure and function of a desulfinase with an acyl-CoA dehydrogenase fold

    PubMed Central

    Schürmann, Marc; Meijers, Rob; Schneider, Thomas R.; Steinbüchel, Alexander; Cianci, Michele

    2015-01-01

    3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase (AcdDPN7; EC 3.13.1.4) was identified during investigation of the 3,3′-dithiodipropionic acid (DTDP) catabolic pathway in the betaproteobacterium Advenella mimigardefordensis strain DPN7T. DTDP is an organic disulfide and a precursor for the synthesis of polythioesters (PTEs) in bacteria, and is of interest for biotechnological PTE production. AcdDPN7 catalyzes sulfur abstraction from 3SP-CoA, a key step during the catabolism of DTDP. Here, the crystal structures of apo AcdDPN7 at 1.89 Å resolution and of its complex with the CoA moiety from the substrate analogue succinyl-CoA at 2.30 Å resolution are presented. The apo structure shows that AcdDPN7 belongs to the acyl-CoA dehydrogenase superfamily fold and that it is a tetramer, with each subunit containing one flavin adenine dinucleotide (FAD) molecule. The enzyme does not show any dehydrogenase activity. Dehydrogenase activity would require a catalytic base (Glu or Asp residue) at either position 246 or position 366, where a glutamine and a glycine are instead found, respectively, in this desulfinase. The positioning of CoA in the crystal complex enabled the modelling of a substrate complex containing 3SP-CoA. This indicates that Arg84 is a key residue in the desulfination reaction. An Arg84Lys mutant showed a complete loss of enzymatic activity, suggesting that the guanidinium group of the arginine is essential for desulfination. AcdDPN7 is the first desulfinase with an acyl-CoA dehydrogenase fold to be reported, which underlines the versatility of this enzyme scaffold. PMID:26057676

  20. Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).

    PubMed

    Venkatraman, Janani; Bhat, Jyothi; Solapure, Suresh M; Sandesh, Jatheendranath; Sarkar, Debasmita; Aishwarya, Sundaram; Mukherjee, Kakoli; Datta, Santanu; Malolanarasimhan, Krishnan; Bandodkar, Balachandra; Das, Kaveri S

    2012-03-01

    The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.

  1. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, but the acyl-galactose acyl composition varies with the plant species and applied stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Head group acylation of monogalactosyldiacylglycerol is a plant lipid modification occurring during bacterial infection. Little is known about the range of stresses that induce this lipid modification, the molecular species induced, and the function of the modification. Lipidomic analysis using trip...

  2. C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight.

    PubMed

    Mera, Paula; Bentebibel, Assia; López-Viñas, Eduardo; Cordente, Antonio G; Gurunathan, Chandrashekaran; Sebastián, David; Vázquez, Irene; Herrero, Laura; Ariza, Xavier; Gómez-Puertas, Paulino; Asins, Guillermina; Serra, Dolors; García, Jordi; Hegardt, Fausto G

    2009-03-15

    Central nervous system administration of C75 produces hypophagia and weight loss in rodents identifying C75 as a potential drug against obesity and type 2 diabetes. However, the mechanism underlying this effect is unknown. Here we show that C75-CoA is generated chemically, in vitro and in vivo from C75 and that it is a potent inhibitor of carnitine palmitoyltranferase 1 (CPT1), the rate-limiting step of fatty-acid oxidation. Three-D docking and kinetic analysis support the inhibitory effect of C75-CoA on CPT1. Central nervous system administration of C75 in rats led to C75-CoA production, inhibition of CPT1 and lower body weight and food intake. Our results suggest that inhibition of CPT1, and thus increased availability of fatty acids in the hypothalamus, contribute to the pharmacological mechanism of C75 to decrease food intake.

  3. A pathogenic fungi diphenyl ether phytotoxin targets plant enoyl (acyl carrier protein) reductase.

    PubMed

    Dayan, Franck E; Ferreira, Daneel; Wang, Yan-Hong; Khan, Ikhlas A; McInroy, John A; Pan, Zhiqiang

    2008-07-01

    Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the pi-pi stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD(+). Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection.

  4. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  5. Regioselective self-acylating cyclodextrins in organic solvent

    PubMed Central

    Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

    2016-01-01

    Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods. PMID:27020946

  6. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    SciTech Connect

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; Kim, Ryeo Jin; Yang, Weili; Shorrosh, Basil; Suh, Mi Chung; Ohlrogge, John

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.

  7. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    DOE PAGES

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes ofmore » mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

  8. Regioselective self-acylating cyclodextrins in organic solvent

    NASA Astrophysics Data System (ADS)

    Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

    2016-03-01

    Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods.

  9. Dietary inhibitors of monoamine oxidase A.

    PubMed

    Dixon Clarke, Sarah E; Ramsay, Rona R

    2011-07-01

    Inhibition of monoamine oxidase is one way to treat depression and anxiety. The information now available on the pharmacokinetics of flavonoids and of the components of tobacco prompted an exploration of whether a healthy diet (with or without smoking) provides active compounds in amounts sufficient to partially inhibit monoamine oxidase. A literature search was used to identify dietary monoamine oxidase inhibitors, the levels of these compounds in foods, the pharmacokinetics of the absorption and distribution, and tissue levels observed. An estimated daily intake and the expected tissue concentrations were compared with the measured efficacies of the compounds as inhibitors of monoamine oxidases. Norharman, harman and quercetin dietary presence, pharmacokinetics, and tissue levels were consistent with significant levels reaching neuronal monoamine oxidase from the diet or smoking; 1,2,3,4-tetrahydroisoquinoline, eugenol, 1-piperoylpiperidine, and coumarin were not. Quercetin was equipotent with norharman as a monoamine oxidase A inhibitor and its metabolite, isorhamnetin, also inhibits. Total quercetin was the highest of the compounds in the sample diet. Although bioavailability was variable depending on the source, a healthy diet contains amounts of quercetin that might give sufficient amounts in brain to induce, by monoamine oxidase A inhibition, a small decrease in neurotransmitter breakdown.

  10. CoaTx-II, a new dimeric Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom with bactericidal potential: Insights into its structure and biological roles.

    PubMed

    Almeida, J R; Lancellotti, M; Soares, A M; Calderon, L A; Ramírez, D; González, W; Marangoni, S; Da Silva, S L

    2016-09-15

    Snake venoms are rich and intriguing sources of biologically-active molecules that act on target cells, modulating a diversity of physiological functions and presenting promising pharmacological applications. Lys49 phospholipase A2 is one of the multifunctional proteins present in these complex secretions and, although catalytically inactive, has a variety of biological activities, including cytotoxic, antibacterial, inflammatory, antifungal activities. Herein, a Lys49 phospholipase A2, denominated CoaTx-II from Crotalus oreganus abyssus, was purified and structurally and pharmacologically characterized. CoaTx-II was isolated with a high degree of purity by a combination of two chromatographic steps; molecular exclusion and reversed-phase high performance liquid chromatography. This toxin is dimeric with a mass of 13868.2 Da (monomeric form), as determined by mass spectrometry. CoaTx-II is rich in Arg and Lys residues and displays high identity with other Lys49 PLA2 homologues, which have high isoelectric points. The structural model of dimeric CoaTx-II shows that the toxin is non-covalently stabilized. Despite its enzymatic inactivity, in vivo CoaTx-II caused local muscular damage, characterized by increased plasma creatine kinase and confirmed by histological alterations, in addition to an inflammatory activity, as demonstrated by mice paw edema induction and pro-inflammatory cytokine IL-6 elevation. CoaTx-II also presents antibacterial activity against gram negative (Pseudomonas aeruginosa 31NM, Escherichia coli ATCC 25922) and positive (Staphyloccocus aureus BEC9393 and Rib1) bacteria. Therefore, data show that this newly purified toxin plays a central role in mediating the degenerative events associated with envenomation, in addition to demonstrating antibacterial properties, with potential for use in the development of strategies for antivenom therapy and combating antibiotic-resistant bacteria.

  11. Structural insights into sulfite oxidase deficiency.

    PubMed

    Karakas, Erkan; Wilson, Heather L; Graf, Tyler N; Xiang, Song; Jaramillo-Busquets, Sandra; Rajagopalan, K V; Kisker, Caroline

    2005-09-30

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  12. Cross sections for production of the CO(A 1 Pi)-(X 1 Sigma) fourth positive band system and O(3 S) by photodissociation of CO2

    NASA Technical Reports Server (NTRS)

    Gentieu, E. P.; Mentall, J. E.

    1972-01-01

    The CO(A 1 Pi) cross sections reported here, along with previously determined electron impact results, establish the basis for calculating CO fourth positive system volume emission rates in the Martian dayglow. Calculated volume emission rates in turn determine relative distribution of photon vs. electron impact as mechanisms for producing CO(A 1 Pi) in the Mars atmosphere. The smallness of the O(1304) cross section confirms previous indirect evidence that photodissociative excitation of CO2 is not an important source of O(3 S) in the upper atmosphere of Mars.

  13. Genistein effect on xanthine oxidase activity.

    PubMed

    Sumbayev, V V

    2001-01-01

    Genistein was defined to be an allosteric xanthine oxidase inhibitor in the concentrations 0.1-4.0 microM and xanthine oxidase activator with superoxide scavenging activity in the concentrations 5.0 microM and higher. But the most effective allosteric binding with the highest affinity was observed in the genistein concentrations 0.1-1.0 microM. Intraperitoneum injections of genistein (500 micrograms/kg) during three days with the interval 24 hours decrease xanthine oxidase activity in the liver, lung and brain of the Vistar rats.

  14. Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

    PubMed Central

    Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

    2016-01-01

    An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

  15. A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.

    PubMed

    Shin, Daniel; Frane, Nicole D; Brecht, Ryan M; Keeler, Jesse; Nagarajan, Rajesh

    2015-12-01

    Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-l-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well-documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA- and acyl-ACP-utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.

  16. Asymmetric Allylboration of Acyl Imines Catalyzed by Chiral Diols

    PubMed Central

    Lou, Sha; Moquist, Philip N.; Schaus, Scott E.

    2008-01-01

    Chiral BINOL-derived diols catalyze the enantioselective asymmetric allylboration of acyl imines. The reaction requires 15 mol% of (S)-3,3′-Ph2-BINOL as the catalyst and allyldiisopropoxyborane as the nucleophile. The reaction products are obtained in good yields (75 – 94%) and high enantiomeric ratios (95:5 – 99.5:0.5) for aromatic and aliphatic imines. High diastereoselectivities (dr > 98:2) and enantioselectivities (er > 98:2) are obtained in the reactions of acyl imines with crotyldiisopropoxyboranes. This asymmetric transformation is directly applied to the synthesis of maraviroc, the selective CCR5 antagonist with potent activity against HIV-1 infection. Mechanistic investigations of the allylboration reaction including IR, NMR, and mass spectrometry study indicate that acyclic boronates are activated by chiral diols via exchange of one of the boronate alkoxy groups with activation of the acyl imine via hydrogen bonding. PMID:18020334

  17. Identification of Unusual Phospholipid Fatty Acyl Compositions of Acanthamoeba castellanii

    PubMed Central

    Palusinska-Szysz, Marta; Kania, Magdalena; Turska-Szewczuk, Anna; Danikiewicz, Witold; Russa, Ryszard; Fuchs, Beate

    2014-01-01

    Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these

  18. Sphingomyelin interfacial behavior: the impact of changing acyl chain composition.

    PubMed Central

    Li, X M; Smaby, J M; Momsen, M M; Brockman, H L; Brown, R E

    2000-01-01

    Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico-mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in-plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes. PMID:10733971

  19. Quantum chemical study of penicillin: Reactions after acylation

    NASA Astrophysics Data System (ADS)

    Li, Rui; Feng, Dacheng; Zhu, Feng

    The density functional theory methods were used on the model molecules of penicillin to determine the possible reactions after their acylation on ?-lactamase, and the results were compared with sulbactam we have studied. The results show that, the acylated-enzyme tetrahedral intermediate can evolves with opening of ?-lactam ring as well as the thiazole ring; the thiazole ring-open products may be formed via ?-lactam ring-open product or from tetrahedral intermediate directly. Those products, in imine or enamine form, can tautomerize via hydrogen migration. In virtue of the water-assisted, their energy barriers are obviously reduced.

  20. Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes.

    PubMed

    Finnegan, Patrick M; Umbach, Ann L; Wilce, Jackie A

    2003-12-18

    The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

  1. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

  2. Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs[S

    PubMed Central

    Li, Qingling; Zhang, Shenghui; Berthiaume, Jessica M.; Simons, Brigitte; Zhang, Guo-Fang

    2014-01-01

    A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80–114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs. PMID:24367045

  3. Copper(II)/amine synergistically catalyzed enantioselective alkylation of cyclic N-acyl hemiaminals with aldehydes.

    PubMed

    Sun, Shutao; Mao, Ying; Lou, Hongxiang; Liu, Lei

    2015-07-07

    The first catalytic asymmetric alkylation of N-acyl quinoliniums with aldehydes has been described. A copper/amine synergistic catalytic system has been developed, allowing the addition of functionalized aldehydes to a wide range of electronically varied N-acyl quinoliniums in good yields with excellent enantiocontrol. The synergistic catalytic system was also effective for N-acyl dihydroisoquinoliniums and β-caboliniums, demonstrating the general applicability of the protocol in the enantioselective alkylation of diverse cyclic N-acyl hemiaminals.

  4. The Level of Circulating Octanoate Does Not Predict Ghrelin O-Acyl Transferase (GOAT)-Mediated Acylation of Ghrelin During Fasting

    PubMed Central

    Nikolayev, Alexander; Liu, Jianhua; Pezzoli, Suzan S.; Farhy, Leon S.; Patrie, James; Gaylinn, Bruce D.; Heiman, Mark; Thorner, Michael O.

    2015-01-01

    Background: Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. Objectives: To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Methods: Eight healthy young men (aged 18–28 years, body mass index range, 20.6–26.2 kg/m2) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. Results: With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14–C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. Conclusions: These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity. PMID:25337923

  5. A Cerulenin Insensitive Short Chain 3-Ketoacyl-Acyl Carrier Protein Synthase in Spinacia oleracea Leaves

    PubMed Central

    Jaworski, Jan G.; Clough, Richard C.; Barnum, Susan R.

    1989-01-01

    A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryl- and hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated. Images Figure 2 Figure 3 PMID:16666765

  6. Characterization of the JWST Pathfinder mirror dynamics using the center of curvature optical assembly (CoCOA)

    NASA Astrophysics Data System (ADS)

    Wells, Conrad; Hadaway, James B.; Olczak, Gene; Cosentino, Joseph; Johnston, John D.; Whitman, Tony; Connolly, Mark; Chaney, David; Knight, J. Scott; Telfer, Randal

    2016-07-01

    The James Webb Space Telescope (JWST) Optical Telescope Element (OTE) consists of a 6.6 m clear aperture, 18 segment primary mirror, all-reflective, three-mirror anastigmat operating at cryogenic temperatures. To verify performance of the primary mirror, a full aperture center of curvature optical null test is performed under cryogenic conditions in Chamber A at the National Aeronautics and Space Administration (NASA) Johnson Space Center (JSC) using an instantaneous phase measuring interferometer. After phasing the mirrors during the JWST Pathfinder testing, the interferometer is utilized to characterize the mirror relative piston and tilt dynamics under different facility configurations. The correlation between the motions seen on detectors at the focal plane and the interferometer validates the use of the interferometer for dynamic investigations. The success of planned test hardware improvements will be characterized by the multi-wavelength interferometer (MWIF) at the Center of Curvature Optical Assembly (CoCOA).

  7. Characterization of the JWST Pathfinder Mirror Dynamics Using the Center of Curvature Optical Assembly (CoCOA)

    NASA Technical Reports Server (NTRS)

    Wells, Conrad; Hadaway, James B.; Olczak, Gene; Cosentino, Joseph; Johnston, John D.; Whitman, Tony; Connolly, Mark; Chaney, David; Knight, J. Scott; Telfer, Randal

    2016-01-01

    The JWST (James Webb Space Telescope) Optical Telescope Element (OTE) consists of a 6.6 meter clear aperture, 18-segment primary mirror, all-reflective, three-mirror anastigmat operating at cryogenic temperatures. To verify performance of the primary mirror, a full aperture center of curvature optical null test is performed under cryogenic conditions in Chamber A at NASA Johnson Space Center using an instantaneous phase measuring interferometer. After phasing the mirrors during the JWST Pathfinder testing, the interferometer is utilized to characterize the mirror relative piston and tilt dynamics under different facility configurations. The correlation between the motions seen on detectors at the focal plane and the interferometer validates the use of the interferometer for dynamic investigations. The success of planned test hardware improvements will be characterized by the multi-wavelength interferometer (MWIF) at the Center of Curvature Optical Assembly (CoCOA).

  8. Sulfite Oxidase Activity Is Essential for Normal Sulfur, Nitrogen and Carbon Metabolism in Tomato Leaves

    PubMed Central

    Brychkova, Galina; Yarmolinsky, Dmitry; Batushansky, Albert; Grishkevich, Vladislav; Khozin-Goldberg, Inna; Fait, Aaron; Amir, Rachel; Fluhr, Robert; Sagi, Moshe

    2015-01-01

    Plant sulfite oxidase [SO; E.C.1.8.3.1] has been shown to be a key player in protecting plants against exogenous toxic sulfite. Recently we showed that SO activity is essential to cope with rising dark-induced endogenous sulfite levels in tomato plants (Lycopersicon esculentum/Solanum lycopersicum Mill. cv. Rheinlands Ruhm). Here we uncover the ramifications of SO impairment on carbon, nitrogen and sulfur (S) metabolites. Current analysis of the wild-type and SO-impaired plants revealed that under controlled conditions, the imbalanced sulfite level resulting from SO impairment conferred a metabolic shift towards elevated reduced S-compounds, namely sulfide, S-amino acids (S-AA), Co-A and acetyl-CoA, followed by non-S-AA, nitrogen and carbon metabolite enhancement, including polar lipids. Exposing plants to dark-induced carbon starvation resulted in a higher degradation of S-compounds, total AA, carbohydrates, polar lipids and total RNA in the mutant plants. Significantly, a failure to balance the carbon backbones was evident in the mutants, indicated by an increase in tricarboxylic acid cycle (TCA) cycle intermediates, whereas a decrease was shown in stressed wild-type plants. These results indicate that the role of SO is not limited to a rescue reaction under elevated sulfite, but SO is a key player in maintaining optimal carbon, nitrogen and sulfur metabolism in tomato plants. PMID:27135342

  9. Azide inhibition of urate oxidase.

    PubMed

    Gabison, Laure; Colloc'h, Nathalie; Prangé, Thierry

    2014-07-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX-UA or UOX-8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site.

  10. Azide inhibition of urate oxidase

    PubMed Central

    Gabison, Laure; Colloc’h, Nathalie; Prangé, Thierry

    2014-01-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX–UA or UOX–8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site. PMID:25005084

  11. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.

    PubMed

    Stout, Jake M; Boubakir, Zakia; Ambrose, Stephen J; Purves, Randy W; Page, Jonathan E

    2012-08-01

    The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.

  12. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-05

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling.

  13. Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

    PubMed Central

    Vick, Jacob E.; Clomburg, James M.; Blankschien, Matthew D.; Chou, Alexander; Kim, Seohyoung

    2014-01-01

    We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli. PMID:25527535

  14. Pre-exercise medium-chain triglyceride application prevents acylcarnitine accumulation in skeletal muscle from very-long-chain acyl-CoA-dehydrogenase-deficient mice.

    PubMed

    Primassin, Sonja; Tucci, Sara; Herebian, Diran; Seibt, Annette; Hoffmann, Lars; ter Veld, Frank; Spiekerkoetter, Ute

    2010-06-01

    Dietary modification with medium-chain triglyceride (MCT) supplementation is one crucial way of treating children with long-chain fatty acid oxidation disorders. Recently, supplementation prior to exercise has been reported to prevent muscular pain and rhabdomyolysis. Systematic studies to determine when MCT supplementation is most beneficial have not yet been undertaken. We studied the effects of an MCT-based diet compared with MCT administration only prior to exercise in very-long-chain acyl-CoA dehydrogenase (VLCAD) knockout (KO) mice. VLCAD KO mice were fed an MCT-based diet in same amounts as normal mouse diet containing long-chain triglycerides (LCT) and were exercised on a treadmill. Mice fed a normal LCT diet received MCT only prior to exercise. Acylcarnitine concentration, free carnitine concentration, and acyl-coenzyme A (CoA) oxidation capacity in skeletal muscle as well as hepatic lipid accumulation were determined. Long-chain acylcarnitines significantly increased in VLCAD-deficient skeletal muscle with an MCT diet compared with an LCT diet with MCT bolus prior to exercise, whereas an MCT bolus treatment significantly decreased long-chain acylcarnitines after exercise compared with an LCT diet. C8-carnitine was significantly increased in skeletal muscle after MCT bolus treatment and exercise compared with LCT and long-term MCT treatment. Increased hepatic lipid accumulation was observed in long-term MCT-treated KO mice. MCT seems most beneficial when given in a single dose directly prior to exercise to prevent acylcarnitine accumulation. In contrast, continuous MCT treatment produces a higher skeletal muscle content of long-chain acylcarnitines after exercise and increases hepatic lipid storage in VLCAD KO mice.

  15. Linear magnetoelectric effect as a signature of long-range collinear antiferromagnetic ordering in the frustrated spinel CoA l2O4

    NASA Astrophysics Data System (ADS)

    Ghara, Somnath; Ter-Oganessian, N. V.; Sundaresan, A.

    2017-03-01

    The ground state of the frustrated A -site magnetic spinel CoA l2O4 has been a controversial issue whether it is a collinear antiferromagnetic ordering or a spiral spin-liquid state, as the ratio of the two competing interactions J2/J1 lies close to the boundary between these two ground states. Here we address the magnetic ground state in CoA l2O4 with different amounts of C o2 +/A l3 + site disorder from the study of magnetoelectric effect and Monte Carlo simulations. CoA l2O4 with low site disorder exhibits a linear magnetoelectric effect below the magnetic ordering temperature. With increasing disorder, the magnetoelectric effect is suppressed and the sample with 14 % disorder exhibits a spin glass behavior without the magnetoelectric effect. Monte Carlo simulations support the experimental findings and suggest that the site disorder suppresses long-range antiferromagnetic order and induces a spin glass state. Since the linear magnetoelectric effect requires a long-range magnetic ordering, we suggest that the ground state of CoA l2O4 with low site disorder is a collinear antiferromagnet.

  16. Acyl migration kinetics of vegetable oil 1,2-diacylglycerols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The acyl migration kinetics of long-chain 1,2-diacylglycerol (1,2-DAG) to form 1,3-diacylglycerol (1,3-DAG) over the temperature range of 25 to 80 degrees Celsius were examined using proton NMR spectroscopy. The 1,2-DAG mole fraction of 0.32 at equilibrium was found to be insensitive to temperature...

  17. One-Step Conversion of Methyl Ketones to Acyl Chlorides.

    PubMed

    Zaragoza, Florencio

    2015-10-16

    Treatment of aromatic and heteroaromatic methyl ketones with sulfur monochloride and catalytic amounts of pyridine in refluxing chlorobenzene leads to the formation of acyl chlorides. Both electron-rich and electron-poor aryl methyl ketones can be used as starting materials. The resulting C1-byproduct depends on the precise reaction conditions chosen.

  18. Lubricity characteristics of seed oils modified by acylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemically modified seed oils via acylation of epoxidized and polyhydroxylated derivatives were investigated for their potential as candidates for lubrication. The native oil was preliminarily epoxidized and ring-opened in a one-pot reaction using formic acid-H2O2 followed by aqueous HCl treatment t...

  19. Mechanistic studies of malonic acid-mediated in situ acylation.

    PubMed

    Chandra, Koushik; Naoum, Johnny N; Roy, Tapta Kanchan; Gilon, Chaim; Gerber, R Benny; Friedler, Assaf

    2015-09-01

    We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules.

  20. Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

    PubMed Central

    Fritz, Kristofer S.; Green, Michelle F.; Petersen, Dennis R.; Hirschey, Matthew D.

    2013-01-01

    Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated metabolism, induce specific

  1. Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate† ,‡

    PubMed Central

    Valley, Michael P.; Fitzpatrick, Paul F.

    2006-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C–H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG‡ for C–H bond cleavage by 1.6–1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase. PMID:12741843

  2. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

    PubMed Central

    Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

  3. Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylating agents.

    PubMed

    Brown, A D; Powers, J C

    1995-08-01

    Acylated derivatives of thrombin have been made using low molecular weight acylating agents, carbamylating agents and carbonylating agents. The compounds used to acylate the active site serine include isatoic anhydrides, benzoxazinones, benzylisocyanate, N-(benzylcarbonyloxy)succinimide and p-(dimethylamino)benzoylimidazolide. The rates of acylation and deacylation were determined. The best overall inhibitors of thrombin are 2-ethoxy-4H-3,1-benzoxazin-4-one, isatoic anhydride and tert-butyl-2,4-dioxo-2H-3,1-benzoxazine-1(4H)-acetate, which have k2/Ki values of 52,700 M-1s-1, 48,900 M-1s-1 and 5400 M-1s-1, respectively. The carbamyl derivative of thrombin formed with benzylisocyanate had the slowest rate of deacylation (2.3 x 10(-7) s-1), while the ester derivative formed with 2-(N,N-dimethylamino)methylimino-4H-3,1-benzoxazin-4-one had the fastest rate of deacylation (1.9 x 10(-4) s-1).

  4. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

    PubMed Central

    Jones, A; Davies, H M; Voelker, T A

    1995-01-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

  5. Parenteral and enteral metabolism of anaplerotic triheptanoin in normal rats. II. Effects on lipolysis, glucose production, and liver acyl-CoA profile

    PubMed Central

    Gu, Lei; Zhang, Guo-Fang; Kombu, Rajan S.; Allen, Frederick; Kutz, Gerd; Brewer, Wolf-Ulrich; Roe, Charles R.

    2010-01-01

    The anaplerotic odd-medium-chain triglyceride triheptanoin is used in clinical trials for the chronic dietary treatment of patients with long-chain fatty acid oxidation disorders. We previously showed (Kinman RP, Kasumov T, Jobbins KA, Thomas KR, Adams JE, Brunengraber LN, Kutz G, Brewer WU, Roe CR, Brunengraber H. Am J Physiol Endocrinol Metab 291: E860–E866, 2006) that the intravenous infusion of triheptanoin increases lipolysis traced by the turnover of glycerol. In this study, we tested whether lipolysis induced by triheptanoin infusion is accompanied by the potentially harmful release of long-chain fatty acids. Rats were infused with heptanoate ± glycerol or triheptanoin. Intravenous infusion of triheptanoin at 40% of caloric requirement markedly increased glycerol endogenous Ra but not oleate endogenous Ra. Thus, the activation of lipolysis was balanced by fatty acid reesterification in the same cells. The liver acyl-CoA profile showed the accumulation of intermediates of heptanoate β-oxidation and C5-ketogenesis and a decrease in free CoA but no evidence of metabolic perturbation of liver metabolism such as propionyl overload. Our data suggest that triheptanoin, administered either intravenously or intraduodenally, could be used for intensive care and nutritional support of metabolically decompensated long-chain fatty acid oxidation disorders. PMID:19903863

  6. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function

    PubMed Central

    Seedorf, Udo; Raabe, Martin; Ellinghaus, Peter; Kannenberg, Frank; Fobker, Manfred; Engel, Thomas; Denis, Simone; Wouters, Fred; Wirtz, Karel W.A.; Wanders, Ronald J.A.; Maeda, Nobuyo; Assmann, Gerd

    1998-01-01

    Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(−/−) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein. PMID:9553048

  7. Biochemical characteristics of AtFAR2, a fatty acid reductase from Arabidopsis thaliana that reduces fatty acyl-CoA and -ACP substrates into fatty alcohols.

    PubMed

    Doan, Thuy T P; Carlsson, Anders S; Stymne, Sten; Hofvander, Per

    2016-01-01

    Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.

  8. Defective Pollen Wall Is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase[C][W][OA

    PubMed Central

    Shi, Jing; Tan, Hexin; Yu, Xiao-Hong; Liu, Yuanyun; Liang, Wanqi; Ranathunge, Kosala; Franke, Rochus Benni; Schreiber, Lukas; Wang, Yujiong; Kai, Guoying; Shanklin, John; Ma, Hong; Zhang, Dabing

    2011-01-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots. PMID:21705642

  9. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  10. Acylation of Glucagon-Like Peptide-2: Interaction with Lipid Membranes and In Vitro Intestinal Permeability

    PubMed Central

    Trier, Sofie; Linderoth, Lars; Bjerregaard, Simon; Andresen, Thomas Lars; Rahbek, Ulrik Lytt

    2014-01-01

    Background Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation as well as increasing enzymatic stability without disrupting biological potency. Acylation has furthermore been shown to increase interactions with the lipid membranes of mammalian cells. The extent to which such interactions hinder or benefit delivery of acylated peptide drugs across cellular barriers such as the intestinal epithelia is currently unknown. The present study investigates the effect of acylating peptide drugs from a drug delivery perspective. Purpose We hypothesize that the membrane interaction is an important parameter for intestinal translocation, which may be used to optimize the acylation chain length for intestinal permeation. This work aims to characterize acylated analogues of the intestinotrophic Glucagon-like peptide-2 by systematically increasing acyl chain length, in order to elucidate its influence on membrane interaction and intestinal cell translocation in vitro. Results Peptide self-association and binding to both model lipid and cell membranes was found to increase gradually with acyl chain length, whereas translocation across Caco-2 cells depended non-linearly on chain length. Short and medium acyl chains increased translocation compared to the native peptide, but long chain acylation displayed no improvement in translocation. Co-administration of a paracellular absorption enhancer was found to increase translocation irrespective of acyl chain length, whereas a transcellular enhancer displayed increased synergy with the long chain acylation. Conclusions These results show that membrane interactions play a prominent role during intestinal translocation of an acylated peptide. Acylation benefits permeation for shorter and medium chains due to increased membrane interactions, however, for longer chains insertion in the membrane becomes dominant and hinders translocation, i.e. the peptides get ‘stuck’ in the cell

  11. Effect of naphthalene on cytochrome oxidase activity

    SciTech Connect

    Harmon, H.J.

    1988-01-01

    Previous reports have demonstrated that naphthalene inhibits oxygen consumption in Daphnia magna tissue culture cells, and intact mitochondria and submitochondrial particles. These studies were extended to algal mitochondrial respiration as well as photosynthetic activity. The authors were able to demonstrate the specific site of apparent respiratory inhibition to be coenzyme Q (ubiquinone, UQ) and later to demonstrate the molecular basis of this inhibition at ubiquinone. The authors previously could not demonstrate an effect of naphthalene on cytochrome oxidase activity. However, the observation that naphthalene can stimulate respiration in algae prompted the reinvestigation of the effect of naphthalene on the kinetics of cytochrome oxidase. Cytochrome oxidase is a multi-subunit membranous protein responsible for the oxidation of cytochrome c and the reduction of molecular oxygen to water. Because of the complicated nature and mechanism of this enzyme, the potential exists for multiple and possibly opposite effects of naphthalene on its function.

  12. Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

    PubMed

    Yuan, L; Voelker, T A; Hawkins, D J

    1995-11-07

    The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good

  13. Importance of hydrophobic interaction between a SoxB-type cytochrome c oxidase with its natural substrate cytochrome c-551 and its mutants.

    PubMed

    Kagekawa, Sayaka; Mizukami, Makoto; Noguchi, Shunsuke; Sakamoto, Junshi; Sone, Nobuhito

    2002-08-01

    Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermophilic bacilli, can be over-expressed in Bacillus thermodenitrificans cells by tranformation with pSTEc551. Several mutant cytochromes c-551 were prepared by site-directed mutagenesis to this expression plasmid. Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their K(m) values were 0.04-0.12 microM, depending on the site replaced. In contrast, the C19A mutant cytochrome, which was produced in Brevibacillus choshinensis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity. The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551. These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxidase, despite the fact that the importance of an electrostatic interaction between cytochrome c and mitochondrial cytochrome aa(3) oxidase has already been established.

  14. [S-Acyl derivatives of thiosalicylamides having antifungal activity. II].

    PubMed

    Mazza, M; Modena, T; Montanari, L; Pavanetto, F

    1978-07-01

    Some S-acyl derivatives of N-alkylthiosalicylamides [Table I: substances (I leads to XXXI)] were prepared and tested for antifungal activity. The substances, most of which had not been previously reported, were prepared by condensation of 2-mercapto-N-alkylbenzamides with suitable acylating agents. The antifungal activity of the compounds was tested in vitro against Candida albicans and Trichophyton mentagrophytes. For some compounds the was tested activity against the above strains fungicidal, Candida tropicalis and Saccharomyces cerevisiae. Many of the compounds proved to have high antifungal activity comparable with that of Clotrimazol. The results extended knowledge on the structure-antifungal activity relationships of this class of compounds. The compounds with the highest antifungal activity were: 2-acetylmercapto-N,n-heptylbenzamide (XXVIII); 2-acetylmercapto-5-Cl-N,n-propylbenzamide (XIV); 2-acetylmercapto-N,n-octylbenzamide (XXXI); 2-acetylmercapto-N,n-pentylbenzamide (XXV); 2-acetylmercapto-N,n-hexylbenzamide (XXVII).

  15. Metabolic Glycoengineering with N-Acyl Side Chain Modified Mannosamines.

    PubMed

    Wratil, Paul R; Horstkorte, Rüdiger; Reutter, Werner

    2016-08-08

    In metabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides. After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates. The feasibility of MGE was first discovered for sialylated glycans, by using N-acyl-modified mannosamines as precursor molecules for unnatural sialic acids. Prerequisite is the promiscuity of the enzymes of the Roseman-Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N-acyl side chain of mannosamine analogues, for example, elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications). Unnatural sialic acids are incorporated into glycoconjugates of cells and organs. MGE has intriguing biological consequences for treated cells (aliphatic MGE) and offers the opportunity to visualize the topography and dynamics of sialylated glycans in vitro, ex vivo, and in vivo (bioorthogonal MGE).

  16. Thiourea-Catalyzed Aminolysis of N-acyl Homoserine Lactones

    DTIC Science & Technology

    2013-01-01

    of N-acyl homoserine lactones (AHLs), molecules integral to bacterial quorum sensing . The catalysts afford rate enhancement of up to 10 times the...SUBJECT TERMS quorum sensing Michael A. Bertucci, Stephen J. Lee, Michel R. Gagné University of North Carolina - Chapel Hill Office of Sponsored... quorum sensing . The catalysts afford rate enhancement of up to 10 times the control in CD3CN. Mild catalysis in other polar aprotic solvents is

  17. Antinociceptive property of new 4-acyl-arylhydrazone pyrazole compounds.

    PubMed

    Matheus, M E; Oliveira, L F; Freitas, A C; Carvalho, A M; Barreiro, E J

    1991-01-01

    A series of new 4-acyl-arylhydrazone pyrazole compounds were tested for antinociceptive activity using the inhibition of abdominal contortions induced by acetylcholine (4 mg/kg, ip) in the mouse. Dipyrone was used for comparison of the antinociceptive potency of the compounds being tested. All drugs were administered po in saline (dipyrone) or in propylene glycol (4-acyl-arylhydrazones). The maximum response induced by dipyrone (86% inhibition) was assigned an efficacy index of 1.0. Although none of the compounds had an efficacy index greater than 1.0, all three reached 1.0. The two most potent compounds, W1d and W1g, which also had an efficacy similar to that of dipyrone, contain a p-N(CH3)2 and m-OH,p-OCH3 group in the aromatic ring of the acyl-hydrazone, respectively. W1d presented the lowest antinociceptive ED50 in the series (1.41 mg/kg) and was eleven times more potent than dipyrone (ED50 = 15.80 mg/kg). Other substitutions at the para position had lower potency than W1d. The present results indicate that the introduction of a group at the para position of the acyl-arylhydrazone ring increases the antinociceptive activity of these compounds to provide compounds of the same efficacy but greater potency than dipyrone to which these new compounds are structurally related. Other assays of nociceptive activity are being used to characterize the mechanism of action of the potential new drugs.

  18. Acylation type determines ghrelin's effects on energy homeostasis in rodents.

    PubMed

    Heppner, Kristy M; Chaudhary, Nilika; Müller, Timo D; Kirchner, Henriette; Habegger, Kirk M; Ottaway, Nickki; Smiley, David L; Dimarchi, Richard; Hofmann, Susanna M; Woods, Stephen C; Sivertsen, Bjørn; Holst, Birgitte; Pfluger, Paul T; Perez-Tilve, Diego; Tschöp, Matthias H

    2012-10-01

    Ghrelin is a gastrointestinal polypeptide that acts through the ghrelin receptor (GHSR) to promote food intake and increase adiposity. Activation of GHSR requires the presence of a fatty-acid (FA) side chain on amino acid residue serine 3 of the ghrelin molecule. However, little is known about the role that the type of FA used for acylation plays in the biological action of ghrelin. We therefore evaluated a series of differentially acylated peptides to determine whether alterations in length or stability of the FA side chain have an impact on the ability of ghrelin to activate GHSR in vitro or to differentially alter food intake, body weight, and body composition in vivo. Fatty acids principally available in the diet (such as palmitate C16) and therefore representing potential substrates for the ghrelin-activating enzyme ghrelin O-acyltransferase (GOAT) were used for dose-, time-, and administration/route-dependent effects of ghrelin on food intake, body weight, and body composition in rats and mice. Our data demonstrate that altering the length of the FA side chain of ghrelin results in the differential activation of GHSR. Additionally, we found that acylation of ghrelin with a long-chain FA (C16) delays the acute central stimulation of food intake. Lastly, we found that, depending on acylation length, systemic and central chronic actions of ghrelin on adiposity can be enhanced or reduced. Together our data suggest that modification of the FA side-chain length can be a novel approach to modulate the efficacy of pharmacologically administered ghrelin.

  19. Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins.

    PubMed

    Owatworakit, Amorn; Townsend, Belinda; Louveau, Thomas; Jenner, Helen; Rejzek, Martin; Hughes, Richard K; Saalbach, Gerhard; Qi, Xiaoquan; Bakht, Saleha; Roy, Abhijeet Deb; Mugford, Sam T; Goss, Rebecca J M; Field, Robert A; Osbourn, Anne

    2013-02-08

    Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

  20. Six new acylated anthocyanins from red radish (Raphanus sativus).

    PubMed

    Tamura, Satoru; Tsuji, Kouji; Yongzhen, Piao; Ohnishi-Kameyama, Mayumi; Murakami, Nobutoshi

    2010-09-01

    Six new acylated anthocyanins (1-6) were isolated along with the three known congeners (7-9) from the fresh roots of red radishes (Raphanus sativus L.) cultivated by our group. Their chemical structures were elucidated by spectroscopic properties. Among the six new anthocyanins, the five constituents (1, 2, 4-6) were shown to contain the malonyl function at 6-OH in the glucopyranosyl residue linked to C-5 in the pelargonidin nucleus.

  1. A new cytotoxic acylated apigenin glucoside from Phyllanthus emblica L.

    PubMed

    El-Desouky, S K; Ryu, Shi Young; Kim, Young-Kyoon

    2008-01-10

    A new acylated apigenin glucoside (apigenin-7-O-(6''-butyryl-beta-glucopyranoside) (1) was isolated from the methanolic extract of the leaves of Phyllanthus emblica L. (Euphorbiaceae) together with the known compounds; gallic acid (2), methyl gallate (3), 1,2,3,4,6-penta-O-galloylglucose (4) and luteolin-4'-O-neohesperiodoside (5). Their chemical structures were elucidated on the basis of spectroscopic studies ((1)H NMR, (13)C NMR, DEPT, HSQC, HMBC).

  2. Purification of the Alpha Glycerophosphate Oxidase From Trypanosomes.

    DTIC Science & Technology

    1992-08-01

    is the purifica- tion of the glycerphosphate oxidase from the terminal oxidase in bloodstream trypanosomes. African trypanosomiasis remains one of the...oxidase from the terminal oxidase in bloodstream trypanosomes. African trypanosomiasis remains one of the major diseases in the world today, affecting...interest as a possible target for drug chemotherapy . At present only suramin and organic arsenicals remain as the mainstay of chemotherapy , despite their

  3. Metabolism of acyl-lipids in Chlamydomonas reinhardtii.

    PubMed

    Li-Beisson, Yonghua; Beisson, Fred; Riekhof, Wayne

    2015-05-01

    Microalgae are emerging platforms for production of a suite of compounds targeting several markets, including food, nutraceuticals, green chemicals, and biofuels. Many of these products, such as biodiesel or polyunsaturated fatty acids (PUFAs), derive from lipid metabolism. A general picture of lipid metabolism in microalgae has been deduced from well characterized pathways of fungi and land plants, but recent advances in molecular and genetic analyses of microalgae have uncovered unique features, pointing out the necessity to study lipid metabolism in microalgae themselves. In the past 10 years, in addition to its traditional role as a model for photosynthetic and flagellar motility processes, Chlamydomonas reinhardtii has emerged as a model organism to study lipid metabolism in green microalgae. Here, after summarizing data on total fatty acid composition, distribution of acyl-lipid classes, and major acyl-lipid molecular species found in C. reinhardtii, we review the current knowledge on the known or putative steps for fatty acid synthesis, glycerolipid desaturation and assembly, membrane lipid turnover, and oil remobilization. A list of characterized or putative enzymes for the major steps of acyl-lipid metabolism in C. reinhardtii is included, and subcellular localizations and phenotypes of associated mutants are discussed. Biogenesis and composition of Chlamydomonas lipid droplets and the potential importance of lipolytic processes in increasing cellular oil content are also highlighted.

  4. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

  5. Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa

    PubMed Central

    Guo, Dianjing; Chen, Fang; Inoue, Kentaro; Blount, Jack W.; Dixon, Richard A.

    2001-01-01

    Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in β-5 linked dimers of G units. Accumulation of soluble caffeic acid β-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis. PMID:11158530

  6. Impact of single-dose nandrolone decanoate on gonadotropins, blood lipids and HMG CoA reductase in healthy men.

    PubMed

    Gårevik, N; Börjesson, A; Choong, E; Ekström, L; Lehtihet, M

    2016-06-01

    The aim was to study the effect and time profile of a single dose of nandrolone decanoate (ND) on gonadotropins, blood lipids and HMG CoA reductase [3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR)] in healthy men. Eleven healthy male participants aged 29-46 years were given a single dose of 150 mg ND as an intramuscular dose of Deca Durabol®, Organon. Blood samples for sex hormones, lipids and HMGCR mRNA analysis were collected prior to ND administration day 0, 4 and 14. A significant suppression of luteinising hormone (LH) and follicle-stimulating hormone (FSH) was seen after 4 days. Total testosterone and bioavailable testosterone level decreased significantly throughout the observed study period. A small but significant decrease in sexual hormone-binding globulin (SHBG) was seen after 4 days but not after 14 days. Total serum (S)-cholesterol and plasma (P)-apolipoprotein B (ApoB) increased significantly after 14 days. In 80% of the individuals, the HMGCR mRNA level was increased 4 days after the ND administration. Our results show that a single dose of 150 mg ND increases (1) HMGCR mRNA expression, (2) total S-cholesterol and (3) P-ApoB level. The long-term consequences on cardiovascular risk that may appear in users remain to be elucidated.

  7. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

    PubMed Central

    2011-01-01

    Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

  8. The First Mammalian Aldehyde Oxidase Crystal Structure

    PubMed Central

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-01-01

    Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

  9. An oxidase road to platelet adhesion.

    PubMed

    Krause, Diane S

    2016-03-17

    Platelet adhesion to collagen via collagen receptors is an important part of thrombosis. In this issue of Blood, Matsuura et al identify collagen receptors as previously unrecognized targets of the extracellular enzyme lysyl oxidase (LOX), the level of which is increased in myeloproliferative neoplasms (MPNs) and other conditions associated with pathological thromboses.

  10. Polyphenol oxidase activity in annual forage clovers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO)-mediated phenol reactions in red clover (Trifolium pratense L.) bind forage protein and reduce proteolysis, producing beneficial effects on forage protein degradability, silage fermentation, and soil-N cycling. We evaluated PPO activity in seven previously untested annual c...

  11. A colorimetric assay for cytokinin oxidase.

    PubMed

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  12. Exploiting algal NADPH oxidase for biophotovoltaic energy.

    PubMed

    Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K; Bombelli, Paolo; Howe, Christopher J; Merchant, Sabeeha S; Davies, Julia M; Smith, Alison G

    2016-01-01

    Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

  13. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development.

    PubMed

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J

    2012-06-01

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT-encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T-DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol-localized, mevalonate-derived isoprenoid biosynthetic pathway.

  14. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed Central

    Stahl, U.; Banas, A.; Stymne, S.

    1995-01-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

  15. Direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase

    SciTech Connect

    Rajasekharan, R.; Ray, T.K.; Cronan, J.E. Jr.

    1988-09-01

    1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of /sup 32/P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, /sup 32/P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of /sup 32/Pi to /sup 32/P-labeled 1-acyl-sn-glycerol-3-phosphate.

  16. Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

    NASA Astrophysics Data System (ADS)

    Crittenden, Christopher M.; Akin, Lucas D.; Morrison, Lindsay J.; Trent, M. Stephen; Brodbelt, Jennifer S.

    2016-12-01

    Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages.

  17. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

    PubMed Central

    Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

    2015-01-01

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

  18. Inhibition of rat fat cell lipolysis by monoamine oxidase and semicarbazide-sensitive amine oxidase substrates.

    PubMed

    Visentin, Virgile; Prévot, Danielle; Marti, Luc; Carpéné, Christian

    2003-04-18

    It has been demonstrated that amine oxidase substrates stimulate glucose transport in cardiomyocytes and adipocytes, promote adipogenesis in pre-adipose cell lines and lower blood glucose in diabetic rats. These insulin-like effects are dependent on amine oxidation by semicarbazide-sensitive amine oxidase or by monoamine oxidase. The present study aimed to investigate whether amine oxidase substrates also exhibit another insulin-like property, the inhibition of lipolysis. We therefore tested the influence of tyramine and benzylamine on lipolytic activity in rat adipocytes. These amines did not modify basal lipolysis but dose-dependently counteracted the stimulation induced by lipolytic agents. The response to 10 nM isoprenaline was totally inhibited by tyramine 1 mM. The blockade produced by inhibition of amine oxidase activity or by 1 mM glutathione suggested that the generation of oxidative species, which occurs during amine oxidation, was involved in tyramine antilipolytic effect. Among the products resulting from amine oxidation, only hydrogen peroxide was antilipolytic in a manner that was potentiated by vanadate, as for tyramine or benzylamine. Antilipolytic responses to tyramine and to insulin were sensitive to wortmannin. These data suggest that inhibition of lipolysis is a novel insulin-like effect of amine oxidase substrates which is mediated by hydrogen peroxide generated during amine oxidation.

  19. ZrCl4-mediated regio- and chemoselective Friedel-Crafts acylation of indole.

    PubMed

    Guchhait, Sankar K; Kashyap, Maneesh; Kamble, Harshad

    2011-06-03

    An efficient method for regio- and chemoselective Friedel-Crafts acylation of indole using acyl chlorides in the presence of ZrCl(4) has been discovered. It minimizes/eliminates common competing reactions that occur due to high and multiatom-nucleophilic character of indole. In this method, a wide range of aroyl, heteroaroyl alkenoyl, and alkanoyl chlorides undergo smooth acylation with various indoles without NH protection and afford 3-acylindoles in good to high yields.

  20. Acute ethanol treatment induces a bimodal response of phospholipid acylation rates in rat red blood cells

    SciTech Connect

    Verine, A.; Valette, A.; Richard, D.; Boyer, J. )

    1991-01-01

    A single intraperitoneal injection of ethanol in rats elicited a bimodal response of acylation rates in phosphatidylcholine and phosphatidylethanolamine of intact red blood cells. Within an initial period, ethanol inhibited acylation rates. The inhibition then reversed, leading to increased values which persisted as long as ethanol was present in plasma. Acylation rates were not correlated to ethanol concentrations in plasma. The authors suggest that red cells first desensitize to, then overcompensate for the inhibitory effect of ethanol on acylation reactions. These adaptive changes may be one of the events mediating membrane tolerance to ethanol.

  1. A novel plasmid for detection of N-acyl homoserine lactones.

    PubMed

    Ling, Elizabeth A; Ellison, Matthew L; Pesci, Everett C

    2009-07-01

    Many bacteria utilize acyl-homoserine lactones as cell to cell signals that can regulate the expression of numerous genes. Structural differences in acyl-homoserine lactones produced by different bacteria, such as acyl side chain length and the presence or absence of an oxy group, make many of the commonly used detection bioassays impractical for broad range detection. Here we present a simple, broad range acyl-homoserine lactone detection bioassay that can be used to detect a wide range of these chemical signals. A plasmid (pEAL01) was constructed and transformed into Pseudomonas aeruginosa strain QSC105 to allow for detection of a broad range of acyl-homoserine lactones through induction of a lasB'-lacZ transcriptional fusion. Monitoring beta-galactosidase activity from this bioassay showed that P. aeruginosa strain QSC105 (pEAL01) could detect the presence of eight acyl-homoserine lactones tested at physiological concentrations. This novel strain could also detect acyl-homoserine lactones from the extracts of four different bacteria that produce different acyl-homoserine lactones signals. These data indicate that strain QSC105 (pEAL01) can be used to detect a wide variety of acyl-homoserine lactones by a simple beta-galactosidase assay and this bioassay could be a useful and inexpensive tool to quickly identify the presence of these signal molecules.

  2. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

    PubMed Central

    Ohura, T; Kraus, J P; Rosenberg, L E

    1989-01-01

    We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2741949

  3. Natural Compounds as Modulators of NADPH Oxidases

    PubMed Central

    2013-01-01

    Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

  4. Lysyl Oxidase and the Tumor Microenvironment

    PubMed Central

    Wang, Tong-Hong; Hsia, Shih-Min; Shieh, Tzong-Ming

    2016-01-01

    The lysyl oxidase (LOX) family of oxidases contains a group of extracellular copper-dependent enzymes that catalyze the cross-linking of collagen and elastin by oxidation, thus maintaining the rigidity and structural stability of the extracellular matrix (ECM). Aberrant expression or activation of LOX alters the cellular microenvironment, leading to many diseases, including atherosclerosis, tissue fibrosis, and cancer. Recently, a number of studies have shown that LOX is overexpressed in most cancers and that it is involved in the regulation of tumor progression and metastasis. In contrast, a few reports have also indicated the tumor-suppressing role of LOX. In this short review, we discuss recent research on the correlations between LOX and cancer. Further, the role of LOX in tumor microenvironment remodeling, tumorigenesis, and metastasis and the underlying mechanisms have also been elucidated. PMID:28036074

  5. Acylated flavonol glycosides from the flower of Elaeagnus angustifolia L.

    PubMed

    Bendaikha, Sarah; Gadaut, Méredith; Harakat, Dominique; Magid, Alabdul

    2014-07-01

    Seven acylated flavonol glycosides named elaeagnosides A-G, in addition to seven known flavonoids were isolated from the flowers of Elaeagnus angustifolia. Their structures were elucidated by different spectroscopic methods including 1D, 2D NMR experiments and HR-ESI-MS analysis. In order to identify natural antioxidant and tyrosinase inhibitor agents, the abilities of these flavonoids to scavenge the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and to inhibit tyrosinase activity were evaluated. Results revealed that two of these compounds had significant anti-oxidant effect and one compound showed weak tyrosinase-inhibitory activity compared with kojic acid, quercetin, or ascorbic acid, which were used as positive control.

  6. Lysyl oxidase mediates hypoxic control of metastasis.

    PubMed

    Erler, Janine T; Giaccia, Amato J

    2006-11-01

    Hypoxic cancer cells pose a great challenge to the oncologist because they are especially aggressive, metastatic, and resistant to therapy. Recently, we showed that elevation of the extracellular matrix protein lysyl oxidase (LOX) correlates with metastatic disease and is essential for hypoxia-induced metastasis. In an orthotopic rodent model of breast cancer, a small-molecule or antibody inhibitor of LOX abolished metastasis, offering preclinical validation of this enzyme as a therapeutic target.

  7. Ligand interactions with galactose oxidase: mechanistic insights.

    PubMed Central

    Whittaker, M M; Whittaker, J W

    1993-01-01

    Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme. PMID:8386015

  8. Imaging Monoamine Oxidase in the Human Brain

    SciTech Connect

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  9. Tetrazolium Oxidase Polymorphism in Rainbow Trout

    PubMed Central

    Cederbaum, Stephen D.; Yoshida, Akira

    1972-01-01

    Tetrazolium oxidase from the blood and liver of rainbow trout was found to be genetically polymorphic. The inheritance pattern of the liver enzyme was compatible only with a one locus-two allele hypothesis. The enzymes in the blood while having an electrophoretically identical polymorphism could differ genotypically from that of the liver in a given fish. The significance of these findings to the understanding of the evolution of the salmonid genome is discussed. PMID:4675090

  10. Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    PubMed Central

    Igarashi, Junsuke; Hashimoto, Takeshi; Kubota, Yasuo; Shoji, Kazuyo; Maruyama, Tokumi; Sakakibara, Norikazu; Takuwa, Yoh; Ujihara, Yoshihiro; Katanosaka, Yuki; Mohri, Satoshi; Naruse, Keiji; Yamashita, Tetsuo; Okamoto, Ryuji; Hirano, Katsuya; Kosaka, Hiroaki; Takata, Maki; Konishi, Ryoji; Tsukamoto, Ikuko

    2014-01-01

    COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca2+ concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [3H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis. PMID:25505610

  11. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  12. Xanthine dehydrogenase to xanthine oxidase conversion in ischemic rat intestine

    SciTech Connect

    McKelvey, T.G.; Engerson, T.D.; Elmore, C.R.; Jones, H.P. )

    1990-02-26

    The ischemic conversion of the NADH-producing xanthine dehydrogenase (XDH) to an oxidase form, that produces both superoxide radical and hydrogen peroxide, has been proposed as an important step in initiating oxygen radical-mediated ischemia-reperfusion injury. It has also been reported that two forms of converted oxidase are produced in ischemic rat liver; a reversible xanthine oxidase produced through sulfhydryl oxidation, that can be reconverted to XDH by incubation with 10mM dithiothreitol (Dtt) at 37{degrees}C, and a Dtt-irreversible oxidase produced via proteolysis. The authors report that increased oxidase in the ischemic rat intestine results from significant increases in both the Dtt-reversible and Dtt-irreversible forms of xanthine oxidase. Total oxidase activity (Irreversible + Dtt-reversible) was 19% of the total enzyme activity (XDH + XO) in control ileum and distal jejunum, increased to 26% after 1 hour of ischemia at 37{degrees}C, and significantly to 36% after 1.5 hours. After 3 hours 73% of the activity was in the oxidase form. Irreversible oxidase comprised 15% of the total activity in control intestine, significantly increased to 25% after 2 hours, and further to 42% after 3 hours. Dtt-reversible oxidase was 3% of the total activity in controls, increased to 13% after 1.5 hours, and significantly to 29% after 2 hours.

  13. Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).

    PubMed

    Hanaoka, Ryuki; Dawid, Igor B; Kawahara, Atsuo

    2007-02-01

    Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.

  14. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives’ Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps

    PubMed Central

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  15. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives' Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps.

    PubMed

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively.

  16. Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes 1

    PubMed Central

    Andrews, Jaen; Keegstra, Kenneth

    1983-01-01

    Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum. PMID:16663076

  17. Stereoelectronic basis for the kinetic resolution of N-heterocycles with chiral acylating reagents.

    PubMed

    Hsieh, Sheng-Ying; Wanner, Benedikt; Wheeler, Philip; Beauchemin, André M; Rovis, Tomislav; Bode, Jeffrey W

    2014-06-10

    The kinetic resolution of N-heterocycles with chiral acylating agents reveals a previously unrecognized stereoelectronic effect in amine acylation. Combined with a new achiral hydroxamate, this effect makes possible the resolution of various N-heterocycles by using easily prepared reagents. A transition-state model to rationalize the stereochemical outcome of this kinetic resolution is also proposed.

  18. Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride.

    PubMed

    Li, Conghu; Tian, Zhenhua; Liu, Wentao; Li, Guoying

    2015-10-01

    The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen.

  19. Ortho C-H Acylation of Aryl Iodides by Palladium/Norbornene Catalysis.

    PubMed

    Dong, Zhe; Wang, Jianchun; Ren, Zhi; Dong, Guangbin

    2015-10-19

    Reported herein is a palladium/norbornene-catalyzed ortho-arene acylation of aryl iodides by a Catellani-type C-H functionalization. This transformation is enabled by isopropyl carbonate anhydrides, which serve as both an acyl cation equivalent and a hydride source.

  20. The Role of Mitochondrial Non-Enzymatic Protein Acylation in Ageing

    PubMed Central

    Hong, Shin Yee; Ng, Li Theng; Ng, Li Fang; Inoue, Takao; Tolwinski, Nicholas S.; Hagen, Thilo; Gruber, Jan

    2016-01-01

    In recent years, various large-scale proteomic studies have demonstrated that mitochondrial proteins are highly acylated, most commonly by addition of acetyl and succinyl groups. These acyl modifications may be enzyme catalysed but can also be driven non-enzymatically. The latter mechanism is promoted in mitochondria due to the nature of the mitochondrial microenvironment, which is alkaline and contains high concentrations of acyl-CoA species. Protein acylation may modify enzyme activity, typically inhibiting it. We posited that organismal ageing might be accompanied by an accumulation of acylated proteins, especially in mitochondria, and that this might compromise mitochondrial function and contribute to ageing. In this study, we used R. norvegicus, C. elegans and D. melanogaster to compare the acylation status of mitochondrial proteins between young and old animals. We observed a specific age-dependent increase in protein succinylation in worms and flies but not in rat. Rats have two substrate-specific mitochondrial deacylases, SIRT3 and SIRT5 while both flies and worms lack these enzymes. We propose that accumulation of mitochondrial protein acylation contributes to age-dependent mitochondrial functional decline and that SIRT3 and SIRT5 enzymes may promote longevity through regulation of mitochondrial protein acylation during ageing. PMID:28033361

  1. Selective acylation of plasma membrane proteins of Mycoplasma agalactiae: the causal agent of agalactia.

    PubMed

    Le Hénaff, M; Guéguen, M M; Fontenelle, C

    2000-01-01

    Revealed by in vivo labeling with (14)C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) approximately palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria.

  2. COA User's Guide

    SciTech Connect

    Fox, B.; Pautz, J.; Sellers, C.

    1999-01-28

    The Department of Energy (DOE) has one of the largest and most complete collections of information on crude oil composition that is available to the public. The computer program that manages this database of crude oil analyses has recently been rewritten to allow easier access to this information. This report describes how the new system can be accessed and how the information contained in the Crude Oil Analysis Data Bank can be obtained.

  3. Impact of intracerebroventricular obestatin on plasma acyl ghrelin, des-acyl ghrelin and nesfatin-1 levels, and on gastric emptying in rats.

    PubMed

    Chen, Chih-Yen; Lee, Wei-Jei; Chong, Keong; Lee, Shou-Dong; Liao, You-Di

    2012-07-01

    Obestatin, which is a putative 23-amino-acid peptide, is derived from the C-terminal part of the mammalian preproghrelin gene. Nesfatin-1 mRNA is co-expressed with ghrelin in gastric endocrine X/A-like cells; therefore, nesfatin-1 may also interact with preproghrelin gene products in the stomach. In this study, we investigated the impact of obestatin on the plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, and on the gastric emptying of a solid nutrient meal 2 h after an intracerebroventricular (ICV) injection in conscious, fasted rats. The rats were implanted with ICV catheters. Plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, expected to be co-expressed with obestatin, were measured, whereas the human/rat corticotropin-releasing factor (h/rCRF) was applied as an inhibitor of gastric emptying. The ICV administration of obestatin (0.1, 0.3 and 1.0 nmol/rat) did not modify the plasma acyl ghrelin and des-acyl ghrelin levels, the acyl ghrelin/des-acyl ghrelin ratio and nesfatin-1 concentrations. The ICV acute administration of obestatin had no influence on the 2-h rate of gastric emptying of a solid nutrient meal, but the ICV h/rCRF injection delayed it. The weight of food ingested 1 h before ICV injection significantly, but negatively correlated with the gastric emptying of a solid nutrient meal. Our study indicates that the ICV injection of obestatin does not change the 2-h rate of gastric emptying of a solid nutrient meal and the relatively weak interrelationships between ghrelin gene products and nesfatin-1. However, the weight of the ingested food negatively affects the gastric emptying of a solid nutrient meal in conscious, fasted rats.

  4. Substrate specificity of guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase for methyl- and nitrobenzaldehydes.

    PubMed

    Veskoukis, Aristidis S; Kouretas, Demetrios; Panoutsopoulos, Georgios I

    2006-01-01

    Both aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are important in the oxidation of N-heterocyclic xenobiotics, whereas their role in the oxidation of xenobiotic aldehydes is usually ignored. The present investigation describes the interaction of methyl- and nitrosubstituted benzaldehydes, in the ortho-, meta- and parapositions, with guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase. The kinetic constants showed that most substituted benzaldehydes are excellent substrates of aldehyde oxidase with lower affinities for xanthine oxidase. Low Km values for aldehyde oxidase were observed with most benzaldehydes tested, with 3-nitrobenzaldehyde having the lowest Km value and 3-methylbenzaldehyde being the best substrate in terms of substrate efficiency (Ks). Additionally, low Km values for xanthine oxidase were found with most benzaldehydes tested. However, all benzaldehydes also had low Vmax values, which made them poor substrates of xanthine oxidase. It is therefore possible that aldehyde oxidase may be critical in the oxidation of xenobiotic and endobiotic derived aldehydes and its role in such reactions should not be ignored.

  5. Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement.

    PubMed

    Trier, Sofie; Linderoth, Lars; Bjerregaard, Simon; Strauss, Holger M; Rahbek, Ulrik L; Andresen, Thomas L

    2015-10-01

    Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may

  6. Non-enzymatic protein acylation as a carbon stress regulated by sirtuin deacylases

    PubMed Central

    Wagner, Gregory R.; Hirschey, Matthew D.

    2014-01-01

    Cellular proteins are decorated with a wide range of acetyl and other acyl modifications. Many studies have demonstrated regulation of site-specific acetylation by acetyltransferases and deacetylases. Acylation is emerging as a new type of lysine modification, but less is known about its overall regulatory role. Furthermore, the mechanisms of lysine acylation, its overlap with protein acetylation, and how it influences cellular function are major unanswered questions in the field. In this review, we discuss the known roles of acetyltransferases and deacetylases, and the sirtuins as a conserved family of NAD+-dependent protein deacylases that are important for response to cellular stress and homeostasis. We also consider the evidence for an emerging idea of non-enzymatic protein acylation. Finally, we put forward the hypothesis that protein acylation is a form of protein “carbon stress”, that the deacylases evolved to remove as a part of a global protein quality control network. PMID:24725594

  7. NAD(P)H oxidase and renal epithelial ion transport

    PubMed Central

    Schreck, Carlos

    2011-01-01

    A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na+ and K+ transport. Finally, we will review the relationship between tubular H+ efflux and NADPH oxidase activity. PMID:21270341

  8. Steady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2014-05-01

    Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100% of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f(a) = 1.0) was significantly higher than cesium ions (f(a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f(a) = 0.54), whereas quenching with iodide ion was decreased (f(a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.

  9. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  10. RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

    PubMed

    Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

    2017-03-18

    Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

  11. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl...

  12. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl...

  13. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting...

  14. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting...

  15. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting...

  16. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting...

  17. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting...

  18. 248-nm laser photolysis of CHBr3/O-atom mixtures: kinetic evidence for UV CO(A) chemiluminescence in the reaction of methylidyne radicals with atomic oxygen.

    PubMed

    Vaghjiani, Ghanshyam L

    2005-03-17

    The 4th positive and Cameron band emissions from electronically excited CO have been observed for the first time in 248-nm pulsed laser photolysis of a trace amount of CHBr(3) vapor in an excess of O atoms. O atoms were produced by dissociation of N(2)O (or O(2)) in a cw-microwave discharge cavity in 2.0 Torr of He at 298 K. The CO emission intensity in these bands showed a quadratic dependence on the laser fluence employed. Temporal profiles of the CO(A) and other excited-state products that formed in the photoproduced precursor + O-atom reactions were measured by recording their time-resolved chemiluminescence in discrete vibronic bands. The CO 4th positive transition (A(1)Pi, v' = 0 --> X(1)Sigma(+), v' ' = 2) near 165.7 nm was monitored in this work to deduce the pseudo-first-order decay kinetics of the CO(A) chemiluminescence in the presence of various added substrates (CH(4), NO, N(2)O, H(2), and O(2)). From this, the second-order rate coefficient values were determined for reactions of these substrates with the photoproduced precursors. The measured reactivity trends suggest that the prominent precursors responsible for the CO(A) chemiluminescence are the methylidyne radicals, CH(X(2)Pi) and CH(a(4)Sigma(-)), whose production requires the absorption of at least 2 laser photons by the photolysis mixture. The O-atom reactions with brominated precursors (CBr, CHBr, and CBr(2)), which also form in the photolysis, are shown to play a minor role in the production of the CO(A or a) chemiluminescence. However, the CBr(2) + O-atom reaction was identified as a significant source for the 289.9-nm Br(2) chemiluminescence that was also observed in this work. The 282.2-nm OH and the 336.2-nm NH chemiluminescences were also monitored to deduce the kinetics of CH(X(2)Pi) and CH(a(4)Sigma(-)) reactions when excess O(2) and NO were present.

  19. A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.

    PubMed

    Kojima, Kouji; Keta, Sumie; Uesaka, Kazuma; Kato, Akihiro; Takatani, Nobuyuki; Ihara, Kunio; Omata, Tatsuo; Aichi, Makiko

    2016-12-01

    Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.

  20. Individual variation in hepatic aldehyde oxidase activity.

    PubMed

    Al-Salmy, H S

    2001-04-01

    Aldehyde oxidase (AO) is a molybdo-flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N-heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver. Cytosolic AO was characterised using both the metabolism of N-[(2-dimethylamino)ethyl] acridine-4-carboxamide (DACA) and benzaldehyde to form DACA-9(10H)-acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18-fold (0.03-0.50 m/min/mg). Vmax ranged from 0.20-3.10 nmol/ min/mg, and Km ranged from 3.5-14.2 microM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5-fold (0.40-1.8 ml/min/mg). Vmax ranged from 3.60-12.6 nmol/min/mg and Km ranged from 3.6-14.6 microM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29-73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.

  1. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  2. Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA.

    PubMed

    Uchino, Keiichi; Saito, Terumi; Gebauer, Birgit; Jendrossek, Dieter

    2007-11-01

    Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from (14)C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD(+), indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD(+)/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

  3. Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

    PubMed Central

    Gahl, W A; Vale, A M; Pitot, H C

    1980-01-01

    1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate. PMID:7406861

  4. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  5. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

  6. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

    PubMed Central

    Peng, Zeyu; Dittmer, Neal T.; Lang, Minglin; Brummett, Lisa M.; Braun, Caroline L.; Davis, Lawrence C.; Kanost, Michael R.; Gorman, Maureen J.

    2015-01-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surpring because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  7. Acyl-homoserine lactone quorum sensing: from evolution to application.

    PubMed

    Schuster, Martin; Sexton, D Joseph; Diggle, Stephen P; Greenberg, E Peter

    2013-01-01

    Quorum sensing (QS) is a widespread process in bacteria that employs autoinducing chemical signals to coordinate diverse, often cooperative activities such as bioluminescence, biofilm formation, and exoenzyme secretion. Signaling via acyl-homoserine lactones is the paradigm for QS in Proteobacteria and is particularly well understood in the opportunistic pathogen Pseudomonas aeruginosa. Despite thirty years of mechanistic research, empirical studies have only recently addressed the benefits of QS and provided support for the traditional assumptions regarding its social nature and its role in optimizing cell-density-dependent group behaviors. QS-controlled public-goods production has served to investigate principles that explain the evolution and stability of cooperation, including kin selection, pleiotropic constraints, and metabolic prudence. With respect to medical application, appreciating social dynamics is pertinent to understanding the efficacy of QS-inhibiting drugs and the evolution of resistance. Future work will provide additional insight into the foundational assumptions of QS and relate laboratory discoveries to natural ecosystems.

  8. Acyl-Homoserine Lactone Quorum Sensing in the Roseobacter Clade

    PubMed Central

    Zan, Jindong; Liu, Yue; Fuqua, Clay; Hill, Russell T.

    2014-01-01

    Members of the Roseobacter clade are ecologically important and numerically abundant in coastal environments and can associate with marine invertebrates and nutrient-rich marine snow or organic particles, on which quorum sensing (QS) may play an important role. In this review, we summarize current research progress on roseobacterial acyl-homoserine lactone-based QS, particularly focusing on three relatively well-studied representatives, Phaeobacter inhibens DSM17395, the marine sponge symbiont Ruegeria sp. KLH11 and the dinoflagellate symbiont Dinoroseobacter shibae. Bioinformatic survey of luxI homologues revealed that over 80% of available roseobacterial genomes encode at least one luxI homologue, reflecting the significance of QS controlled regulatory pathways in adapting to the relevant marine environments. We also discuss several areas that warrant further investigation, including studies on the ecological role of these diverse QS pathways in natural environments. PMID:24402124

  9. Acylation in trypanosomatids: an essential process and potential drug target

    PubMed Central

    Goldston, Amanda M.; Sharma, Aabha I.; Paul, Kimberly S.; Engman, David M.

    2014-01-01

    Fatty acylation—the addition of fatty acid moieties such as myristate and palmitate to proteins—is essential for the survival, growth, and infectivity of the trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. Myristoylation and palmitoylation are critical for parasite growth, targeting and localization, and the intrinsic function of some proteins. The trypanosomatids possess a single N-myristoyltransferase (NMT) and multiple palmitoyl acyltransferases, and these enzymes and their cellular targets are only now being characterized. Global inhibition of either process leads to cell death in trypanosomatids, and genetic ablation of NMT compromises virulence. Moreover, NMT inhibitors effectively cure T. brucei infection in rodents. Thus, protein acylation represents an attractive target for the development of trypanocidal drugs. PMID:24954795

  10. Tunable Oleo-Furan Surfactants by Acylation of Renewable Furans

    PubMed Central

    2016-01-01

    An important advance in fluid surface control was the amphiphilic surfactant composed of coupled molecular structures (i.e., hydrophilic and hydrophobic) to reduce surface tension between two distinct fluid phases. However, implementation of simple surfactants has been hindered by the broad range of applications in water containing alkaline earth metals (i.e., hard water), which disrupt surfactant function and require extensive use of undesirable and expensive chelating additives. Here we show that sugar-derived furans can be linked with triglyceride-derived fatty acid chains via Friedel–Crafts acylation within single layer (SPP) zeolite catalysts. These alkylfuran surfactants independently suppress the effects of hard water while simultaneously permitting broad tunability of size, structure, and function, which can be optimized for superior capability for forming micelles and solubilizing in water. PMID:27924310

  11. Acylated quercetagetin glycosides with antioxidant activity from Tagetes maxima.

    PubMed

    Parejo, Irene; Bastida, Jaume; Viladomat, Francesc; Codina, Carles

    2005-10-01

    The fractionation of a methanolic extract of Tagetes maxima guided for antioxidant activity resulted in the isolation of three acylated quercetagetin glycosides, quercetagetin-7-O-(6-O-caffeoyl-beta-D-glucopyranoside), quercetagetin-7-O-(6-O-p-coumaroyl-beta-D-glucopyranoside) and quercetagetin-7-O-(6-O-galloyl-beta-D-glucopyranoside), as well as four known flavonoid glycosides. The structural elucidation was accomplished by spectroscopic methods (ESI-MS/MS and NMR). The antioxidant activity of fractions and isolated compounds was determined by checking the scavenging activity against three different radicals: 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH*), hydroxyl (*OH), and superoxide (O2*-). The three isolated compounds exhibited a high radical scavenging activity in comparison with reference compounds.

  12. Synthesis and evaluation of novel acyl derivatives from jatropha oil as potential lubricant basestocks.

    PubMed

    Sammaiah, Arukali; Padmaja, Korlipara V; Prasad, Rachapudi B N

    2014-05-21

    A novel class of jatropha oil-based acylated derivatives from hydroxy alkyl esters of jatropha fatty acids (C1, C3, C4, and C8) and various anhydrides (C2, C3, C4, and C6) were synthesized and their physicochemical and lubricant properties reported. Jatropha fatty acid alkyl esters were dihydroxylated using the in situ performic acid method and further acylated with different anhydrides to produce acylated derivatives. Acylated derivatives of dihydroxy jatropha fatty acid alkyl esters were charaterized by NMR, FTIR, GC, and GC-MS analysis and were evaluated for their viscosity, viscosity index, pour and flash points, and oxidation stability. Most of the derivatives are either in ISO VG 22 or 32 viscosity grade with good viscosity index. It was observed that increase in acyl chain length and branching in the end-chain ester improved the pour point of the diacyl derivatives. All of the hexanoylated esters exhibited better oxidation stability compared to other acylated products, and their pour points are comparable to those of synthetic esters such as TMP trioleates. In general, isoalcohol esters with longer acyl chains showed promise as potential candidates for hydraulic fluids and metal-working fluids in ISO VG 22 and 32 viscosity range.

  13. The TIP GROWTH DEFECTIVE1 S-acyl transferase regulates plant cell growth in Arabidopsis.

    PubMed

    Hemsley, Piers A; Kemp, Alison C; Grierson, Claire S

    2005-09-01

    TIP GROWTH DEFECTIVE1 (TIP1) of Arabidopsis thaliana affects cell growth throughout the plant and has a particularly strong effect on root hair growth. We have identified TIP1 by map-based cloning and complementation of the mutant phenotype. TIP1 encodes an ankyrin repeat protein with a DHHC Cys-rich domain that is expressed in roots, leaves, inflorescence stems, and floral tissue. Two homologues of TIP1 in yeast (Saccharomyces cerevisiae) and human (Homo sapiens) have been shown to have S-acyl transferase (also known as palmitoyl transferase) activity. S-acylation is a reversible hydrophobic protein modification that offers swift, flexible control of protein hydrophobicity and affects protein association with membranes, signal transduction, and vesicle trafficking within cells. We show that TIP1 binds the acyl group palmitate, that it can rescue the morphological, temperature sensitivity, and yeast casein kinase2 localization defects of the yeast S-acyl transferase mutant akr1Delta, and that inhibition of acylation in wild-type Arabidopsis roots reproduces the Tip1- mutant phenotype. Our results demonstrate that S-acylation is essential for normal plant cell growth and identify a plant S-acyl transferase, an essential research tool if we are to understand how this important, reversible lipid modification operates in plant cells.

  14. N-Acylation During Glidobactin Biosynthesis by the Tridomain Nonribosomal Peptide Synthetase Module GlbF

    PubMed Central

    Imker, Heidi J.; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T.

    2011-01-01

    Summary Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on co-expression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr1 amino group and generate the fatty acyl-Thr1-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis. PMID:21035730

  15. Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

    PubMed

    Kite, Geoffrey C; Larsson, Sonny; Veitch, Nigel C; Porter, Elaine A; Ding, Ning; Simmonds, Monique S J

    2013-04-10

    LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

  16. Metabolism of Acyl-Homoserine Lactone Quorum-Sensing Signals by Variovorax paradoxus

    PubMed Central

    Leadbetter, Jared R.; Greenberg, E. P.

    2000-01-01

    Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-l-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus. PMID:11092851

  17. Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus.

    PubMed

    Leadbetter, J R; Greenberg, E P

    2000-12-01

    Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus.

  18. Preparation and characterization of O-acylated fucosylated chondroitin sulfate from sea cucumber.

    PubMed

    Gao, Na; Wu, Mingyi; Liu, Shao; Lian, Wu; Li, Zi; Zhao, Jinhua

    2012-08-01

    Fucosylated chondroitin sulfate (FuCS), a kind of complex glycosaminoglycan from sea cucumber, has potent anticoagulant activity. In order to understand the relationship between structures and activity, the depolymerized FuCS (dFuCS) was chosen to prepare its derivates by selective substitution at OH groups. Its O-acylation was carried out in a homogeneous way using carboxylic acid anhydrides. The structures of O-acylated derivatives were characterized by NMR. The results indicated that the 4-O-sulfated fucose residues may be easier to be acylated than the other ones in the sulfated fucose branches. But the O-acylation was always accompanied by the β-elimination, and the degree of elimination was higher as that of acylation was higher. The results of clotting assay indicated that the effect of partial O-acylation of the dFuCS on their anticoagulant potency was not significant and the O-acylation of 2-OH groups of 4-O-sulfated fucose units did not affect the anticoagulant activity.

  19. Cytokinin Receptors Are Involved in Alkamide Regulation of Root and Shoot Development in Arabidopsis1[C][OA

    PubMed Central

    López-Bucio, José; Millán-Godínez, Mayra; Méndez-Bravo, Alfonso; Morquecho-Contreras, Alina; Ramírez-Chávez, Enrique; Molina-Torres, Jorge; Pérez-Torres, Anahí; Higuchi, Masayuki; Kakimoto, Tatsuo; Herrera-Estrella, Luis

    2007-01-01

    Alkamides and N-acilethanolamides are a class of lipid compounds related to animal endocannabinoids of wide distribution in plants. We investigated the structural features required for alkamides to regulate plant development by comparing the root responses of Arabidopsis (Arabidopsis thaliana) seedlings to a range of natural and synthetic compounds. The length of the acyl chain and the amide moiety were found to play a crucial role in their biological activity. From the different compounds tested, N-isobutyl decanamide, a small saturated alkamide, was found to be the most active in regulating primary root growth and lateral root formation. Proliferative-promoting activity of alkamide treatment was evidenced by formation of callus-like structures in primary roots, ectopic blades along petioles of rosette leaves, and disorganized tumorous tissue originating from the leaf lamina. Ectopic organ formation by N-isobutyl decanamide treatment was related to altered expression of the cell division marker CycB1:uidA and an enhanced expression of the cytokinin-inducible marker ARR5:uidA both in roots and in shoots. The involvement of cytokinins in mediating the observed activity of alkamides was tested using Arabidopsis mutants lacking one, two, or three of the putative cytokinin receptors CRE1, AHK2, and AHK3. The triple cytokinin receptor mutant was insensitive to N-isobutyl decanamide treatment, showing absence of callus-like structures in roots, the lack of lateral root proliferation, and absence of ectopic outgrowths in leaves under elevated levels of this alkamide. Taken together our results suggest that alkamides and N-acylethanolamides may belong to a class of endogenous signaling compounds that interact with a cytokinin-signaling pathway to control meristematic activity and differentiation processes during plant development. PMID:17965178

  20. Can HMG Co-A reductase inhibitors (“statins”) slow the progression of age-related macular degeneration? The Age-Related Maculopathy Statin Study (ARMSS)

    PubMed Central

    Guymer, Robyn H; Dimitrov, Peter N; Varsamidis, Mary; Lim, Lyndell L; Baird, Paul N; Vingrys, Algis J; Robman, Luba

    2008-01-01

    Age-related macular degeneration (AMD) is responsible for the majority of visual impairment in the Western world. The role of cholesterol-lowering medications, HMG Co-A reductase inhibitors or statins, in reducing the risk of AMD or of delaying its progression has not been fully investigated. A 3-year prospective randomized controlled trial of 40 mg simvastatin per day compared to placebo in subjects at high risk of AMD progression is described. This paper outlines the primary aims of the Age-Related Maculopathy Statin Study (ARMSS), and the methodology involved. Standardized clinical grading of macular photographs and comparison of serial macular digital photographs, using the International grading scheme, form the basis for assessment of primary study outcomes. In addition, macular function is assessed at each visit with detailed psychophysical measurements of rod and cone function. Information collected in this study will assist in the assessment of the potential value of HMG Co-A reductase inhibitors (statins) in reducing the risk of AMD progression. PMID:18982929

  1. Monolignol acylation and lignin structure in some nonwoody plants: a 2D NMR study.

    PubMed

    Martínez, Angel T; Rencoret, Jorge; Marques, Gisela; Gutiérrez, Ana; Ibarra, David; Jiménez-Barbero, Jesús; del Río, José C

    2008-11-01

    Lignins from three nonwoody angiosperms were analyzed by 2D NMR revealing important differences in their molecular structures. The Musa textilis milled-wood-lignin (MWL), with a syringyl-to-guaiacyl (S/G) ratio of 9, was strongly acylated (near 85% of side-chains) at the gamma-carbon by both acetates and p-coumarates, as estimated from (1)H-(13)C correlations in C(gamma)-esterified and C(gamma)-OH units. The p-coumarate H(3,5)-C(3,5) correlation signal was completely displaced by acetylation, and disappeared after alkali treatment, indicating that p-coumaric acid was esterified maintaining its free phenolic group. By contrast, the Cannabis sativa MWL (S/G approximately 0.8) was free of acylating groups, and the Agave sisalana MWL (S/G approximately 4) showed high acylation degree (near 80%) but exclusively with acetates. Extensive C(gamma)-acylation results in the absence (in M. textilis lignin) or low abundance (4% in A. sisalana lignin) of beta-beta' resinol linkages, which require free C(gamma)-OH to form the double tetrahydrofuran ring. However, minor signals revealed unusual acylated beta-beta' structures confirming that acylation is produced at the monolignol level, in agreement with chromatographic identification of gamma-acetylated sinapyl alcohol among the plant extractives. In contrast, resinol substructures involved 22% side-chains in the C.sativa MWL. The ratio between beta-beta' and beta-O-4' side-chains in these and other MWL varied from 0.32 in C.sativa MWL to 0.02 in M. textilis MWL, and was inversely correlated with the degree of acylation. The opposite was observed for the S/G ratio that was directly correlated with the acylation degree. Monolignol acylation is discussed as a mechanism potentially involved in the control of lignin structure.

  2. Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions*

    PubMed Central

    Lager, Ida; Yilmaz, Jenny Lindberg; Zhou, Xue-Rong; Jasieniecka, Katarzyna; Kazachkov, Michael; Wang, Peng; Zou, Jitao; Weselake, Randall; Smith, Mark A.; Bayon, Shen; Dyer, John M.; Shockey, Jay M.; Heinz, Ernst; Green, Allan; Banas, Antoni; Stymne, Sten

    2013-01-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism. PMID:24189065

  3. Xanthine oxidase inhibitors from Garcinia esculenta twigs.

    PubMed

    Zhu, Lun-Lun; Fu, Wen-Wei; Watanabe, Shimpei; Shao, Yi-Nuo; Tan, Hong-Sheng; Zhang, Hong; Tan, Chang-Heng; Xiu, Yan-Feng; Norimoto, Hisayoshi; Xu, Hong-Xi

    2014-12-01

    The EtOAc-soluble portion of the 80 % (v/v) EtOH extract from the twigs of Garcinia esculenta exhibited strong xanthine oxidase inhibition in vitro. Bioassay-guided purification led to the isolation of 1,3,6,7-tetrahydroxyxanthone (3) and griffipavixanthone (8) as the main xanthine oxidase inhibitors, along with six additional compounds (1, 2, 4-7), including two new compounds (1 and 2). This enzyme inhibition was dose dependent with an IC50 value of approximately 1.2 µM for 3 and 6.3 µM for 8. The inhibitory activity of 3 was stronger than the control allopurinol (IC50 value: 5.3 µM). To our knowledge, compound 8 is the first bixanthone that demonstrated potent XO inhibitory activity in vitro. The structures of the new compounds were established by spectroscopic analysis, and the optical properties and absolute stereochemistry of racemic (±) esculentin A (2) were further determined by the calculation of the DP4 probability and analysis of its MTPA ester derivatives.

  4. Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

    PubMed

    Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

    1998-12-01

    Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated

  5. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.

    PubMed

    Mashek, Douglas G; Bornfeldt, Karin E; Coleman, Rosalind A; Berger, Johannes; Bernlohr, David A; Black, Paul; DiRusso, Concetta C; Farber, Steven A; Guo, Wen; Hashimoto, Naohiro; Khodiyar, Varsha; Kuypers, Frans A; Maltais, Lois J; Nebert, Daniel W; Renieri, Alessandra; Schaffer, Jean E; Stahl, Andreas; Watkins, Paul A; Vasiliou, Vasilis; Yamamoto, Tokuo T

    2004-10-01

    By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

  6. Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

    PubMed

    Jurtshuk, P; Mueller, T J; Wong, T Y

    1981-09-14

    A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

  7. Molecular Evolution of Cytochrome bd Oxidases across Proteobacterial Genomes

    PubMed Central

    Degli Esposti, Mauro; Rosas-Pérez, Tania; Servín-Garcidueñas, Luis Eduardo; Bolaños, Luis Manuel; Rosenblueth, Monica; Martínez-Romero, Esperanza

    2015-01-01

    This work is aimed to resolve the complex molecular evolution of cytochrome bd ubiquinol oxidase, a nearly ubiquitous bacterial enzyme that is involved in redox balance and bioenergetics. Previous studies have created an unclear picture of bd oxidases phylogenesis without considering the existence of diverse types of bd oxidases. Integrated approaches of genomic and protein analysis focused on proteobacteria have generated a molecular classification of diverse types of bd oxidases, which produces a new scenario for interpreting their evolution. A duplication of the original gene cluster of bd oxidase might have occurred in the ancestors of extant α-proteobacteria of the Rhodospirillales order, such as Acidocella, from which the bd-I type of the oxidase might have diffused to other proteobacterial lineages. In contrast, the Cyanide-Insensitive Oxidase type may have differentiated into recognizable subtypes after another gene cluster duplication. These subtypes are widespread in the genomes of α-, β-, and γ-proteobacteria, with occasional instances of lateral gene transfer. In resolving the evolutionary pattern of proteobacterial bd oxidases, this work sheds new light on the basal taxa of α-proteobacteria from which the γ-proteobacterial lineage probably emerged. PMID:25688108

  8. Detection and characterization of a multicopper oxidase from Nitrosomonas europaea.

    PubMed

    Lawton, Thomas J; Rosenzweig, Amy C

    2011-01-01

    Blue copper oxidase (BCO) is a multicopper oxidase (MCO) found in Nitrosomonas europaea as well as in other ammonia-oxidizing organisms. In this chapter, we detail methods used to detect, isolate, and characterize BCO from N. europaea. A method for identifying and classifying MCOs commonly found in nitrifiers based on primary sequence is also described.

  9. Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

    PubMed Central

    Lambowitz, A M; Sabourin, J R; Bertrand, H; Nickels, R; McIntosh, L

    1989-01-01

    Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species. Images PMID:2524649

  10. Lysyl oxidase activity in human normal skins and postburn scars.

    PubMed

    Hayakawa, T; Hino, N; Fuyamada, H; Nagatsu, T; Aoyama, H

    1976-09-06

    Lysyl oxidase activity of human normal skins derived from the frontal thighs of 33 subjects showed large variations and the mean value was 11 455 +/- 7 172 (S.D.) cpm/g of wet weight tissue. The age of lesion affected the lysyl oxidase activity in postburn scars. Granulation tissues showed a fairly low activity; however, the activity increased sharply within 2--3 months, and reached a significantly higher value than that of normal skin. The high level of activity continued for up to 2--3 years, then gradually decreased to normal range after 5 years or so. Lysyl oxidase activity was detected only after 4 M urea treatment of tissues. Benzylamine oxidase activity also showed large variations in both normal skins and postburn scars, with mean values of: 0.128 +/- 0.077 (S.D.) and 0.145 +/- 0.090 (S.D.) mmol/g of wet weight/h, respectively. No correlation was observed between lysyl oxidase and benzylamine oxidase activities. The granulation tissues showed significantly high values of benzylamine oxidase activity in contrast to the low values of lysyl oxidase activity.

  11. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.

    PubMed Central

    Faergeman, N J; Knudsen, J

    1997-01-01

    The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase. PMID:9173866

  12. The complex roles of NADPH oxidases in fungal infection

    PubMed Central

    Hogan, Deborah; Wheeler, Robert T.

    2014-01-01

    Summary NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signaling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signaling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

  13. The NADH oxidase-Prx system in Amphibacillus xylanus.

    PubMed

    Niimura, Youichi

    2007-01-01

    Amphibacillus NADH oxidase belongs to a growing new family of peroxiredoxin-linked oxidoreductases including alkyl hydroperoxide reductase F (AhpF). Like AhpF it displays extremely high hydroperoxide reductase activity in the presence of a Prx, thus making up the NADH oxidase-Prx system. The NADH oxidase primarily catalyzes the reduction of oxygen by NADH to form H2O2, while the Prx immediately reduces H2O2 (or ROOH) to water (or ROH). Consequently, the NADH oxidase-Prx system catalyzes the reduction of both oxygen and hydrogen peroxide to water with NADH as the preferred electron donor. The NADH oxidase-Prx system is widely distributed in aerobically growing bacteria lacking a respiratory chain and catalase, and plays an important role not only in scavenging hydroperoxides but also in regenerating NAD in these bacteria.

  14. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  15. Genotypic Variation in Cytokinin Oxidase from Phaseolus Callus Cultures 1

    PubMed Central

    Kaminek, Miroslav; Armstrong, Donald J.

    1990-01-01

    Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues. Images Figure 6 PMID:16667652

  16. Expression of the alternative oxidase complements cytochrome c oxidase deficiency in human cells

    PubMed Central

    Dassa, Emmanuel P; Dufour, Eric; Gonçalves, Sérgio; Paupe, Vincent; Hakkaart, Gertjan A J; Jacobs, Howard T; Rustin, Pierre

    2009-01-01

    Cytochrome c oxidase (COX) deficiency is associated with a wide spectrum of clinical conditions, ranging from early onset devastating encephalomyopathy and cardiomyopathy, to neurological diseases in adulthood and in the elderly. No method of compensating successfully for COX deficiency has been reported so far. In vitro, COX-deficient human cells require additional glucose, pyruvate and uridine for normal growth and are specifically sensitive to oxidative stress. Here, we have tested whether the expression of a mitochondrially targeted, cyanide-resistant, alternative oxidase (AOX) from Ciona intestinalis could alleviate the metabolic abnormalities of COX-deficient human cells either from a patient harbouring a COX15 pathological mutation or rendered deficient by silencing the COX10 gene using shRNA. We demonstrate that the expression of the AOX, well-tolerated by the cells, compensates for both the growth defect and the pronounced oxidant-sensitivity of COX-deficient human cells. PMID:20049701

  17. Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C

    2005-04-11

    Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.

  18. Acyl tunichlorins: a new class of nickel chlorins isolated from the Caribbean tunicate Trididemnum solidum.

    PubMed Central

    Sings, H L; Bible, K C; Rinehart, K L

    1996-01-01

    A new class of nickel-containing chlorins (acyl tunichlorins) has been isolated from the Caribbean tunicate Trididemnum solidum. The structures of 28 of these nickel (II) hydroporphyrins were elucidated using mass spectrometry, one- and two-dimensional NMR spectroscopy, and chemical degradation/derivatization. Unique structural features of these compounds include the diversity of aliphatic side chains, which are derived from C14:0 to C22:6 fatty acids, and their location at an unprecedented position at C-2a on the hydroporphyrin nucleus. No chlorins with ester-linked acyl side chains at C-2a have been reported previously. Although the exact biological role that these compounds play in T. solidum remains unknown, acyl tunichlorins represent the only nickel-containing chlorins to be isolated from a living system and are the C-2a acyl derivatives of tunichlorin, a nickel chlorin reported by this laboratory in 1988. PMID:8855217

  19. Enhanced Activity of Nanocrystalline Beta Zeolite for Acylation of Veratrole with Acetic Anhydride.

    PubMed

    Aisha Mahmood Abdulkareem, Al-Turkustani; Selvin, Rosilda

    2016-04-01

    Friedel-Craft acylation of veratrole using homogeneous acid catalysts such as AlCl3, FeCl3, ZnCl2, and HF etc. produces acetoveratrone, (3',4'-dimethoxyacetophenone), which is the intermediate for synthesis of papavarine alkaloids. The problems associated with these homogeneous catalysts can be overcome by using heterogeneous solid catalysts. Since acetoveratrone is a larger molecule, large pore Beta zeolites with smaller particle sizes are beneficial for the liquid-phase acylation of veratrole, for easy diffusion of reactants and products. The present study aims in the acylation of veratrole with acetic anhydride using nanocrystalline Beta Zeolite catalyst. A systematic investigation of the effects of various reaction parameters was done. The catalysts were characterized for their structural features by using XRD, TEM and DLS analyses. The catalytic activity of nanocrystalline Beta zeolite was compared with commercial Beta zeolite for the acylation and was found that nanocrystalline Beta zeolite possessed superior activity.

  20. [NADPH oxidases, Nox: new isoenzymes family].

    PubMed

    Chuong Nguyen, Minh Vu; Lardy, Bernard; Paclet, Marie-Hélène; Rousset, Francis; Berthier, Sylvie; Baillet, Athan; Grange, Laurent; Gaudin, Philippe; Morel, Françoise

    2015-01-01

    NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.

  1. Visualization of monoamine oxidase in human brain

    SciTech Connect

    Fowler, J.S.; Volkow, N.D.; Wang, G.J.; Pappas, N.; Shea, C.; MacGregor, R.R.; Logan, J.

    1996-12-31

    Monoamine oxidase is a flavin enzyme which exists in two subtypes, MAO A and MAO B. In human brain MAO B predominates and is largely compartmentalized in cell bodies of serotonergic neurons and glia. Regional distribution of MAO B was determined by positron computed tomography with volunteers after the administration of deuterium substituted [11C]L-deprenyl. The basal ganglia and thalamus exhibited the greatest concentrations of MAO B with intermediate levels in the frontal cortex and cingulate gyrus while lowest levels were observed in the parietal and temporal cortices and cerebellum. We observed that brain MAO B increases with are in health normal subjects, however the increases were generally smaller than those revealed with post-mortem studies.

  2. NADPH Oxidases in Lung Health and Disease

    PubMed Central

    Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R.; Cheng, Guangjie

    2014-01-01

    Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838–2853. PMID:24093231

  3. New N-acyl taurine from the sea urchin Glyptocidaris crenularis.

    PubMed

    Zhou, Xuefeng; Xu, Tunhai; Wen, Kewei; Yang, Xian-Wen; Xu, Shi-Hai; Liu, Yonghong

    2010-01-01

    A new N-acyl taurine (1), together with a new natural product, l-(beta-D-ribofuranosyl)-1,2,4-triazole (4), and two known compounds (2 and 3), were isolated from the sea urchin, Glyptocidaris crenularis. The new N-acyl taurine was elucidated as 2-(5R,15S-dihydroxyeicosanoylamino) ethanesulfonic acid on the basis of spectroscopic (NMR, MS) analyses and the modified Mosher ester method. Compound 2 showed significant toxicity against brine shrimp larvae.

  4. Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

    PubMed

    Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

    2016-10-01

    Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

  5. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  6. Plant fatty acyl reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications.

    PubMed

    Rowland, Owen; Domergue, Frédéric

    2012-09-01

    Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols.

  7. Structural basis for acyl-group discrimination by human Gcn5L2

    PubMed Central

    Ringel, Alison E.; Wolberger, Cynthia

    2016-01-01

    Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications in vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group. PMID:27377381

  8. Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

    PubMed Central

    Morbidoni, H R; de Mendoza, D; Cronan, J E

    1996-01-01

    A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome. PMID:8759840

  9. Rheological behavior of acylated pepsin-solubilized collagen solutions: Effects of concentration

    NASA Astrophysics Data System (ADS)

    Li, Conghu; Duan, Lian; Tian, Zhenhua; Liu, Wentao; Li, Guoying; Huang, Xiaoping

    2015-11-01

    Effects of concentration on the rheological behavior of acylated pepsin-solubilized collagen solutions were investigated by steady shear tests, dynamic frequency sweep, creep tests and thixotropic loop measurements in this paper. The results showed that both acylated collagen and native collagen solutions exhibited the typical pseudoplastic behavior and displayed shear thinned behavior with the increase of shear rate. With the increase of acylated collagen concentrations from 5 to 10 mg/mL, shear viscosity, elasticity modulus ( G'), viscous modulus ( G″), complex viscosity ( η*), and the ability to resist deformation increased due to the physical entanglement, whilst loss tangent (tan δ) decreased. Additionally, with the increase of acylated collagen concentrations, the area of thixotropic loop increased from 6.94 to 44.40 watts/m3, indicating that the thixotropy of acylated collagen increased. Compared with native collagen solution, acylated collagen solution had stronger shear viscosity, η*, thixotropy, and ability to resist deformation. Furthermore, Power law model, Carreau model, Cross model, Leonov model and Burger model, were suitable for the fitting of the experimental data.

  10. Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

    SciTech Connect

    Sato, Miho; Nakahara, Keiko; Goto, Shintaro; Kaiya, Hiroyuki; Miyazato, Mikiya . E-mail: a0d201u@cc.miyazaki-u.ac.jp; Date, Yukari; Nakazato, Masamitsu; Kangawa, Kenji; Murakami, Noboru

    2006-11-24

    Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [{sup 125}I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.

  11. Lipopolysaccharides with acylation defects potentiate TLR4 signaling and shape T cell responses.

    PubMed

    Martirosyan, Anna; Ohne, Yoichiro; Degos, Clara; Gorvel, Laurent; Moriyón, Ignacio; Oh, Sangkon; Gorvel, Jean-Pierre

    2013-01-01

    Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+) T and CD8(+) T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

  12. Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

    SciTech Connect

    Atlow, S.C.; Bonadonna-Aparo, L.; Klibanov, A.M.

    1984-01-01

    A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

  13. Characterization of two amine oxidases from Aspergillus carbonarius AIU 205.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2015-06-01

    We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263-268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A. carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.

  14. THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

    PubMed Central

    Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

    1944-01-01

    1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

  15. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    PubMed

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a

  16. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

    PubMed

    Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A M; Ruggiero, Carmelina

    2014-01-01

    Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties.

  17. Enhancement of cardenolide and phytosterol levels by expression of an N-terminally truncated 3-hydroxy-3-methylglutaryl CoA reductase in Transgenic digitalis minor.

    PubMed

    Sales, Ester; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2007-06-01

    Pathway engineering in medicinal plants attains a special significance in Digitalis species, the main industrial source of cardiac glycosides, steroidal metabolites derived from mevalonic acid via the triterpenoid pathway. In this work, the Arabidopsis thaliana HMG1 cDNA, coding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the MVA pathway, was expressed in the cardenolide-producing plant Digitalis minor. Transgenic plants were morphologically indistinguishable from control wild plants and displayed the same developmental pattern. Constitutive expression of HMG1 resulted in an increased sterol and cardenolide production in both in vitro- and greenhouse-grown plants. This work demonstrates that transgenic D. minor plants are a valuable system to study and achieve metabolic engineering of the cardenolide pathway and in consequence for the genetic improvement of Digitalis species.

  18. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis.

    PubMed

    Liu, Ying; Xu, Qiao-Xian; Xi, Pei-Yu; Chen, Hong-Hao; Liu, Chun-Sheng

    2013-05-01

    The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

  19. The thermodynamic characteristics of ferrocene alkyl and acyl derivatives

    NASA Astrophysics Data System (ADS)

    Emel'Yanenko, V. N.; Krol', O. V.; Varushchenko, R. M.; Druzhinina, A. I.; Verevkin, S. P.

    2010-07-01

    The heat capacity of iso-butylferrocene C5H5FeC5H4-C4H9- i was measured over the temperature range 7-372 K in an adiabatic vacuum calorimeter. Substance purity and the thermodynamic characteristics of fusion (temperature, enthalpy, and entropy) were determined. Saturated vapor pressures and the enthalpies of vaporization of n-propylferrocene C5H5FeC5H4-C3H7- n, propionylferrocene C5H5FeC5H4-COC2H5, and iso-butylferrocene were measured by the dynamic method of substance transfer in an inert gas flow. The entropy, enthalpy, and Gibbs energy of the substances in the ideal gas state at 298.15 K were calculated. The thermodynamic values obtained in this work and reported in the literature for ferrocene alkyl and acyl derivatives were critically analyzed. The mutual consistency of the data on both homologous series was checked.

  20. The solution structure of acyl carrier protein from Mycobacterium tuberculosis.

    PubMed

    Wong, Hing C; Liu, Gaohua; Zhang, Yong-Mei; Rock, Charles O; Zheng, Jie

    2002-05-03

    Acyl carrier protein (ACP) performs the essential function of shuttling the intermediates between the enzymes that constitute the type II fatty acid synthase system. Mycobacterium tuberculosis is unique in producing extremely long mycolic acids, and tubercular ACP, AcpM, is also unique in possessing a longer carboxyl terminus than other ACPs. We determined the solution structure of AcpM using protein NMR spectroscopy to define the similarities and differences between AcpM and the typical structures. The amino-terminal region of the structure is well defined and consists of four helices arranged in a right-handed bundle held together by interhelical hydrophobic interactions similar to the structures of other bacterial ACPs. The unique carboxyl-terminal extension from helix IV has a "melted down" feature, and the end of the molecule is a random coil. A comparison of the apo- and holo-forms of AcpM revealed that the 4'-phosphopantetheine group oscillates between two states; in one it is bound to a hydrophobic groove on the surface of AcpM, and in another it is solvent-exposed. The similarity between AcpM and other ACPs reveals the conserved structural motif that is recognized by all type II enzymes. However, the function of the coil domain extending from helix IV to the carboxyl terminus remains enigmatic, but its structural characteristics suggest that it may interact with the very long chain intermediates in mycolic acid biosynthesis or control specific protein-protein interactions.

  1. Endogenous N-acyl taurines regulate skin wound healing

    PubMed Central

    Sasso, Oscar; Pontis, Silvia; Armirotti, Andrea; Cardinali, Giorgia; Kovacs, Daniela; Migliore, Marco; Summa, Maria; Moreno-Sanz, Guillermo; Picardo, Mauro; Piomelli, Daniele

    2016-01-01

    The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs—N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]—as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy. PMID:27412859

  2. Defluoridation potential of jute fibers grafted with fatty acyl chain

    NASA Astrophysics Data System (ADS)

    Manna, Suvendu; Saha, Prosenjit; Roy, Debasis; Sen, Ramkrishna; Adhikari, Basudam

    2015-11-01

    Waterborne fluoride is usually removed from water by coagulation, adsorption, ion exchange, electro dialysis or reverse osmosis. These processes are often effective over narrow pH ranges, release ions considered hazardous to human health or produce large volumes of toxic sludge that are difficult to handle and dispose. Although plant matters have been shown to remove waterborne fluoride, they suffer from poor removal efficiency. Following from the insight that interaction between microbial carbohydrate biopolymers and anionic surfaces is often facilitated by lipids, an attempt has been made to enhance fluoride adsorption efficiency of jute by grafting the lignocellulosic fiber with fatty acyl chains found in vegetable oils. Fluoride removal efficiency of grafted jute was found to be comparable or higher than those of alternative defluoridation processes. Infrared and X-ray photoelectron spectroscopic evidence indicated that hydrogen bonding, protonation and C-F bonding were responsible for fluoride accumulation on grafted jute. Adsorption based on grafted jute fibers appears to be an economical, sustainable and eco-friendly alternative technique for removing waterborne fluoride.

  3. Acylation and metabolism of (n-6) fatty acids in hepatocytes

    SciTech Connect

    Voss, A.C.; Sprecher, H.

    1986-05-01

    Isolated hepatocytes (5 x 10/sup 6/ in 2ml) from chow fed rats were incubated from 20 to 60 min. with increasing concentrations of (1-/sup 14/C) labeled 18:2 (n-6), 18:3 (n-6) or 20:3 (n-6) to define optimum conditions for measuring acylation and metabolism to other (n-6) acids with subsequent incorporation into lipids. The triglycerides (TG) and phospholipids (PL) contained 157 and 80 nmols of 18:2 (n-6) and 6.0 and 6.1 nmols of other (n-6) acids, respectively, when cells were incubated with 0.3mM (1-/sup 14/C) 18:2 (n-6) for 40 min. When cells were incubated with 0.3mM (1-/sup 14/C) 18:2 (n-6) plus 0.15 to 0.45mM 18:3 (n-6) or 20:3 (n-6), the metabolism of 18:2 (n-6) to other (n-6) acids was inhibited but not totally abolished. These results may suggest that (n-6) acid made from linoleate do not totally equilibrate with exogenous 18:3 (n-6) or 20:3 (n-6).

  4. Total and acylated ghrelin levels in children with poor growth.

    PubMed

    Pinsker, Jordan E; Ondrasik, Deborah; Chan, Debora; Fredericks, Gregory J; Tabisola-Nuesca, Eludrizza; Fernandez-Aponte, Minela; Focht, Dean R; Poth, Merrily

    2011-06-01

    Ghrelin, an enteric hormone with potent appetite stimulating effects, also stimulates growth hormone release. We hypothesized that altered levels of total ghrelin (TG) or acylated ghrelin (AG) could affect growth by altering growth hormone secretion, subsequently affecting insulin-like growth factor-1 (IGF-1) generation or by altering appetite and food intake. After institutional review board approval, 52 children presenting for evaluation of chronic gastrointestinal symptoms (group 1), poor weight gain (group 2), or poor linear growth (group 3) were evaluated for fasting TG and AG levels in addition to their regular evaluation. Serum ghrelin, IGF-1, and prealbumin were compared between groups. No difference was observed for mean fasting TG between groups. However, mean fasting AG was highest in patients in group 2 (465 ± 128 pg/mL) versus group 1 (176 ± 37 pg/mL) and group 3 (190 ± 34 pg/mL). IGF-1 was lowest in patients in group 2 despite similar prealbumin levels among the three groups. We conclude that serum AG levels are highest in children with isolated poor weight gain compared with children with short stature or chronic gastrointestinal symptoms, suggesting the possibility of resistance to AG in underweight children. Additional studies are needed to further clarify ghrelin's role in growth and appetite.

  5. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    PubMed

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination.

  6. Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae).

    PubMed

    Kite, Geoffrey C; Rowe, Emily R; Lewis, Gwilym P; Veitch, Nigel C

    2011-04-01

    The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar

  7. A special acyl carrier protein for transferring long hydroxylated fatty acids to lipid A in Rhizobium.

    PubMed

    Brozek, K A; Carlson, R W; Raetz, C R

    1996-12-13

    Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4'. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4'-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24

  8. The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A sup 2 H and sup 31 P NMR study

    SciTech Connect

    Jordi, W.; de Kroon, A.I.P.M.; Killian, A.; de Kruijff, B. )

    1990-03-06

    Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-(2-{sup 2}H{sub 1})serine) and acyl chain deuterated (1,2-(11,11-{sup 2}H{sub 2})dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. {sup 2}H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.

  9. Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

    PubMed

    Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

    2016-02-26

    We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition.

  10. [An endogenous inhibitor of monoamine oxidase A (tribulin A) from brain: purification and structure identification].

    PubMed

    Medvedev, A E; Kamyshanskaia, N S; Halket, J; Glover, V; Sandler, A

    1995-05-01

    The endogenous monoamine oxidase inhibitor, tribulin, contains several components which selectively (or nonselectively) inhibit monoamine oxidases A and B. The pig brain tribulin component selectively inhibiting monoamine oxidase A was purified and identified as 4-hydroxyphenylethanol using gas chromatography-mass spectrometry. This compound was also found in the rabbit brain tribulin fraction which selectively inhibits monoamine oxidase A but has no influence on monoamine oxidase B. 4-Hydroxyphenylethanol inhibits monoamine oxidase A in an incompetitive manner with respect to the substrate, serotonin (Ki = 1.4 mM). Possible pathways of 4-hydroxyphenylethanol synthesis and its biological importance as the monoamine oxidase A inhibiting component of tribulin are discussed.

  11. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (surfactants) at levels not to exceed 10% in pesticide formulations containing glyphosate: Name CAS Reg. No. N... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  12. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (surfactants) at levels not to exceed 10% in pesticide formulations containing glyphosate: Name CAS Reg. No. N... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  13. Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation

    PubMed Central

    Hopkins, Anna L.; Nelson, Timothy A. S.; Guschina, Irina A.; Parsons, Lydia C.; Lewis, Charlotte L.; Brown, Richard C.; Christian, Helen C.; Davies, Jeffrey S.; Wells, Timothy

    2017-01-01

    Despite being unable to activate the cognate ghrelin receptor (GHS-R), unacylated ghrelin (UAG) possesses a unique activity spectrum that includes promoting bone marrow adipogenesis. Since a receptor mediating this action has not been identified, we re-appraised the potential interaction of UAG with GHS-R in the regulation of bone marrow adiposity. Surprisingly, the adipogenic effects of intra-bone marrow (ibm)-infused acylated ghrelin (AG) and UAG were abolished in male GHS-R-null mice. Gas chromatography showed that isolated tibial marrow adipocytes contain the medium-chain fatty acids utilised in the acylation of UAG, including octanoic acid. Additionally, immunohistochemistry and immunogold electron microscopy revealed that tibial marrow adipocytes show prominent expression of the UAG-activating enzyme ghrelin O-acyl transferase (GOAT), which is located in the membranes of lipid trafficking vesicles and in the plasma membrane. Finally, the adipogenic effect of ibm-infused UAG was completely abolished in GOAT-KO mice. Thus, the adipogenic action of exogenous UAG in tibial marrow is dependent upon acylation by GOAT and activation of GHS-R. This suggests that UAG is subject to target cell-mediated activation – a novel mechanism for manipulating hormone activity. PMID:28361877

  14. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  15. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  16. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1207 N-acyl sarcosines and sodium...

  17. Facile synthesis of N-acyl 2-aminobenzothiazoles by NHC-catalyzed direct oxidative amidation of aldehydes.

    PubMed

    Premaletha, Sethulekshmy; Ghosh, Arghya; Joseph, Sumi; Yetra, Santhivardhana Reddy; Biju, Akkattu T

    2017-01-26

    A mild, general, and high yielding synthesis of N-acyl 2-aminobenzothiazoles has been demonstrated by N-heterocyclic carbene (NHC)-organocatalyzed direct amidation of aldehydes with 2-aminobenzothiazoles proceeding via acyl azolium intermediates. The carbene generated from the triazolium salt under oxidative conditions was the key for the success of this reaction. The method was subsequently applied to the synthesis of various biologically important N-acyl 2-aminobenzothiazoles.

  18. Diametric Stereocontrol in Dynamic Catalytic Reduction of Racemic Acyl Phosphonates: Divergence from α-Keto Ester Congeners

    PubMed Central

    Corbett, Michael T.; Johnson, Jeffrey S.

    2013-01-01

    An unexpected dichotomy was observed in the Ru-catalyzed asymmetric transfer hydrogenation of acyl phosphonates: reduction proceeded from the opposite face relative to that observed in the analogous reduction of α-keto esters. The first highly selective catalytic hydrogenation of acyl phosphonates was utilized in the dynamic kinetic resolution of α-aryl acyl phosphonates providing β-stereogenic α-hydroxy phosphonic acid derivatives. PMID:23297694

  19. Aldehyde-induced xanthine oxidase activity in raw milk.

    PubMed

    Steffensen, Charlotte L; Andersen, Henrik J; Nielsen, Jacob H

    2002-12-04

    In the present study, the aldehyde-induced pro-oxidative activity of xanthine oxidase was followed in an accelerated raw milk system using spin-trap electron spin resonance (ESR) spectroscopy. The aldehydes acetaldehyde, propanal, hexanal, trans-2-hexenal, trans-2-heptenal, trans-2-nonenal, and 3-methyl-2-butenal were all found to initiate radical reactions when added to milk. Formation of superoxide through aldehyde-induced xanthine oxidase activity is suggested as the initial reaction, as all tested aldehydes were shown to trigger superoxide formation in an ultrahigh temperature (UHT) milk model system with added xanthine oxidase. It was found that addition of aldehydes to milk initially increased the ascorbyl radical concentration with a subsequent decay due to ascorbate depletion, which renders the formation of superoxide in milk with added aldehyde. The present study shows for the first time potential acceleration of oxidative events in milk through aldehyde-induced xanthine oxidase activity.

  20. Isolation of oxidase-negative Pseudomonas aeruginosa from sputum culture.

    PubMed

    Hampton, K D; Wasilauskas, B L

    1979-05-01

    Two isolates of Pseudomonas aeruginosa lacking characteristic indophenol oxidase were recovered from a sputum specimen. A discussion of the characteristic biochemical tests and antibiograms along with a possible explanation for this phenomenon is presented.

  1. Polyphenol oxidase produced during encystation of Acanthamoeba castellanii.

    PubMed

    Sykes, D E; Band, R N

    1985-08-01

    Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.

  2. Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism.

    PubMed

    Sullivan, Michael L

    2014-01-01

    Most cloned and/or characterized plant polyphenol oxidases (PPOs) have catechol oxidase activity (i.e., they oxidize o-diphenols to o-quinones) and are localized or predicted to be localized to plastids. As a class, they have broad substrate specificity and are associated with browning of produce and other plant materials. Because PPOs are often induced by wounding or pathogen attack, they are most generally believed to play important roles in plant defense responses. However, a few well-characterized PPOs appear to have very specific roles in the biosynthesis of specialized metabolites via both tyrosinase (monophenol oxidase) and catechol oxidase activities. Here we detail a few examples of these and explore the possibility that there may be many more "biosynthetic" PPOs.

  3. Stimulation of cellular XTT reduction by cytochrome oxidase inhibitors.

    PubMed

    Kunimoto, S; Nosaka, C; Takeuchi, T

    1999-06-01

    XTT reducing activity by CHO and L1210 cells was found to be stimulated by the presence of cytochrome oxidase inhibitors such as NaN3 or KCN. Among the other respiratory chain inhibitors, antimycin A (a complex III inhibitor) and chlorpromazine inhibited cellular XTT reduction, and rotenone and malonate showed slight inhibition and no effect, respectively. It is suggested that XTT reduction is coupled with the respiratory chain via cytochrome c, which is located between complexes III and IV (cytochrome oxidase).

  4. A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases.

    PubMed

    Christensen, Quin H; Grove, Tyler L; Booker, Squire J; Greenberg, E Peter

    2013-08-20

    Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a Ki of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti-quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach.

  5. Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

    PubMed

    Voelker, T A; Davies, H M

    1994-12-01

    The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.

  6. Cloning, characterization, and expression analysis of acyl-acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis).

    PubMed

    Dong, Shubin; Huang, Jiacong; Li, Yannan; Zhang, Jing; Lin, Shanzhi; Zhang, Zhixiang

    2014-05-25

    Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)△FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.

  7. The Impact of Single Nucleotide Polymorphisms on Human Aldehyde OxidaseS

    PubMed Central

    Hartmann, Tobias; Terao, Mineko; Garattini, Enrico; Teutloff, Christian; Alfaro, Joshua F.; Jones, Jeffrey P.; Leimkühler, Silke

    2012-01-01

    Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 μg/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs. PMID:22279051

  8. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    SciTech Connect

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E. )

    1990-09-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with ({sup 14}C)iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 ({plus minus} 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.

  9. Arabidopsis PIZZA Has the Capacity to Acylate Brassinosteroids

    PubMed Central

    Schneider, Katja; Breuer, Christian; Kawamura, Ayako; Jikumaru, Yusuke; Hanada, Atsushi; Fujioka, Shozo; Ichikawa, Takanari; Kondou, Youichi; Matsui, Minami; Kamiya, Yuji; Yamaguchi, Shinjiro; Sugimoto, Keiko

    2012-01-01

    Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis. PMID:23071642

  10. Characterization of monomeric substates of ascorbate oxidase.

    PubMed

    Di Venere, Almerinda; Nicolai, Eleonora; Rosato, Nicola; Rossi, Antonello; Finazzi Agrò, Alessandro; Mei, Giampiero

    2011-05-01

    Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV ∼ -55 mL·mol(-1), is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits' interface.

  11. The function of ascorbate oxidase in tobacco.

    PubMed

    Pignocchi, Cristina; Fletcher, John M; Wilkinson, Joy E; Barnes, Jeremy D; Foyer, Christine H

    2003-07-01

    The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, L-galactono-gamma-lactone dehydrogenase, stromal APX, and thylakoid APX transcripts remained constant over the day/night cycle. Salicylic acid inhibited growth, increased expression of the pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance. In contrast, the application of auxin enhanced growth and increased AO and PR 1a gene expression. Therefore, AO transcript abundance varied in a manner similar to hormone-mediated changes in plant growth. To study the effects of modified AO expression on growth, transformed tobacco plants expressing AO in the sense and antisense orientations were generated. The resultant large changes in apoplastic AO activity in the transformed tobacco plants had little effect on whole leaf ascorbate (AA) content, but they had dramatic effects on apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool, whereas decreased AO activity increased the amount of AA compared with dehydroascorbate. A relationship was observed between AO activity and plant height and biomass. Native AO transcript levels were no longer subject to light/dark regulation in AO sense and antisense plants. Taken together, these data show that there is an interaction between hormone, redox, and light signals at the level of the apoplast via modulation of ion of AA content.

  12. Monoamine oxidase: Radiotracer chemistry and human studies

    DOE PAGES

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; ...

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variablesmore » which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.« less

  13. Monoamine oxidase: Radiotracer chemistry and human studies

    SciTech Connect

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; Alia-Klein, Nelly; Wang, Gene-Jack; Volkow, Nora D.

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variables which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.

  14. Modular assembly of yeast cytochrome oxidase.

    PubMed

    McStay, Gavin P; Su, Chen Hsien; Tzagoloff, Alexander

    2013-02-01

    Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

  15. Polyphenol oxidase from yacon roots (Smallanthus sonchifolius).

    PubMed

    Neves, Valdir Augusto; da Silva, Maraiza Aparecida

    2007-03-21

    Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490+/-3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.

  16. Origin and evolution of lysyl oxidases

    PubMed Central

    Grau-Bové, Xavier; Ruiz-Trillo, Iñaki; Rodriguez-Pascual, Fernando

    2015-01-01

    Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize primary amine substrates to reactive aldehydes. The best-studied role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals by cross-linking collagens and elastin, although intracellular functions have been reported as well. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence. Here we have surveyed a wide selection of genomes in order to infer the evolutionary history of LOX. We identified LOX proteins not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea – which reveals a pre-metazoan origin for this gene family. LOX genes expanded during metazoan evolution resulting in two superfamilies, LOXL2/L3/L4 and LOX/L1/L5. Considering the current knowledge on the function of mammalian LOX isoforms in ECM remodeling, we propose that LOXL2/L3/L4 members might have preferentially been involved in making cross-linked collagen IV-based basement membrane, whereas the diversification of LOX/L1/L5 forms contributed to chordate/vertebrate-specific ECM innovations, such as elastin and fibronectin. Our work provides a novel view on the evolution of this family of enzymes. PMID:26024311

  17. Leflunomide, a Reversible Monoamine Oxidase Inhibitor.

    PubMed

    Petzer, Jacobus P; Petzer, Anél

    2016-01-01

    A screening study aimed at identifying inhibitors of the enzyme, monoamine oxidase (MAO), among clinically used drugs have indicated that the antirheumatic drug, leflunomide, is an inhibitor of both MAO isoforms. Leflunomide inhibits human MAO-A and MAO-B and exhibits IC50 values of 19.1 μM and 13.7 μM, respectively. The corresponding Ki values are 17.7 μM (MAO-A) and 10.1 μM (MAO-B). Dialyses of mixtures of the MAO enzymes and leflunomide show that inhibition of the MAOs by leflunomide is reversible. The principal metabolite of leflunomide, teriflunomide (A77 1726), in contrast is not an MAO inhibitor. This study concludes that, although leflunomide is only moderately potent as an MAO inhibitor, isoxazole derivatives may represent a general class of MAO inhibitors and this heterocycle may find application in MAO inhibitor design. In this respect, MAO inhibitors are used in the clinic for the treatment of depressive illness and Parkinson's disease, and are under investigation as therapy for certain types of cancer, Alzheimer's disease and age-related impairment of cardiac function.

  18. Molecular "wiring" glucose oxidase in supramolecular architecture.

    PubMed

    Deng, Liu; Liu, Ying; Yang, Guocheng; Shang, Li; Wen, Dan; Wang, Fuan; Xu, Zhiai; Dong, Shaojun

    2007-07-01

    Supramolecular organized multilayers were constructed by multiwalled carbon nanotubes modified with ferrocene-derivatized poly(allylamine) redox polymer and glucose oxidase by electrostatic self-assembly. From the analysis of voltammetric signals and fluorescence results, a linear increment of the coverage of enzyme per bilayer was estimated, which demonstrated that the multilayer is constructed in a spatially ordered manner. The cyclic voltammograms obtained from the indium tin oxide (ITO) electrodes coated by the (Fc-PAH@CNT/GOx)n multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers; that is, the sensitivity is tunable by controlling the number of bilayers associated with ITO electrodes. The incorporation of redox-polymer-functionalized carbon nanotubes (CNT) into enzyme films resulted in a 6-10-fold increase in the glucose electrocatalytic current; the bimolecular rate constant of FADH2 oxidation (wiring efficiency) was increased up to 12-fold. Impedance spectroscopy data have yielded the electron diffusion coefficient (De) of this nanostructure to be over 10(-8) cm2 s(-1), which is typically higher than those systems without CNT by at least a factor of 10, indicating that electron transport in the new supramolecular architecture was enhanced by communication of the redox active site of enzyme, redox polymer, and CNT.

  19. MONOAMINE OXIDASE: RADIOTRACER DEVELOPMENT AND HUMAN STUDIES.

    SciTech Connect

    FOWLER,J.S.; LOGAN,J.; VOLKOW,N.D.; WANG,G.J.; MACGREGOR,R.R.; DING,Y.S.

    2000-09-28

    PET is uniquely capable of providing information on biochemical transformations in the living human body. Although most of the studies of monoamine oxidase (MAO) have focused on measurements in the brain, the role of peripheral MAO as a phase 1 enzyme for the metabolism of drugs and xenobiotics is gaining attention (Strolin Benedetti and Tipton, 1998; Castagnoli et al., 1997.). MAO is well suited for this role because its concentration in organs such as kidneys, liver and digestive organs is high sometimes exceeding that in the brain. Knowledge of the distribution of the MAO subtypes within different organs and different cells is important in determining which substrates (and which drugs and xenobiotics) have access to which MAO subtypes. The highly variable subtype distribution with different species makes human studies even more important. In addition, the deleterious side effects of combining MAO inhibitors with other drugs and with foodstuffs makes it important to know the MAO inhibitory potency of different drugs both in the brain and in peripheral organs (Ulus et al., 2000). Clearly PET can play a role in answering these questions, in drug research and development and in discovering some of the factors which contribute to the highly variable MAO levels in different individuals.

  20. Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia.

    PubMed

    Yamada, Takahiro; Takemura, Yoshizumi; Niisato, Naomi; Mitsuyama, Etsuko; Iwasaki, Yoshinobu; Marunaka, Yoshinori

    2009-03-13

    We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

  1. The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure[S

    PubMed Central

    Son, Mijin; London, Erwin

    2013-01-01

    Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains. PMID:23093551

  2. Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

    SciTech Connect

    Carey, L.M.; Rodriguez, A.; Meighen, E.

    1984-08-25

    The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of > 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.

  3. Computational Prediction of acyl-coA Binding Proteins Structure in Brassica napus.

    PubMed

    Raboanatahiry, Nadia Haingotiana; Lu, Guangyuan; Li, Maoteng

    2015-01-01

    Acyl-coA binding proteins could transport acyl-coA esters from plastid to endoplasmic reticulum, prior to fatty acid biosynthesis, leading to the formation of triacylglycerol. The structure and the subcellular localization of acyl-coA binding proteins (ACBP) in Brassica napus were computationally predicted in this study. Earlier, the structure analysis of ACBPs was limited to the small ACBPs, the current study focused on all four classes of ACBPs. Physicochemical parameters including the size and the length, the intron-exon structure, the isoelectric point, the hydrophobicity, and the amino acid composition were studied. Furthermore, identification of conserved residues and conserved domains were carried out. Secondary structure and tertiary structure of ACBPs were also studied. Finally, subcellular localization of ACBPs was predicted. The findings indicated that the physicochemical parameters and subcellular localizations of ACBPs in Brassica napus were identical to Arabidopsis thaliana. Conserved domain analysis indicated that ACBPs contain two or three kelch domains that belong to different families. Identical residues in acyl-coA binding domains corresponded to eight amino acid residues in all ACBPs of B. napus. However, conserved residues of common ACBPs in all species of animal, plant, bacteria and fungi were only inclusive in small ACBPs. Alpha-helixes were displayed and conserved in all the acyl-coA binding domains, representing almost the half of the protein structure. The findings confirm high similarities in ACBPs between A. thaliana and B. napus, they might share the same functions but loss or gain might be possible.

  4. Altered hepatic retinyl ester concentration and acyl composition in response to alcohol consumption

    PubMed Central

    Clugston, Robin D.; Jiang, Hongfeng; Lee, Man Xia; Berk, Paul D.; Goldberg, Ira J.; Huang, Li-Shin; Blaner, William S.

    2013-01-01

    Retinoids (vitamin A and its metabolites) are essential micronutrients that regulate many cellular processes. Greater than 70% of the body’s retinoid reserves are stored in the liver as retinyl ester (RE). Chronic alcohol consumption induces depletion of hepatic retinoid stores, and the extent of this has been correlated with advancing stages of alcoholic liver disease. The goal of this study was to analyze the mechanisms responsible for depletion of hepatic RE stores by alcohol consumption. A change in the fatty-acyl composition of RE in alcohol-fed mice was observed within two weeks after the start of alcohol consumption. Specifically, alcohol-feeding was associated with a significant decline in hepatic retinyl palmitate levels; however, total RE levels were maintained by a compensatory increase in levels of usually minorRE species, particularly retinyl oleate. Our data suggests that alcohol feeding initially stimulates a futile cycle of RE hydrolysis and synthesis, and that the change in RE acyl composition is associated with a change in the acyl composition of hepatic phosphatidylcholine. The alcohol-induced change in RE acyl composition was specific to the liver, and was not seen in lung or white adipose tissue. This shift in hepatic RE fatty acyl composition is a sensitive indicator of alcohol consumption and may be an early biomarker for events associated with the development of alcoholic liver disease. PMID:23583843

  5. Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson’s disease.

    PubMed

    Abbott, Sarah K; Li, Hongyun; Muñoz, Sonia Sanz; Knoch, Bianca; Batterham, Marijka; Murphy, Karen E; Halliday, Glenda M; Garner, Brett

    2014-04-01

    Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson’s disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.

  6. Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

    PubMed Central

    Watkins, Erik B.; Gao, Haifei; Dennison, Andrew J.C.; Chopin, Nathalie; Struth, Bernd; Arnold, Thomas; Florent, Jean-Claude; Johannes, Ludger

    2014-01-01

    Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding. PMID:25185550

  7. Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans.

    PubMed

    Sugio, Tsuyoshi; Hisazumi, Tomohiro; Kanao, Tadayoshi; Kamimura, Kazuo; Takeuchi, Fumiaki; Negishi, Atsunori

    2006-07-01

    It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

  8. Conversion of Escherichia coli pyruvate oxidase to an 'alpha-ketobutyrate oxidase'.

    PubMed Central

    Chang, Y Y; Cronan, J E

    2000-01-01

    Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate ('alpha-ketobutyrate'), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380 had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K(m) for pyruvate and a 10-fold lower V(max) with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme. PMID:11104678

  9. Identification of the alternative terminal oxidase of higher plant mitochondria

    PubMed Central

    Elthon, Thomas E.; McIntosh, Lee

    1987-01-01

    In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed. Images PMID:16593898

  10. Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues 1

    PubMed Central

    Chatfield, J. Mark; Armstrong, Donald J.

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N6-(Δ2-isopentenyl)-adenine-2,8-3H (i6 Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N6-side chain of i6Ade. PMID:16665511

  11. Pentamines as substrate for human spermine oxidase

    PubMed Central

    Takao, Koichi; Shirahata, Akira; Samejima, Keijiro; Casero, Robert A.; Igarashi, Kazuei; Sugita, Yoshiaki

    2013-01-01

    Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO. PMID:23449327

  12. Monoamine oxidase inhibitory components from Cayratia japonica.

    PubMed

    Han, Xiang Hua; Hong, Seong Su; Hwang, Ji Sang; Lee, Myung Koo; Hwang, Bang Yeon; Ro, Jai Seup

    2007-01-01

    Seven flavonoids were isolated from the whole plants and fruits of Cayratia japonica through the activity-guided isolation of a methanol extract using a monoamine oxidase (MAO) inhibition assay as a monitor. The chemical structures of the isolates were assigned as apigenin-7-O-beta-D-glucuronopyranoside (1), apigenin (2), luteolin (3), luteolin-7-O-beta-D-glucopyranoside (4), (+)-dihydroquercetin (taxifolin) (5), (+)-dihydrokaempferol (aromadendrin) (6) and quercetin (7). Among the isolated compounds, flavones such as apigenin (2) and luteolin (3), as well as the flavonol, quercetin (7) showed potent inhibitory effects against the MAO activity with IC50 values of 6.5, 22.6, and 31.6 microM, respectively. However, the flavone glycosides, apigenin-7-O-beta-D-glucuronopyranoside (1) and luteolin-7-O-beta-D-glucopyranoside (4), showed mild MAO inhibition (IC50 values: 81.7 and 118.6 microM, respectively). The flavanonol derivatives, taxifolin (5) and aromadendrin (6), also showed weak inhibition (IC50 values: 154.7 and 153.1 microM, respectively). Furthermore, quercetin (7) had a more potent inhibitory effect on MAO-A (IC50 value: 2.8 microM) than MAO-B (IC50 value: 90.0 microM). Apigenin (2) and luteolin (3) also preferentially inhibited MAO-A (IC50 values: 1.7 and 4.9 microM, respectively) compared with MAO-B (IC50 values: 12.8 and 59.7 microM, respectively).

  13. A novel sodium N-fatty acyl amino acid surfactant using silkworm pupae as stock material

    NASA Astrophysics Data System (ADS)

    Wu, Min-Hui; Wan, Liang-Ze; Zhang, Yu-Qing

    2014-03-01

    A novel sodium N-fatty acyl amino acid (SFAAA) surfactant was synthesized using pupa oil and pupa protein hydrolysates (PPH) from a waste product of the silk industry. The aliphatic acids from pupa oil were modified into N-fatty acyl chlorides by thionyl chloride (SOCl2). SFAAA was synthesized using acyl chlorides and PPH. GC-MS analysis showed fatty acids from pupa oil consist mainly of unsaturated linolenic and linoleic acids and saturated palmitic and stearic acids. SFAAA had a low critical micelle concentration, great efficiency in lowering surface tension and strong adsorption at an air/water interface. SFAAA had a high emulsifying power, as well as a high foaming power. The emulsifying power of PPH and SFAAA in an oil/water emulsion was better with ethyl acetate as the oil phase compared to n-hexane. The environment-friendly surfactant made entirely from silkworm pupae could promote sustainable development of the silk industry.

  14. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  15. Synthesis of amino acyl adenylates using the tert-butoxycarbonyl protecting group

    NASA Technical Reports Server (NTRS)

    Armstrong, D. W.; Seguin, R.; Saburi, M.; Fendler, J. H.

    1979-01-01

    The synthesis of amino acyl adenylates using N-tert-butoxycarbonyl-protected amino acids is reported. Anhydrous solutions containing N-tert-butoxycarbonyl alanine, phenylalanine, and methionine were combined with the anhydrous mono (tri-n-octylammonium) salt of adenosine 5'-phosphate and the resultant amino acyl adenylates were characterized by means of elemental analysis, and infrared and proton NMR spectroscopy. Amino acyl adenylate yields of up to 60% were obtained with high purity at room temperatures. The reported synthesis is considered to represent a large improvement over previous methods due to the purity of the products, normal temperature requirements, and the stability of the starting compounds, which suggests its use in investigations of prebiotic oligo- and polypeptide synthesis.

  16. Synthesis, Surface Active Properties and Cytotoxicity of Sodium N-Acyl Prolines.

    PubMed

    Sreenu, Madhumanchi; Narayana Prasad, Rachapudi Badari; Sujitha, Pombala; Kumar, Chityal Ganesh

    2015-01-01

    Sodium N-acyl prolines (NaNAPro) were synthesized using mixture of fatty acids obtained from coconut, palm, karanja, Sterculia foetida and high oleic sunflower oils via Schotten-Baumann reaction in 58-75% yields to study the synergetic effect of mixture of hydrophobic fatty acyl functionalities like saturation, unsaturation and cyclopropene fatty acids with different chain lengths and aliphatic hetero cyclic proline head group on their surface and cytotoxicity activities. The products were characterized by chromatographic and spectral techniques. The synthesized products were evaluated for their surface active properties such as surface tension, wetting power, foaming characteristics, emulsion stability, calcium tolerance, critical micelle concentration (CMC) and thermodynamic properties. The results revealed that all the products exhibited superior surface active properties like CMC, calcium tolerance and emulsion stability as compared to the standard surfactant, sodium lauryl sulphate (SLS). In addition, palm, Sterculia foetida and high oleic sunflower fatty N-acyl prolines exhibited promising cytotoxicity against different tumor cell lines.

  17. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    PubMed Central

    Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. PMID:26190825

  18. Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI

    SciTech Connect

    Watson, W.T.; Murphy IV, Frank V.; Gould, Ty A.; Jambeck, Per; Val, Dale L.; Cronan, Jr., John E.; Beck von Bodman, Susan; Churchill, Mair E.A.

    2009-04-22

    Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 66.40, c = 47.33 {angstrom}. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 {angstrom} and the perrhenate ion binding sites and liganding residues have been identified.

  19. Ghrelin, des-acyl ghrelin and obestatin on the gastrointestinal motility.

    PubMed

    Fujimiya, Mineko; Ataka, Koji; Asakawa, Akihiro; Chen, Chih-Yen; Kato, Ikuo; Inui, Akio

    2011-11-01

    Ghrelin, des-acyl ghrelin and obestatin are derived from a common prohormone, preproghrelin by posttranslational processing, originating from endocrine cells in the stomach. Ghrelin exerts stimulatory effects on the motility of antrum and duodenum in both fed and fasted state of animals. On the other hand, des-acyl ghrelin exerts inhibitory effects on the motility of antrum but not on the motility of duodenum in the fasted state of animals. Obestatin exerts inhibitory effects on the motility of antrum and duodenum in the fed state but not in the fasted state of animals. NPY Y2 and Y4 receptors in the brain may mediate the action of ghrelin, CRF type 2 receptor in the brain may mediate the action of des-acyl ghrelin, whereas CRF type 1 and type 2 receptors in the brain may mediate the action of obestatin.

  20. A novel sodium N-fatty acyl amino acid surfactant using silkworm pupae as stock material.

    PubMed

    Wu, Min-Hui; Wan, Liang-Ze; Zhang, Yu-Qing

    2014-03-21

    A novel sodium N-fatty acyl amino acid (SFAAA) surfactant was synthesized using pupa oil and pupa protein hydrolysates (PPH) from a waste product of the silk industry. The aliphatic acids from pupa oil were modified into N-fatty acyl chlorides by thionyl chloride (SOCl2). SFAAA was synthesized using acyl chlorides and PPH. GC-MS analysis showed fatty acids from pupa oil consist mainly of unsaturated linolenic and linoleic acids and saturated palmitic and stearic acids. SFAAA had a low critical micelle concentration, great efficiency in lowering surface tension and strong adsorption at an air/water interface. SFAAA had a high emulsifying power, as well as a high foaming power. The emulsifying power of PPH and SFAAA in an oil/water emulsion was better with ethyl acetate as the oil phase compared to n-hexane. The environment-friendly surfactant made entirely from silkworm pupae could promote sustainable development of the silk industry.

  1. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    PubMed Central

    Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

    2012-01-01

    We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

  2. Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics

    PubMed Central

    Serwa, Remigiusz A.; Abaitua, Fernando; Krause, Eberhard; Tate, Edward W.; O’Hare, Peter

    2015-01-01

    Summary Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions. PMID:26256475

  3. Natural variability in acyl moieties of sugar esters produced by certain tobacco and other Solanaceae species.

    PubMed

    Kroumova, Antoaneta B M; Zaitlin, Dave; Wagner, George J

    2016-10-01

    A unique feature of glandular trichomes of plants in the botanical family Solanaceae is that they produce sugar esters (SE), chemicals that have been shown to possess insecticidal, antifungal, and antibacterial properties. Sugar esters of tobacco (Nicotiana tabacum) provide pest resistance, and are important flavor precursors in oriental tobacco cultivars. Acyl moieties of SEs in Nicotiana spp., petunia, and tomato are shown to vary with respect to carbon length and isomer structure (2-12 carbon chain length; anteiso-, iso-, and straight-chain). Sugar esters and their acyl groups could serve as a model to explore the basis of phenotypic diversity and adaptation to natural and agricultural environments. However, information on the diversity of acyl composition among species, cultivars, and accessions is lacking. Herein, described is the analysis of SE acyl groups found in 21 accessions of Nicotiana obtusifolia (desert tobacco), six of Nicotiana occidentalis subsp. hesperis, three of Nicotiana alata, two of N. occidentalis, four modern tobacco cultivars, five petunia hybrids, and one accession each of a primitive potato (Solanum berthaultii) and tomato (Solanum pennellii). A total of 20 different acyl groups was observed that were represented differently among cultivars, species, and accessions. In Nicotiana species, acetate and iso- and anteiso-branched acids prevailed. Straight-chain groups (2-8 carbons) were prominent in petunias, while octanoic acid was prominent in N. alata and N. × sanderae. Two unexpected acyl groups, 8-methyl nonanoate and decanoate were found in N. occidentalis subsp. hesperis. Longer chain groups were found in the petunia, tomato, and potato species studied.

  4. The Effects of Exercise on Food Intake and Hunger: Relationship with Acylated Ghrelin and Leptin

    PubMed Central

    Vatansever-Ozen, Serife; Tiryaki-Sonmez, Gul; Bugdayci, Guler; Ozen, Guclu

    2011-01-01

    This study investigated the effects of a long bout of aerobic exercise on hunger and energy intake and circulating levels of leptin and acylated ghrelin. Ten healthy male subjects undertook two, 4 h trials in a randomized crossover design. In the exercise trial subjects ran for 105 min at 50% of maximal oxygen uptake and the last 15 min at 70% of maximal oxygen uptake followed by a 120 min rest period. In the control trial, subjects rested for 4 h. Subjects consumed a buffet test meal at 180 min during each trial. Hunger ratings, acylated ghrelin, leptin, glucose and insulin concentrations were measured at 0, 1, 2, 3 and 4 h. No differences were found at baseline values for hunger, acylated ghrelin, leptin, insulin and glucose for both trials (p > 0.05). The estimated energy expenditure of the exercise trial was 1550 ± 136 kcal. Exercise did not change subsequent absolute energy intake, but produced a significant decrease (p < 0.05) in relative energy intake. A two-way ANOVA revealed a significant (p < 0. 05) interaction effect for hunger and acylated ghrelin. In conclusion, this exercise regimen had a positive effect on reducing appetite which is related to reduced acylated ghrelin responses over time. This finding lends support for a role of exercise in weight management. Key points Physical exercise is a strategy used to counteract obesity, since it lowers the energetic balance by increasing energy expenditure. However, because any energy expended in exercise elevates the intensity of hunger and drives food consumption, it is pertinent to ask how effective exercise could be in helping people to lose weight or to prevent weight gain. The effects of exercise on hunger sensations and food intake are fairly controversial and depend on the intensity and duration of exercise. 120 min prolonged treadmill exercise with mix intensity, temporarily decreased hunger sensations, acylated ghrelin and relative energy intake. Variations in exercise intensity should

  5. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-06

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

  6. Location and biosynthesis of monoterpenyl fatty acyl esters in rose petals.

    PubMed

    Dunphy, Patrick J

    2006-06-01

    The upper epidermal layer of cells and the epicuticular wax surface of Lady Seton rose petals are sites of biosynthesis and accumulation, respectively, of a family of terpenyl fatty acyl esters. These esters are based mainly on the acyclic monoterpene alcohol geraniol coupled primarily to fatty acids of chain lengths 16-20 and in mass terms represent from 14% to 64% of the total monoterpenes present in the petals. The lipophilic nature of these non-volatile esters of the monoterpene alcohols contrasts with that of the lipophilic volatile parent alcohols themselves and with the hydrophilic, non-volatile, glucoside derivative of the other principal petal fragrant compounds, the phenylpropanoids, beta-phenyl ethanol and benzyl alcohol. These latter compounds are also synthesised and are resident in the petal. Biosynthetic studies confirmed that the petal upper epidermal cell layer has the capacity to incorporate mevalonic acid into the monoterpene component of the fatty acyl ester. The biosynthesis of the monoterpene component of the fatty acyl ester occurs via the mevalonic acid pathway in Lady Seton as well as in the hybrid tea rose Fragrant Cloud. In the latter flower the biosynthesis of geraniol was biosynthetically trans as was the formation of nerol and citronellol. Both geraniol and nerol were shown to be precursors of citronellol via an NADPH dependent reductase reaction. Oleic acid is assimilated into the acyl moiety of the terpenyl ester in Lady Seton isolated petal discs. It is probable that the lipophilic non-volatile terpenyl fatty acyl esters represent a stable storage form of the corresponding alcohols from their residency within the epicuticular wax layer. These acyl esters may realise, on hydrolysis, additional aroma notes from the living flower and potentially commercially significant quantities of the fragrant terpenols during oil of rose essence production.

  7. Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: identification of a novel variant of isoform 6.

    PubMed

    Van Horn, Cynthia G; Caviglia, Jorge M; Li, Lei O; Wang, Shuli; Granger, Deborah A; Coleman, Rosalind A

    2005-02-08

    The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.

  8. Bezafibrate induces acyl-CoA oxidase mRNA levels and fatty acid peroxisomal beta-oxidation in rat white adipose tissue.

    PubMed

    Vázquez, M; Roglans, N; Cabrero, A; Rodríguez, C; Adzet, T; Alegret, M; Sánchez, R M; Laguna, J C

    2001-01-01

    Rats treated with bezafibrate, a PPAR activator, gain less body weight and increase daily food intake. Previously, we have related these changes to a shift of thermogenesis from brown adipose tissue to white adipose tissue attributable to bezafibrate, which induces uncoupling proteins (UCP), UCP-1 and UCP-3, in rat white adipocytes. Nevertheless, UCP induction was weak, implying additional mechanisms in the change of energy homeostasis produced by bezafibrate. Here we show that bezafibrate, in addition to inducing UCPs, modifies energy homeostasis by directly inducing aco gene expression and peroxisomal fatty acid beta-oxidation in white adipose tissue. Further, bezafibrate significantly reduced plasma triglyceride and leptin concentrations, without modifying the levels of PPARgamma or ob gene in white adipose tissue. These results indicate that bezafibrate reduces the amount of fatty acids available for triglyceride synthesis in white adipose tissue.

  9. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1...., chloride (PMN P-01-646; CAS No. 391232-99-8) is subject to reporting under this section for the...

  10. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1...., chloride (PMN P-01-646; CAS No. 391232-99-8) is subject to reporting under this section for the...

  11. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1...., chloride (PMN P-01-646; CAS No. 391232-99-8) is subject to reporting under this section for the...

  12. Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition.

    PubMed

    Moreno-Pérez, Antonio J; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2011-01-01

    The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results.

  13. Thermoregulation of N-acyl homoserine lactone-based quorum sensing in the soft rot bacterium Pectobacterium atrosepticum.

    PubMed

    Latour, Xavier; Diallo, Stéphanie; Chevalier, Sylvie; Morin, Danièle; Smadja, Bruno; Burini, Jean-François; Haras, Dominique; Orange, Nicole

    2007-06-01

    The psychrotolerant bacterium Pectobacterium atrosepticum produces four N-acyl homoserine lactones under a wide range of temperatures. Their thermoregulation differs from that of the exoenzyme production, described as being under quorum-sensing control. A mechanism involved in this thermoregulation consists of controlling N-acyl homoserine lactones synthase production at a transcriptional level.

  14. Investigation of acyl migration in mono- and dicaffeoylquinic acids under aqueous basic, aqueous acidic, and dry roasting conditions.

    PubMed

    Deshpande, Sagar; Jaiswal, Rakesh; Matei, Marius Febi; Kuhnert, Nikolai

    2014-09-17

    Acyl migration in chlorogenic acids describes the process of migration of cinnamoyl moieties from one quinic acid alcohol group to another, thus interconverting chlorogenic acid regioisomers. It therefore constitutes a special case of transesterification reaction. Acyl migration constitutes an important reaction pathway in both coffee roasting and brewing, altering the structure of chlorogenic acid initially present in the green coffee bean. In this contribution we describe detailed and comprehensive mechanistic studies comparing inter- and intramolecular acyl migration involving the seven most common chlorogenic acids in coffee. We employe aqueous acidic and basic conditions mimicking the brewing of coffee along with dry roasting conditions. We show that under aqueous basic conditions intramolecular acyl migration is fully reversible with basic hydrolysis competing with acyl migration. 3-Caffeoylquinic acid was shown to be most labile to basic hydrolysis. We additionally show that the acyl migration process is strongly pH dependent with increased transesterification taking place at basic pH. Under dry roasting conditions acyl migration competes with dehydration to form lactones. We argue that acyl migration precedes lactonization, with 3-caffeoylquinic acid lactone being the predominant product.

  15. Acylation, Diastereoselective Alkylation, and Cleavage of an Oxazolidinone Chiral Auxiliary: A Multistep Asymmetric Synthesis Experiment for Advanced Undergraduates

    ERIC Educational Resources Information Center

    Smith, Thomas E.; Richardson, David P.; Truran, George A.; Belecki, Katherine; Onishi, Megumi

    2008-01-01

    An introduction to the concepts and experimental techniques of diastereoselective synthesis using a chiral auxiliary is described. The 4-benzyl-2-oxazolidinone chiral auxiliary developed by Evans is acylated with propionic anhydride under mild conditions using DMAP as an acyl transfer catalyst. Deprotonation with NaN(TMS)[subscript 2] at -78…

  16. Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles inacyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for se...

  17. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells.

    PubMed

    Pedroza, Diego A; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J; Mito, Shizue

    2014-01-15

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death.

  18. Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25

    SciTech Connect

    Chang, C.C.Y.; Chang, W.; Chang, T.Y. ); Noll, W.W.; Nutile-McMenemy, N. ); Lindsay, E.A.; Baldini, A. )

    1994-01-01

    Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently the authors' laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. They have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human-hamster somatic cell hybrid panels.

  19. Synthesis of multi ring-fused 2-pyridones via an acyl-ketene imine cyclocondensation.

    PubMed

    Pemberton, Nils; Jakobsson, Lotta; Almqvist, Fredrik

    2006-03-02

    Polycyclic ring-fused 2-pyridones (5a-e and 9a-e) have been prepared via a microwave-assisted acyl-ketene imine cyclocondensation. Starting from 3,4-dihydroisoquinolines (4a-b) or 3,4-dihydroharman (8), fused 2-pyridones could be prepared in a one-step procedure. By using either Meldrum's acid derivatives (1a-d) or 1,3-dioxine-4-ones (7a-b) as acyl-ketene sources, mono- or disubstitution of the fused 2-pyridone ring could be accomplished. As an application of the method, a formal synthesis of the indole alkaloid sempervilam was performed.

  20. Macrocyclic prolinyl acyl guanidines as inhibitors of β-secretase (BACE).

    PubMed

    Boy, Kenneth M; Guernon, Jason M; Wu, Yong-Jin; Zhang, Yunhui; Shi, Joe; Zhai, Weixu; Zhu, Shirong; Gerritz, Samuel W; Toyn, Jeremy H; Meredith, Jere E; Barten, Donna M; Burton, Catherine R; Albright, Charles F; Good, Andrew C; Grace, James E; Lentz, Kimberley A; Olson, Richard E; Macor, John E; Thompson, Lorin A

    2015-11-15

    The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.

  1. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells

    PubMed Central

    Pedroza, Diego A.; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J.; Mito, Shizue

    2014-01-01

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death. PMID:24368029

  2. Kinetic resolution of acids in acylation reactions in the presence of chiral tertiary amines

    SciTech Connect

    Potapov, V.M.; Dem'yanovich, V.M.; Khlebnikov, V.A.

    1988-07-10

    Asymmetric synthesis has now become an important method for the production of optically active compounds, and its most attractive form is asymmetric catalysis. This work was devoted to an investigation into asymmetric catalysis with chiral tertiary amines in acylation reactions. During the acylation of alcohols and amines by the action of racemic 2-phenylpropionic and 2-methyl-3-phenylpropionic acids in the presence of S-nicotine the initial acids are resolved kinetically. The (R)-2-phenylpropionic acid obtained in this way had an optical purity of 0.5-1.5%.

  3. A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines

    PubMed Central

    Ogura, Yuji; Parsons, William H.; Kamat, Siddhesh S.; Cravatt, Benjamin F.

    2016-01-01

    More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells. PMID:27399000

  4. Cation binding site of cytochrome c oxidase: progress report.

    PubMed

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  5. Crystal Structure of Alcohol Oxidase from Pichia pastoris

    PubMed Central

    Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

    2016-01-01

    FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

  6. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

  7. Matrix-assisted ultraviolet laser desorption/ionization time-of-flight (UV-MALDI-TOF) mass spectra of N-acylated and N,O-acylated glycosylamines.

    PubMed

    Sato, Yasuto; Fukuyama, Yuko; Nonami, Hiroshi; Erra-Balsells, Rosa; Stortz, Carlos A; Cerezo, Alberto S; Matulewicz, María C

    2007-12-10

    Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.

  8. The Acyl-Acyl Carrier Protein Synthetase from Synechocystis sp. PCC 6803 Mediates Fatty Acid Import1[C][W][OA

    PubMed Central

    von Berlepsch, Simon; Kunz, Hans-Henning; Brodesser, Susanne; Fink, Patrick; Marin, Kay; Flügge, Ulf-Ingo; Gierth, Markus

    2012-01-01

    The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[14C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [14C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane. PMID:22535424

  9. Heterologous expression of the acyl-acyl carrier protein thioesterase gene from the plant Umbellularia californica mediates polyhydroxyalkanoate biosynthesis in recombinant Escherichia coli.

    PubMed

    Rehm, B H; Steinbüchel, A

    2001-03-01

    The acyl-acyl carrier protein (ACP) thioesterase cDNA from the plant Umbellularia californica was functionally expressed in various recombinant Escherichia coli strains in order to establish a new metabolic route toward medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis from non-related carbon sources. Coexpression of the PHA synthase genes from Ralstonia eutropha and Pseudomonas aeruginosa, or only the PHA synthase gene from P. aeruginosa, respectively, showed PHA(MCL) accumulation when the type II PHA synthase from P. aeruginosa was produced. Both wild-type E. coli and various fad mutants were investigated; and only when the beta-oxidation pathway was impaired PHA(MCL) accumulation from gluconate was observed, contributing to about 6% of cellular dry weight. Thus coexpression of type II PHA synthase gene with cDNA encoding the medium-chain acyl-ACP thioesterase from U. californica established a new PHA(MCL) biosynthesis pathway, connecting fatty acid de novo biosynthesis with fatty acid beta-oxidation, using a non-related carbon source.

  10. Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases.

    PubMed

    Tong, Fumin; Black, Paul N; Coleman, Rosalind A; DiRusso, Concetta C

    2006-03-01

    Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.

  11. Red clover polyphenol oxidase and lipid metabolism.

    PubMed

    Van Ranst, G; Lee, M R F; Fievez, V

    2011-02-01

    Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vit