WorldWideScience

Sample records for acetyl coa carboxylase

  1. Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

    Jang Eun Lee

    Full Text Available Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2 regulates fatty acid oxidation (FAO by inhibiting carnitine palmitoyltransferase 1 (CPT1, a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

  2. Biotin augments acetyl CoA carboxylase 2 gene expression in the hypothalamus, leading to the suppression of food intake in mice.

    Sone, Hideyuki; Kamiyama, Shin; Higuchi, Mutsumi; Fujino, Kaho; Kubo, Shizuka; Miyazawa, Masami; Shirato, Saya; Hiroi, Yuka; Shiozawa, Kota

    2016-07-29

    It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment. PMID:27181349

  3. Role of acetyl CoA

    Existence of an acetyltransferase, which catalizes acetylation of deacetylcephalosporin C to cephalosporin C, was demonstrated for the first time in cell-free extracts of Cephalosporium acremonium. The pH optimum of the enzyme appeared to be 7.0 to 7.5 and the enzyme required essentially Mg2+ as a cofactor for its reaction. The activity of this enzyme was not observed in the cell-free extracts of deacetylcephalosporin C-producing mutants Nos. 20, 29, 36 and 40, but was recovered in a revertant obtained from the mutant No. 40. These results indicate that deacetylcephalosporin C accumulation by these mutants was due to the lack of the acetyltransferase and made it reasonable that the terminal reaction of cephalosporin C biosynthesis in Cephalosporium acremonium proceeded by the catalytic action of acetyltransferase. (auth.)

  4. Chemical Genetics of Acetyl-CoA Carboxylases

    Xuyu Zu

    2013-01-01

    Full Text Available Chemical genetic studies on acetyl-CoA carboxylases (ACCs, rate-limiting enzymes in long chain fatty acid biosynthesis, have greatly advanced the understanding of their biochemistry and molecular biology and promoted the use of ACCs as targets for herbicides in agriculture and for development of drugs for diabetes, obesity and cancers. In mammals, ACCs have both biotin carboxylase (BC and carboxyltransferase (CT activity, catalyzing carboxylation of acetyl-CoA to malonyl-CoA. Several classes of small chemicals modulate ACC activity, including cellular metabolites, natural compounds, and chemically synthesized products. This article reviews chemical genetic studies of ACCs and the use of ACCs for targeted therapy of cancers.

  5. The dynamic organization of fungal acetyl-CoA carboxylase

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  6. The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

    P. Marini(GANIL); Li, S J; Gardiol, D; Cronan, J E; De Mendoza, D

    1995-01-01

    The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.

  7. Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

    Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

    1995-06-01

    Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

  8. Inhibition of acetyl-CoA carboxylase by cystamine may mediate the hypotriglyceridemic activity of pantethine.

    McCarty, M F

    2001-03-01

    Pantethine is a versatile and well-tolerated hypolipidemic agent whose efficacy in this regard appears to be mediated by its catabolic product cystamine, a nucleophile which avidly attacks disulfide groups. An overview of pantethine research suggests that the hypotriglyceridemic activity of pantethine reflects cystamine-mediated inhibition of the hepatic acetyl-CoA carboxylase, which can be expected to activate hepatic fatty acid oxidation. Inhibition of HMG-CoA reductase as well as a more distal enzyme in the cholesterol synthetic pathway may account for pantethine's hypocholesterolemic effects. If pantethine does indeed effectively inhibit hepatic acetyl-CoA carboxylase, it may have adjuvant utility in the hepatothermic therapy of obesity. As a safe and effective compound of natural origin, pantethine merits broader use in the management of hyperlipidemias. PMID:11359352

  9. Wheat cytosolic acetyl-CoA carboxylase complements an ACC1 null mutation in yeast

    Joachimiak, M.; Tevzadze, G.; Podkowinski, J; Haselkorn, R.; Gornicki, P.

    1997-01-01

    Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow. A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3′ tail was used to complement a yeast ACC1 mutation. The complementation demonstrates that active wheat ACCase can be produced in yeast. At low concentrations of galactose,...

  10. Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer.

    Wei, Jia; Tong, Liang

    2015-10-29

    Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis. PMID:26458104

  11. Synthesis of O-[11C]acetyl CoA, O-[11C]acetyl-L-carnitine, and L-[11C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey

    The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with 11C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1-11C]acetyl CoA and O-[2-11C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1-11C]acetyl-L-carnitine and O-[2-11C]acetyl-L-carnitine in 70-80% yield, based on [1-11C]acetate or [2-11C]acetate, respectively. By an N-methylation reaction with [11C]methyl iodide, L-[methyl-11C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl-11C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [11C]methyl iodide. Initial data of the kinetics of the different 11C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented

  12. Synthesis of O-[{sup 11}C]acetyl CoA, O-[{sup 11}C]acetyl-L-carnitine, and L-[{sup 11}C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey

    Jacobson, Gunilla B.; Watanabe, Yasuyoshi; Valind, Sven; Kuratsune, Hirohiko; Laangstroem, Bengt

    1997-07-01

    The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with {sup 11}C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1-{sup 11}C]acetyl CoA and O-[2-{sup 11}C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1-{sup 11}C]acetyl-L-carnitine and O-[2-{sup 11}C]acetyl-L-carnitine in 70-80% yield, based on [1-{sup 11}C]acetate or [2-{sup 11}C]acetate, respectively. By an N-methylation reaction with [{sup 11}C]methyl iodide, L-[methyl-{sup 11}C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl-{sup 11}C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [{sup 11}C]methyl iodide. Initial data of the kinetics of the different {sup 11}C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented.

  13. Cloning and characterization of cotton heteromeric acetyl-CoA carboxylase genes

    2007-01-01

    Heteromeric acetyl-coanzyme A(CoA)carboxylese(ACCase)catalyzes the formation of malonyl-CoA from acetyl-CoA.It plays an essential role in fatty acid synthesis in prokaryotes and most of plants.The heteromeric ACCase is composed of four subunits:biotin carboxylase (BC),biotin carboxyl carrier protein (BCCP),and α-and β-subunits of carboxyltransferese(α-andβ-CT).In this study,we cloned five novel genes encoding the subunits of heteromeric ACCese(one BC,BCCP and β-CT,and two α-CTs) from cotton (Gossypium hirsutum cv.zhongmian 35)by RACE-PCR.The deduced amino acid sequence of these cDNAs shares high similarity with other reported heteromeric ACCese subunits.The phylogenetic analysis indicated that the different subunits of heteromeric ACCase were grouped in a similar pattern.Southern blot analysis revealed the milti-copy patterns of these heteromeric ACCase genes in cotton genome.Semi-quantitative RT-PCR demonstrated that heteromeric ACCese genes were constitutively expressed in all of the cotton tissues,but the transcripts accumulated at a relatively low level in roots.To our knowledge,this is the first report about characterization of the heteromeric ACCase genes in cotton.

  14. Improving polyketide and fatty acid synthesis by engineering of the yeast acetyl-CoA carboxylase.

    Choi, Jin Wook; Da Silva, Nancy A

    2014-10-10

    Polyketides and fatty acids are important in the production of pharmaceuticals, industrial chemicals, and biofuels. The synthesis of the malonyl-CoA building block, catalyzed by acetyl-CoA carboxylase (Acc1), is considered a limiting step to achieving high titers of polyketides and fatty acids in Saccharomyces cerevisiae. Acc1 is deactivated by AMP-activated serine/threonine protein kinase (Snf1) when glucose is depleted. To prevent this deactivation, the enzyme was aligned with the Rattus norvegicus (rat) Acc1 to identify a critical amino acid (Ser-1157) for phosphorylation and deactivation. Introduction of a S1157A mutation into Acc1 resulted in 9-fold higher specific activity following glucose depletion. The enzyme was tested in yeast engineered to produce the polyketide 6-methylsalisylic acid (6-MSA). Both 6-MSA and native fatty acid levels increased by 3-fold. Utilization of this modified Acc1 enzyme will also be beneficial for other products built from malonyl-CoA. PMID:25078432

  15. Determination of ploidy level and isolation of genes encoding acetyl-CoA carboxylase in Japanese Foxtail (Alopecurus japonicus.

    Hongle Xu

    Full Text Available Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.

  16. Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E.coil

    WANG Rui-jian; YANG Xue-ying; ZHENG Liang-yu; YANG Ye; GAO Gui; CAO Shu-gui

    2008-01-01

    The entire gene of carboxyltransferase(CT) domain of acetyl-CoA carboxylase(ACCase) from Chinese Spring wheat(CSW) plastid was cloned firstly,and the 2.3 kb gene was inserted into PET28a+ vector and expressed in E.coil in a soluble state.The (His)6 fusion protein was identified by SDS-PAGE and Western blot.The recombinant protein was purified by affinity chromatography,and the calculated molecular mass(Mr) was 88000.The results of the sequence analysis indicate that the cloned gene(GeneBank accession No.EU124675) was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid.The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides,and further to screen new herbicides.

  17. Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides

    Fernández, Pablo; Alcántara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo; Osuna, María D.; Prado, Rafael

    2016-01-01

    Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor)...

  18. Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides.

    Fernández, Pablo; Alcántara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo; Osuna, María D; De Prado, Rafael

    2016-01-01

    Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha(-1)) compared with the GR50 (1484.6 g ai ha(-1)) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar (14)C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 h after treatment (HAT). The metabolism of (14)C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg. PMID:27148285

  19. [The protective effect of pantothenic acid derivatives and changes in the system of acetyl CoA metabolism in acute ethanol poisoning].

    Moiseenok, A G; Dorofeev, B F; Omel'ianchik, S N

    1988-01-01

    Calcium pantothenate (CaP), calcium 4'-phosphopantothenate (CaPP), pantethine, panthenol, sulfopantetheine and CoA decrease acute toxicity of acetaldehyde in mice. All studied compounds diminish duration of the narcotic action of ethanol--ET (3.5 g/kg intraperitoneally) in mice and rats. In the latter this effect is realized at the expense of "long sleeping" and "middle sleeping" animals. CaP (150 mg/kg subcutaneously) and CaPP (100 mg/kg subcutaneously) prevent hypothermia and a decrease of oxygen consumption in rats induced by ET administration. Combined administration of ET, CaP and CaPP leads to a characteristic increase of acid-soluble CoA fractions in the rat liver and a relative decrease of acetyl CoA synthetase and N-acetyltransferase reactions. The antitoxic effect of preparations of pantothenic acid is not mediated by CoA-dependent reactions of detoxication, but most probably is due to intensification of ET oxidation and perhaps to its elimination from the organism. PMID:2905277

  20. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    Research highlights: → AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. → Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. → AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACCβ activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid β-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACCβ promoter activity via AMPK activation. A human ACCβ promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes ± a NRF-1 expression construct. NRF-1 overexpression decreased ACCβ gene promoter activity by 71 ± 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACCβ was abolished with a pPIIβ-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACCβ promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCβ gene promoter. Here NRF-1 blunted USF1-dependent induction of ACCβ promoter activity by 58 ± 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 ± 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACCβ gene promoter in the mammalian heart. Our data extends AMPK regulation of ACCβ to the transcriptional level.

  1. Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats.

    Harriman, Geraldine; Greenwood, Jeremy; Bhat, Sathesh; Huang, Xinyi; Wang, Ruiying; Paul, Debamita; Tong, Liang; Saha, Asish K; Westlin, William F; Kapeller, Rosana; Harwood, H James

    2016-03-29

    Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein-protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease. PMID:26976583

  2. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  3. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  4. Metabolic regulation of invadopodia and invasion by acetyl-CoA carboxylase 1 and de novo lipogenesis.

    Kristen E N Scott

    Full Text Available Invadopodia are membrane protrusions that facilitate matrix degradation and cellular invasion. Although lipids have been implicated in several aspects of invadopodia formation, the contributions of de novo fatty acid synthesis and lipogenesis have not been defined. Inhibition of acetyl-CoA carboxylase 1 (ACC1, the committed step of fatty acid synthesis, reduced invadopodia formation in Src-transformed 3T3 (3T3-Src cells, and also decreased the ability to degrade gelatin. Inhibition of fatty acid synthesis through AMP-activated kinase (AMPK activation and ACC phosphorylation also decreased invadopodia incidence. The addition of exogenous 16∶0 and 18∶1 fatty acid, products of de novo fatty acid synthesis, restored invadopodia and gelatin degradation to cells with decreased ACC1 activity. Pharmacological inhibition of ACC also altered the phospholipid profile of 3T3-Src cells, with the majority of changes occurring in the phosphatidylcholine (PC species. Exogenous supplementation with the most abundant PC species, 34∶1 PC, restored invadopodia incidence, the ability to degrade gelatin and the ability to invade through matrigel to cells deficient in ACC1 activity. On the other hand, 30∶0 PC did not restore invadopodia and 36∶2 PC only restored invadopodia incidence and gelatin degradation, but not cellular invasion through matrigel. Pharmacological inhibition of ACC also reduced the ability of MDA-MB-231 breast, Snb19 glioblastoma, and PC-3 prostate cancer cells to invade through matrigel. Invasion of PC-3 cells through matrigel was also restored by 34∶1 PC supplementation. Collectively, the data elucidate the novel metabolic regulation of invadopodia and the invasive process by de novo fatty acid synthesis and lipogenesis.

  5. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    Adam, Tasneem; Opie, Lionel H. [Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Observatory 7925 (South Africa); Essop, M. Faadiel, E-mail: mfessop@sun.ac.za [Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600 (South Africa)

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  6. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified

  7. Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior

    Bergauer, Kristin; Sintes, Eva; van Bleijswijk, Judith; Witte, Harry; Herndl, Gerhard J.; Lueders, Tillmann

    2013-01-01

    Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating dark CO2 fixation in the tropical Atlantic, we quantified functional genes indicative for CO2 fixation. We used a Q-PCR-based assay targeting the bifunctional acetyl-CoA/propionyl-CoA carboxylase (a...

  8. Dinuclear nickel complexes modeling the structure and function of the acetyl CoA synthase active site

    Ito, Mikinao; Kotera, Mai; Matsumoto, Tsuyoshi; Tatsumi, Kazuyuki

    2009-01-01

    A dinuclear nickel complex with methyl and thiolate ligands, Ni(dadtEt)Ni(Me)(SDmp) (2), has been synthesized as a dinuclear Nid–Nip-site model of acetyl-CoA synthase (ACS) (dadtEt is N,N′-diethyl-3,7-diazanonane-1,9-dithiolate; Dmp is 2,6-dimesitylphenyl). Complex 2 was prepared via 2 methods: (i) ligand substitution of a dinuclear Ni(II)–Ni(II) cation complex [Ni(dadtEt) Ni(tmtu)2] (OTf)2(1) with MeMgBr and KSDmp (tmtu is tetramethylthiourea), (ii) methyl transfer from methylcobaloxime Co(d...

  9. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense

    Leyva, Luis A.; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E.

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  10. Genes encoding the alpha-carboxyltransferase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    Li, Zhi-Guo; Yin, Wei-Bo; Guo, Huan; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2010-05-01

    Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape. PMID:20616867

  11. Inhibition of acetyl-CoA carboxylases by soraphen A prevents lipid accumulation and adipocyte differentiation in 3T3-L1 cells.

    Cordonier, Elizabeth L; Jarecke, Sarah K; Hollinger, Frances E; Zempleni, Janos

    2016-06-01

    Acetyl-CoA carboxylases (ACC) 1 and 2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA and depend on biotin as a coenzyme. ACC1 localizes in the cytoplasm and produces malonyl-CoA for fatty acid (FA) synthesis. ACC2 localizes in the outer mitochondrial membrane and produces malonyl-CoA that inhibits FA import into mitochondria for subsequent oxidation. We hypothesized that ACCs are checkpoints in adipocyte differentiation and tested this hypothesis using the ACC1 and ACC2 inhibitor soraphen A (SA) in murine 3T3-L1 preadipocytes. When 3T3-L1 cells were treated with 100nM SA for 8 days after induction of differentiation, the expression of PPARγ mRNA and FABP4 mRNA decreased by 40% and 50%, respectively, compared with solvent controls; the decrease in gene expression was accompanied by a decrease in FABP4 protein expression and associated with a decrease in lipid droplet accumulation. The rate of FA oxidation was 300% greater in SA-treated cells compared with vehicle controls. Treatment with exogenous palmitate restored PPARγ and FABP4 mRNA expression and FABP4 protein expression in SA-treated cells. In contrast, SA did not alter lipid accumulation if treatment was initiated on day eight after induction of differentiation. We conclude that loss of ACC1-dependent FA synthesis and loss of ACC2-dependent inhibition of FA oxidation prevent lipid accumulation in adipocytes and inhibit early stages of adipocyte differentiation. PMID:27041646

  12. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    Griffith, David A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Kung, Daniel W. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Esler, William P. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Amor, Paul A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Bagley, Scott W. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Beysen, Carine [KineMed Inc., Emeryville, CA (United States); Carvajal-Gonzalez, Santos [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Doran, Shawn D. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Limberakis, Chris [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Mathiowetz, Alan M. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); McPherson, Kirk [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Price, David A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Ravussin, Eric [Louisiana State Univ., Baton Rouge, LA (United States); Sonnenberg, Gabriele E. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Southers, James A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Sweet, Laurel J. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Turner, Scott M. [KineMed Inc., Emeryville, CA (United States); Vajdos, Felix F. [Pfizer Worldwide Research and Development, Cambridge, MA (United States)

    2014-12-26

    We found that Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. Here, we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.

  13. Increased expression of fatty acid synthase and acetyl-CoA carboxylase in the prefrontal cortex and cerebellum in the valproic acid model of autism

    Chen, Jianling; Wu, Wei; Fu, Yingmei; Yu, Shunying; Cui, Donghong; Zhao, Min; Du, Yasong; Li, Jijun; Li, Xiaohong

    2016-01-01

    The primary aim of the present study was to investigate alterations in enzymes associated with fatty acid synthesis, namely fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), in the prefrontal cortex and cerebellum of the valproic acid (VPA)-induced animal model of autism. In this model, pregnant rats were given a single intraperitoneal injection of VPA, and prefrontal cortex and cerebellum samples from their pups were analyzed. The results of western blotting and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that the protein and mRNA expression levels of FASN, ACC and phospho-ACC (pACC) were increased in the prefrontal cortex and cerebellum of the VPA model of autism. Furthermore, in the prefrontal cortex and cerebellum of the VPA model of autism, AMPK expression is increased, whereas PI3K and Akt expression are unchanged. This suggests that disorder of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/FASN and/or adenosine 5′-monophosphate-activated protein kinase (AMPK)/ACC pathway may be involved in the pathogenesis of autism. It is hypothesized that fatty acid synthesis participates in autism through PI3K/Akt/FASN and AMPK/ACC pathways. PMID:27602061

  14. The glossyhead1 Allele of ACC1 Reveals a Principal Role for Multidomain Acetyl-Coenzyme A Carboxylase in the Biosynthesis of Cuticular Waxes by Arabidopsis

    Lu, S.; Xu, C.; Zhao, H.; Parsons, E. P.; Kosma, D. K.; Xu, X.; Chao, D.; Lohrey, G.; Bangarusamy, D. K.; Wang, G.; Bressan, R. A.; Jenks, M. A.

    2011-11-01

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C{sub 20:0} or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling.

  15. The glossyhead1 allele of acc1 reveals a principal role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by Arabidopsis

    Lu, Shiyou

    2011-09-23

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C 20:0 or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling. © 2011 American Society of Plant Biologists. All Rights Reserved.

  16. Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl-CoA carboxylase as a PII binding partner.

    Rodrigues, Thiago E; Gerhardt, Edileusa C M; Oliveira, Marco A; Chubatsu, Leda S; Pedrosa, Fabio O; Souza, Emanuel M; Souza, Gustavo A; Müller-Santos, Marcelo; Huergo, Luciano F

    2014-02-01

    The PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII -BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII -BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII ; however, it was completely dependent on the post-translational biotinylation of BCCP. PMID:24329683

  17. Computational simulations of structural role of the active-site W374C mutation of acetyl-coenzyme-A carboxylase: multi-drug resistance mechanism.

    Zhu, Xiao-Lei; Yang, Wen-Chao; Yu, Ning-Xi; Yang, Sheng-Gang; Yang, Guang-Fu

    2011-03-01

    Herbicides targeting grass plastidic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) are selectively effective against graminicides. The intensive worldwide use of this herbicide family has selected for resistance genes in a number of grass weed species. Recently, the active-site W374C mutation was found to confer multi-drug resistance toward haloxyfop (HF), fenoxaprop (FR), Diclofop (DF), and clodinafop (CF) in A. myosuroides. In order to uncover the resistance mechanism due to W374C mutation, the binding of above-mentioned four herbicides to both wild-type and the mutant-type ACCase was investigated in the current work by molecular docking and molecular dynamics (MD) simulations. The binding free energies were calculated by molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) method. The calculated binding free energy values for four herbicides were qualitatively consistent with the experimental order of IC(50) values. All the computational model and energetic results indicated that the W374C mutation has great effects on the conformational change of the binding pocket and the ligand-protein interactions. The most significant conformational change was found to be associated with the aromatic amino acid residues, such as Phe377, Tyr161' and Trp346. As a result, the π-π interaction between the ligand and the residue of Phe377 and Tyr161', which make important contributions to the binding affinity, was decreased after mutation and the binding affinity for the inhibitors to the mutant-type ACCase was less than that to the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural role and mechanistic insights obtained from computational simulations will provide a new starting point for the rational design of novel inhibitors to overcome drug resistance associated with W374C mutation. PMID:20499260

  18. Expression and methylation of microsomal triglyceride transfer protein and acetyl-CoA carboxylase are associated with fatty liver syndrome in chicken.

    Liu, Zhen; Li, Qinghe; Liu, Ranran; Zhao, Guiping; Zhang, Yonghong; Zheng, Maiqing; Cui, Huanxian; Li, Peng; Cui, Xiaoyan; Liu, Jie; Wen, Jie

    2016-06-01

    The typical characteristic of fatty liver syndrome (FLS) is an increased hepatic triacylglycerol content, and a sudden decline in egg production often occurs. FLS may develop into fatty liver hemorrhagic syndrome (FLHS), characterized by sudden death from hepatic rupture and hemorrhage. DNA methylation is associated with transcriptional silencing, leading to the etiology and pathogenesis of some animal diseases. The roles of DNA methylation in the genesis of FLS, however, are largely unknown. The lipogenic methyl-deficient diet (MDD) caused FLS similar to human nonalcoholic steatohepatitis (NASH). After 16 Jingxing-Huang (JXH) hens were fed MDD for 10 wk, eight exhibited FLS (designated as FLS-susceptible birds); the remainder, without FLS, served as controls (NFLS). Physiological and biochemical variables, gene expression levels, and DNA methylation were determined in the liver. The development of FLS in JXH hens was accompanied by abnormal lipid accumulation. Relative expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and microsomal triglyceride transfer protein (MTTP) were significantly up-regulated in the FLS group in comparison with the NFLS group. The transcript abundance of sterol regulatory element binding protein 1 (SREBP-1c), stearoyl-CoA desaturase (SCD), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptor alpha (PPARα), and peroxisome proliferator-activated receptor gamma (PPARγ) did not differ between the two groups. Interestingly, MTTP and ACC mRNA abundance were negatively correlated with the level of promoter methylation. The extent of DNA methylation of the cytosine-guanine (CpG) sites in the SREBP-1c, FAS, PPARα, and LXRα promoter regions was also analyzed by direct sequencing but none differed between FLS and NFLS birds. Taken together, these results specify link DNA methylation to the pathogenesis of FLS in chickens. PMID:27083546

  19. Associations of polymorphisms in the promoter I of bovine acetyl-CoA carboxylase-alpha gene with beef fatty acid composition.

    Zhang, S; Knight, T J; Reecy, J M; Wheeler, T L; Shackelford, S D; Cundiff, L V; Beitz, D C

    2010-08-01

    The objectives of this study were to identify single nucleotide polymorphisms (SNPs) in the promoter I (PI) region of the bovine acetyl-CoA carboxylase-alpha (ACACA) gene and to evaluate the extent to which they were associated with lipid-related traits. Eight novel SNPs were identified, which were AJ276223:g.2064T>A (SNP1), g.2155C>T (SNP2), g.2203G>T (SNP3), g.2268T>C (SNP4), g.2274G>A (SNP5), g.2340A>G (SNP6), g.2350T>C (SNP7) and g.2370A>G (SNP8). Complete linkage disequilibrium was observed among SNP1, 2, 4, 5, 6 and 8. Phenotypic data were collected from 573 cross-bred steers with six sire breeds, including Hereford, Angus, Brangus, Beefmaster, Bonsmara and Romosinuano. The genotypes of SNP1/2/4/5/6/8 were significantly associated with adjusted backfat thickness. The genotypes of SNP3 were significantly associated with triacylglycerol (TAG) content and fatty acid composition of longissimus dorsi muscle (LM) in Brangus-, Romosinuano- and Bonsmara-sired cattle. Cattle with g.2203GG genotype had greater concentrations of TAG, total lipid, total saturated fatty acid and total monounsaturated fatty acid than did cattle with g.2203GT genotype. The genotypes of SNP7 were significantly associated with fatty acid composition of LM. Cattle with genotype g.2350TC had greater amounts of several fatty acids in LM than did cattle with genotype g.2350CC. Our results suggested that the SNPs in the PI region of ACACA gene are associated with variations in the fatty acid contents in LM. PMID:20002363

  20. A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A

    Yu, L.; Xiang, S; Lasso, G; Gil, D; Valle, M; Tong, L

    2009-01-01

    Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

  1. Characterization of acetyl-CoA and propionyl-CoA carboxylases encoded by Leptospira interrogans serovar Lai: an initial biochemical study for leptospiral gluconeogenesis via anaplerotic CO2 assimilation

    Nanqiu Peng; Yi Zhong; Qing Zhang; Mingyue Zheng; Wei Zhao; Hualiang Jiang; Chen Yang; Xiaokui Guo; Guoping Zhao

    2012-01-01

    Leptospira interrogans is the causative agent of leptospirosis.The in vitro growth of L.interrogans requires CO2 and a partial 3-hydroxypropionate pathway involving two acyl-CoA carboxylases was suggested by genomic analysis to assimilate CO2.Either set of the candidate genes heterologously co-expressed in Escherichia coli was able to demonstrate both acetyl-CoA carboxylase (ACC)and propionyl-CoA carboxylase (PCC) activities.The trisubunit holoenzyme (LA_2736-LA_2735 and LA_3803),although failed to be purified,was designated ACC based on its substrate preference toward acetyl-CoA.The partially purified bi-subunit holoenzyme (LA_2432-LA_2433) has a considerably higher activity against propionyi-CoA as the substrate than that of acetyl-CoA,and thus,designated PCC.Native polyacrylamide gel electrophoresis indicated that this PCC has a molecular mass of around 669 kDa,suggesting an α4β4 quaternary structure and both structural homology modeling and site-directed mutagenesis analysis of its carboxyltransferase subunit (LA_2433) indicated that the A431 residue located at the bottom of the putative substrate binding pocket may play an important role in substrate specificity determination.Both transcriptomic and proteomic data indicated that enzymes involved in the suggested partial 3-hydroxypropionate pathway were expressed in vivo in addition to ACC/PCC and the homologous genes in genomes of other Leptospira species were re-annotated accordingly.However,as the in vitro detected specific activity of ACC in the crude cell extract was too low to account for the growth of the bacterium in Ellinghausen-McCulloughJohnson-Harris minimal medium,further systematic analysis is required to unveil the mechanism of gluconeogenesis via anaplerotic CO2 assimilation in Leptospira species.

  2. The bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl-CoA carboxylase.

    Gerhardt, Edileusa C M; Rodrigues, Thiago E; Müller-Santos, Marcelo; Pedrosa, Fabio O; Souza, Emanuel M; Forchhammer, Karl; Huergo, Luciano F

    2015-03-01

    Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate-limiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the PII family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria. PMID:25557370

  3. Design of small molecule inhibitors of acetyl-CoA carboxylase 1 and 2 showing reduction of hepatic malonyl-CoA levels in vivo in obese Zucker rats.

    Bengtsson, Christoffer; Blaho, Stefan; Saitton, David Blomberg; Brickmann, Kay; Broddefalk, Johan; Davidsson, Ojvind; Drmota, Tomas; Folmer, Rutger; Hallberg, Kenth; Hallén, Stefan; Hovland, Ragnar; Isin, Emre; Johannesson, Petra; Kull, Bengt; Larsson, Lars-Olof; Löfgren, Lars; Nilsson, Kristina E; Noeske, Tobias; Oakes, Nick; Plowright, Alleyn T; Schnecke, Volker; Ståhlberg, Pernilla; Sörme, Pernilla; Wan, Hong; Wellner, Eric; Oster, Linda

    2011-05-15

    Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats. PMID:21515056

  4. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes

    Maeda, Shiro; Kobayashi, Masa-aki; Araki, Shin-ichi;

    2010-01-01

    It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A ca...

  5. Computational study of the three-dimensional structure of N-acetyltransferase 2-acetyl coenzyme a complex.

    Oda, Akifumi; Kobayashi, Kana; Takahashi, Ohgi

    2010-01-01

    N-Acetyltransferase 2 (NAT2) is one of the most important polymorphic drug-metabolizing enzymes and plays a significant role in individual differences of drug efficacies and/or side effects. Coenzyme A (CoA) is a cofactor in the experimentally determined crystal structure of NAT2, although the acetyl source of acetylation reactions catalyzed by NAT is not CoA, but rather acetyl CoA. In this study, the three-dimensional structure of NAT2, including acetyl CoA, was calculated using molecular dynamics simulation. By substituting acetyl CoA for CoA the amino acid residue Gly286, which is known to transform into a glutamate residue by NAT2*7A and NAT2*7B, comes close to the cofactor binding site. In addition, the binding pocket around the sulfur atom of acetyl CoA expanded in the NAT2-acetyl CoA complex. PMID:20930369

  6. A comparative study of drug resistance mechanism associated with active site and non-active site mutations: I388N and D425G mutants of acetyl-coenzyme-A carboxylase.

    Zhu, Xiao-Lei; Yang, Guang-Fu

    2012-03-01

    A major concern in the development of acetyl-CoA carboxylase-inhibiting (ACCase; EC 6.4.1.2) herbicides is the emergence of resistance as a result of the selection of distinct mutations within the CT domain. Mutations associated with resistance have been demonstrated to include both active sites and non-active sites, including Ile-1781-Leu, Trp- 2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala (numbered according to the Alopecurus myosuroides plastid ACCase). In the present study, extensive computational simulations, including molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) calculations, were carried out to compare the molecular mechanisms of active site mutation (I388N) and non-active site mutation (D425G) in Alopecurus myosuroides resistance to some commercial herbicides targeting ACCase, including haloxyfop (HF), diclofop (DF) and fenoxaprop (FR). All of the computational model and energetic results indicated that both I388N and D425G mutations have effects on the conformational change of the binding pocket. The π-π interaction between ligand and Phe377 and Tyr161' residues, which make an important contribution to the binding affinity, was decreased after mutation. As a result, the mutant-type ACCase has a lower affinity for the inhibitor than the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural and mechanistic insights obtained from the present study will deepen our understanding of the interactions between ACCase and herbicides, which provides a molecular basis for the future design of a promising inhibitor with low resistance risk. PMID:22242795

  7. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian;

    2015-01-01

    -CoA (or pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong...

  8. Rapid switch of hepatic fatty acid metabolism from oxidation to esterification during diurnal feeding of meal-fed rats correlates with changes in the properties of acetyl-CoA carboxylase, but not of carnitine palmitoyltransferase I.

    Moir, A M; Zammit, V A

    1993-01-01

    The effects of the ingestion of a meal on the partitioning of hepatic fatty acids between oxidation and esterification were studied in vivo for meal-fed rats. The time course for the reversal of the starved state was extremely rapid and the process was complete within 2 h, in marked contrast with the reversal of the effects of starvation in rats fed ad libitum [A. M. B. Moir and V. A. Zammit (1993) Biochem. J. 289, 49-55]. This rapid reversal occurred in spite of the fact that, in the liver of the meal-fed animals before feeding, a similar degree of partitioning of fatty acids in favour of oxidation was observed as in 24 h-starved rats (previously fed ad libitum). This suggested that the lower degree of ketonaemia observed in meal-fed rats before a meal is not due to the inability of acylcarnitine formation to compete successfully with esterification of fatty acids to the glycerol moiety. Investigation of the possible mechanisms that could contribute towards the rapid switching-off of fatty acid oxidation revealed that this was correlated with a very rapid rise and overshoot in hepatic malonyl-CoA concentration, but not with any change in the activity, or sensitivity to malonyl-CoA, of the mitochondrial overt carnitine palmitoyltransferase (CPT I). The role of these two parameters in the reversal of fasting-induced hepatic fatty acid oxidation was thus the inverse of that observed previously for refed 24 h-starved rats. The rapid increase in [malonyl-CoA] was accompanied by an immediate and complete reversion of the kinetic characteristics (Ka for citrate, expressed/total activity ratio) of acetyl-CoA carboxylase to those found in the post-meal animals, again in contrast with the time course observed in refed 24 h-starved rats [A. M. B. Moir and V. A. Zammit (1990) Biochem. J. 272, 511-517]. The rapidity with which these changes occurred was specific to the partitioning of acyl-CoA; the meal-induced diversion of glycerolipids towards phospholipid synthesis and the

  9. Intracellular Acetyl Unit Transport in Fungal Carbon Metabolism

    Strijbis, K.; Distel, B.

    2010-01-01

    Acetyl coenzyme A (acetyl-CoA) is a central metabolite in carbon and energy metabolism. Because of its amphiphilic nature and bulkiness, acetyl-CoA cannot readily traverse biological membranes. In fungi, two systems for acetyl unit transport have been identified: a shuttle dependent on the carrier carnitine and a (peroxisomal) citrate synthase-dependent pathway. In the carnitine-dependent pathway, carnitine acetyltransferases exchange the CoA group of acetyl-CoA for carnitine, thereby forming...

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

    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 β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β 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 β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β 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

  11. SIRT3 deacetylates mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 and regulates ketone body production

    Shimazu, Tadahiro; Hirschey, Matthew D; Hua, Lan; Dittenhafer-Reed, Kristin E; Schwer, Bjoern; Lombard, David B; Li, Yu; Bunkenborg, Jakob; Alt, Frederick W; Denu, John M; Jacobson, Matthew P; Verdin, Eric

    2010-01-01

    The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3(...

  12. 高脂饮食对老年大鼠骨骼肌脂肪酸含量及乙酰辅酶A羧化酶表达和活性的影响%Effects of high-fat diet on fatty acid metabolism, expression and activity of acetyl-coenzyme A carboxylase in skeletal muscle in aged rats

    胡淑国; 宋光耀; 王敬; 高宇

    2010-01-01

    目的 探讨增龄和高脂饮食对大鼠骨骼肌脂肪酸含量及乙酰辅酶A羧化酶(acetyl-coenzyme A carboxylase,ACC)表达和活性的影响.方法 将22~24月龄雄性Wistar大鼠随机分为老年对照组和高脂组;4~5月龄大鼠作为青年对照组.老年对照组和青年对照组给基础饲料,高脂组给予高脂饲料,喂养8周.用高胰岛素-正葡萄糖钳夹实验评价各组大鼠胰岛素敏感性,用全自动生化分析仪测定骨骼肌三酰甘油,用荧光分光光度计测定骨骼肌总的长链脂酰辅酶A含量,用Western-blot方法测定骨骼肌ACC、和磷酸化ACC(P-ACC)蛋白表达.结果 (1)老年对照组空腹血糖、胰岛索和游离脂肪酸高于青年对照组,高脂组上述几项指标进一步升高,并且出现血清三酰甘油和总胆固醇水平增高;(2)老年对照组葡萄糖输注率(glucose infusion rates,GIR)低于青年对照组,高脂组GIR低于老年对照组,高脂组GIR在8周末低于4周末;(3)老年对照组骨骼肌三酰甘油及长链脂酰辅酶A含量高于青年对照组,高脂组与老年对照组比较进一步升高;(4)老年对照组与青年对照组之间、高脂组与老年对照组之间骨骼肌ACC蛋白表达均无明显变化(P>0.05);骨骼肌P-ACC蛋白水平在老年对照组低于青年对照组,高脂组与老年对照组比较进一步降低(P0.05). The protein levels of P-ACC in skeletal muscle were lower in OC group, and much lower in HF group than in YC group (P<0.05 or P<0.01). Conclusions Compared with young rats, abnormal fatty acid metabolism and insulin resistance always exist in aged rats. High-fat feeding results in a significant increase in lipid content in skeletal muscle. Alterations of ACC activity may contribute to fat accumulation in skeletal muscle and insulin resistance.

  13. Crescimento e competitividade de biótipos de capim-colchão resistente e suscetível aos herbicidas inibidores da acetil coenzima A carboxilase Growth and competitiveness of biotypes of crabgrass resistant and susceptible to acetyl coenzyme A carboxylase inhibiting herbicides

    Ramiro Fernando López Ovejero

    2007-01-01

    Full Text Available O objetivo deste trabalho foi comparar o crescimento e a habilidade competitiva de dois biótipos de capim-colchão (Digitaria ciliaris, um resistente (R e outro suscetível (S aos herbicidas inibidores da acetil coenzima A carboxilase. O crescimento dos biótipos foi determinado pela coleta da matéria seca das plantas, aos 14, 21, 25, 28, 34, 42, 49, 57, 65, 72, 78, 86, 101, 111 e 118 dias após emergência (DAE. Os dados de massa de matéria seca foram ajustados ao modelo logístico e, também, utilizados para a obtenção da taxa de crescimento absoluto. Para avaliar a habilidade competitiva intra-específica e interespecífica, foram instalados cinco experimentos com o uso do método substitutivo. Compararam-se os biótipos R e S entre si e cada um destes com a cultura da soja, quando semeada no mesmo dia ou sete dias após a semeadura das plantas daninhas. As proporções de plantas entre as espécies ou biótipos utilizados foram: 5:0; 4:1; 3:2; 2:3; 1:4 e 0:5. Os biótipos de capim-colchão apresentaram acúmulo de matéria seca, crescimento absoluto e competição interespecífica semelhantes, e a redução da matéria seca da soja foi similar na presença dos biótipos R e S, o que sugere que ambos os biótipos de capim-colchão possuem a mesma adaptabilidade ecológica.The objective of this work was to compare the growth and the competitive ability of two crabgrass (Digitaria ciliaris biotypes, one resistant (R and other susceptible (S to the acetyl coenzyme A carboxylase inhibiting herbicides. Biotypes growth was determined by collecting the plant dry mass at 14, 21, 25, 28, 34, 42, 49, 57, 65, 72, 78, 86, 101, 111, and 118 days after emergence (DAE. Data of dry mass were adjusted to a logistic model, and were also used to calculate the absolute growth rate. Five experiments were installed to evaluate the intraspecific and interspecific competitive ability, using the substitutive method. The biotypes R and S were compared between

  14. Differences among Adult COAs and Adult Non-COAs on Levels of Self-Esteem, Depression, and Anxiety.

    Dodd, David T.; Roberts, Richard L.

    1994-01-01

    Examined self-esteem, depression, and anxiety among 60 adult children of alcoholics (COAs) and 143 adult non-COAs. Subjects completed Children of Alcoholics Screening Test, demographic questionnaire, Beck Depression Inventory, State-Trait Anxiety Inventory, and Coopersmith Self-Esteem Inventory. Found no significant differences between COAs and…

  15. Autotrophic growth: the methyl binding site of CO dehydrogenase in the synthesis of acetyl-CoA

    A pathway in which CO or CO2 and H2 is used as a source of energy and carbon to synthesize acetyl-CoA is used for autotrophic growth of acetogenes, methanogens and some sulfate-reducing bacteria. All enzymes involved in this pathway have been purified from C. thermoaceticum. Five of them: CO dehydrogenase (CODH), corrinoid protein, methyltransferase, CODH disulfide reductase (SSRd) and ferredoxin catalyzed synthesis of acetyl-CoA from methyltetrahydrofolate, CO and CoA. CODH is a central enzyme catalyzing the condensation of CH3, CO and CoA and per se it catalyzes a reversible exchange of CO with acetyl-CoA. Thus, CODH must have binding sites for CH3, CO and CoA. They have succeeded in methylating β subunits of CODH using 14CH3I or 14CH-corrinoid protein, a native donor of the CH3 group in synthesis of acetyl-CoA. With resulting [14CH3]CODH, only SSRd is required for synthesis of [14C]acetyl-CoA from CO and CoA. The kinetic studies show that CH3I is a competitive inhibitor for exchange reaction between CO and acetyl-CoA. Acetaldehyde and acetyl-CoA but not acetic acid and CoA protected CODH against methylation by CH3I. Methyl group bound to CODH is very slowly removed by CO and CoA and acetyl-CoA accelerated this process. These data confirm that CH3 group from CH3I and CH3-corrinoid protein is bound to the methyl binding site of CODH

  16. Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

    Sirobhushanam, Sirisha; Galva, Charitha; Sen, Suranjana; Wilkinson, Brian J; Gatto, Craig

    2016-09-01

    Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis. PMID:27320015

  17. Molecular evolution of urea amidolyase and urea carboxylase in fungi

    Harris Steven D; Nickerson Kenneth W; Strope Pooja K; Moriyama Etsuko N

    2011-01-01

    Abstract Background Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green al...

  18. ADP-bildende Acetyl-CoA Synthetasen aus hyperthermophilen Archaea: Molekularbiologische und biochemische Charakterisierung von neuartigen Enzymen der Acetat-Bildung und ATP-Synthese

    Musfeldt, Meike

    2001-01-01

    Keine deutschsprachige Zusammenfassung vorhanden. Acetyl-CoA synthetase (ADP-forming) (ADP-ACS) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + Pi -> acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ADP-ACS in Archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145 kDa heterotetramers (a2, b2). By using the N-terminal amino acid sequences of both subunits, which are located at different ...

  19. Effect of [L-Carnitine] on acetyl-L-carnitine production by heart mitochondria

    The authors recently reported a large efflux of acetyl-L-carnitine from rat heart mitochondria during state 3 respiration with pyruvate as substrate both in the presence and absence of malate. In this series of experiments, the effect of the concentration of L-carnitine on the efflux of acetyl-L-carnitine and on the production of 14CO2 from 2-14C-pyruvate was determined. Maximum acetylcarnitine production (approximately 25 n moles/min/mg protein) was obtained at 3-5 mM L-carnitine in the absence of added malate. 14CO2 production decreased as the concentration of L-carnitine increased; it plateaued at 3-5 mM L-carnitine. These data indicate carnitine can stimulate flux of pyruvate through pyruvate dehydrogenase and can reduce flux of acetyl CoA through the Krebs cycle by acting as an acceptor of the acetyl moieties of acetyl CoA generated by pyruvate dehydrogenase

  20. Effect of (L-Carnitine) on acetyl-L-carnitine production by heart mitochondria

    Bieber, L.L.; Lilly, K.; Lysiak, W.

    1986-05-01

    The authors recently reported a large efflux of acetyl-L-carnitine from rat heart mitochondria during state 3 respiration with pyruvate as substrate both in the presence and absence of malate. In this series of experiments, the effect of the concentration of L-carnitine on the efflux of acetyl-L-carnitine and on the production of /sup 14/CO/sub 2/ from 2-/sup 14/C-pyruvate was determined. Maximum acetylcarnitine production (approximately 25 n moles/min/mg protein) was obtained at 3-5 mM L-carnitine in the absence of added malate. /sup 14/CO/sub 2/ production decreased as the concentration of L-carnitine increased; it plateaued at 3-5 mM L-carnitine. These data indicate carnitine can stimulate flux of pyruvate through pyruvate dehydrogenase and can reduce flux of acetyl CoA through the Krebs cycle by acting as an acceptor of the acetyl moieties of acetyl CoA generated by pyruvate dehydrogenase.

  1. AMPK/Snf1 signaling regulates histone acetylation: Impact on gene expression and epigenetic functions.

    Salminen, Antero; Kauppinen, Anu; Kaarniranta, Kai

    2016-08-01

    AMP-activated protein kinase (AMPK) and its yeast homolog, Snf1, are critical regulators in the maintenance of energy metabolic balance not only stimulating energy production but also inhibiting energy-consuming processes. The AMPK/Snf1 signaling controls energy metabolism by specific phosphorylation of many metabolic enzymes and transcription factors, enhancing or suppressing their functions. The AMPK/Snf1 complexes can be translocated from cytoplasm into nuclei where they are involved in the regulation of transcription. Recent studies have indicated that AMPK/Snf1 activation can control histone acetylation through different mechanisms affecting not only gene transcription but also many other epigenetic functions. For instance, AMPK/Snf1 enzymes can phosphorylate the histone H3S10 (yeast) and H2BS36 (mammalian) sites which activate specific histone acetyltransferases (HAT), consequently enhancing histone acetylation. Moreover, nuclear AMPK can phosphorylate type 2A histone deacetylases (HDAC), e.g. HDAC4 and HDAC5, triggering their export from nuclei thus promoting histone acetylation reactions. AMPK activation can also increase the level of acetyl CoA, e.g. by inhibiting fatty acid and cholesterol syntheses. Acetyl CoA is a substrate for HATs, thus increasing their capacity for histone acetylation. On the other hand, AMPK can stimulate the activity of nicotinamide phosphoribosyltransferase (NAMPT) which increases the level of NAD(+). NAD(+) is a substrate for nuclear sirtuins, especially for SIRT1 and SIRT6, which deacetylate histones and transcription factors, e.g. those regulating ribosome synthesis and circadian clocks. Histone acetylation is an important epigenetic modification which subsequently can affect chromatin remodeling, e.g. via bromodomain proteins. We will review the signaling mechanisms of AMPK/Snf1 in the control of histone acetylation and subsequently clarify their role in the epigenetic regulation of ribosome synthesis and circadian clocks

  2. COA based robust output feedback UPFC controller design

    In this paper, a novel method for the design of output feedback controller for unified power flow controller (UPFC) using chaotic optimization algorithm (COA) is developed. Chaotic optimization algorithms, which have the features of easy implementation, short execution time and robust mechanisms of escaping from the local optimum, is a promising tool for the engineering applications. The selection of the output feedback gains for the UPFC controllers is converted to an optimization problem with the time domain-based objective function which is solved by a COA based on Lozi map. Since chaotic mapping enjoys certainty, ergodicity and the stochastic property, the proposed chaotic optimization problem introduces chaos mapping using Lozi map chaotic sequences which increases its convergence rate and resulting precision. To ensure the robustness of the proposed stabilizers, the design process takes into account a wide range of operating conditions and system configurations. The effectiveness of the proposed controller for damping low frequency oscillations is tested and demonstrated through non-linear time-domain simulation and some performance indices studies. The results analysis reveals that the designed COA based output feedback UPFC damping controller has an excellent capability in damping power system low frequency oscillations and enhance greatly the dynamic stability of the power systems.

  3. Molecular evolution of urea amidolyase and urea carboxylase in fungi

    Harris Steven D

    2011-03-01

    Full Text Available Abstract Background Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms. Results Among the 64 fungal species we examined, only those in two Ascomycota classes (Sordariomycetes and Saccharomycetes had the urea amidolyase sequences. Urea carboxylase was found in many but not all of the species in the phylum Basidiomycota and in the subphylum Pezizomycotina (phylum Ascomycota. It was completely absent from the class Saccharomycetes (phylum Ascomycota; subphylum Saccharomycotina. Four Sordariomycetes species we examined had both the urea carboxylase and the urea amidolyase sequences. Phylogenetic analysis showed that these two enzymes appeared to have gone through independent evolution since their bacterial origin. The amidase domain and the urea carboxylase domain sequences from fungal urea amidolyases clustered strongly together with the amidase and urea carboxylase sequences, respectively, from a small number of beta- and gammaproteobacteria. On the other hand, fungal urea carboxylase proteins clustered together with another copy of urea carboxylases distributed broadly among bacteria. The urease proteins were found in all the fungal species examined except for those of the subphylum Saccharomycotina. Conclusions We conclude that the urea amidolyase genes currently found only in fungi are the results of a horizontal

  4. Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

    Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

    2011-01-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of bioti...

  5. Autotrophic growth: methylated carbon monoxide dehydrogenase as an intermediate of acetyl-CoA synthesis

    A new pathway of autotrophic growth has been discovered in certain anaerobic bacteria in which acetyl-CoA is the product formed from CO2 for initiation of anabolism rather than 3-phosphoglycerate as in the Calvin Cycle. CO2 is reduced in combination with tetrahydrofolate to methyltetrahydrofolate (CH3THF) and is the source of the CH3 group. CO2 or CO is the source of the carbonyl group. CO dehydrogenase (CODH), corrinoid enzyme, methyltransferase, ferredoxin and CODH disulfide reductase have been isolated from Clostridium thermoaceticum and shown to catalyze the synthesis of acetyl-CoA from CH3THF, CO and CoA. The methyltransferase catalyzes transfer of the CH3 group from CH3THF to the corrinoid enzyme from which the methyl is transferred to CODH. CO is bound to the Ni of CODH forming a Ni-Fe-C center. When CO2 is the source of carbon, H2 and hydrogenase are required for reduction of the CO2 by CODH. CODH disulfide reductase is required for the addition of CoA to the CODH (Pezacka, E. and Wood, H.G. J. Biol. Chem., in press). Then, CODH catalyzes the combination of the three groups forming acetyl-CoA. The authors have now succeeded in methylating CODH using 14CH3I or 14CH3-B12. With the resulting 14CH3-CODH, only CODH disulfide reductase is required for synthesis of [14C]acetyl-CoA from CO and CoA. The amino acid sequence at the CH3-site is being investigated

  6. Crystal Structure of the N-Acetyltransferase Domain of Human N-Acetyl-L-Glutamate Synthase in Complex with N-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNA...

  7. Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila

    The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria

  8. Regulation of phosphoenolpyruvate carboxylase in PVYNTN-infected tobacco plants

    Müller, Karel; Doubnerová, V.; Synková, Helena; Čeřovská, Noemi; Ryšlavá, H.

    2009-01-01

    Roč. 390, č. 3 (2009), s. 245-251. ISSN 1431-6730 R&D Projects: GA ČR GA206/03/0310; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50380511 Keywords : biotic stress * Nicotiana tabacum * phosphoenolpyruvate carboxylase (PEPC) Subject RIV: ED - Physiology Impact factor: 2.732, year: 2009

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

    Hynes, Michael J; Murray, Sandra L

    2010-07-01

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

  10. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author)

  11. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    Ferreira, R.M.B. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia]|[Universidade Nova de Lisboa, Oeiras (Portugal). Instituto de Tecnologia Quimica e Biologica; Franco, E.; Teixeira, A.R.N. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of {sup 35}S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author).

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

    U.S. Environmental Protection Agency — The COA_Mosaic33 layer designates areas with potential for forest/grassland mosaic conservation. These are areas of natural or semi-natural forest/grassland mosaic...

  13. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

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

    1996-04-01

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

  14. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chuna Ram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  15. Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate

    Sirithanakorn, Chaiyos; Adina-Zada, Abdussalam; Wallace, John C.; Jitrapakdee, Sarawut; Attwood, Paul V.

    2014-01-01

    l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to ...

  16. Cryogenic Optical Assembly (COA) cooldown analysis for the Cosmic Background Explorer (COBE)

    Coladonato, Robert J.; Irish, Sandra M.; Mosier, Carol L.

    1990-01-01

    The Cosmic Background Explorer (COBE) spacecraft, developed by Goddard Space Flight Center (GSFC), was successfully launched on November 18, 1989 aboard a Delta expendable launch vehicle. Two of the three instruments for this mission were mounted inside a liquid helium (LHe) dewar which operates at a temperature of 2 K. These two instruments are the Diffuse Infrared Background Experiment (DIRBE) and the Far Infrared Absolute Spectrophotometer (FIRAS). They are mounted to a common Instrument Interface Structure (IIS) and the entire assembly is called the Cryogenic Optical Assembly (COA). As part of the structural verification requirement, it was necessary to show that the entire COA exhibited adequate strength and would be capable of withstanding the launch environment. This requirement presented an unique challenge for COBE because the COA is built and assembled at room temperature (300 K), cooled to 2 K, and then subjected to launch loads. However, strength testing of the entire COA at 2 K could not be done because of facility limitations. Therefore, it was decided to perform the strength verification of the COA by analysis.

  17. Acetyltransferase and human hemoglobin acetylation

    A minor component of human fetal hemoglobin (Hb F) is acetylated at the amino-terminus of the γ-globin chains. A similar minor component of Hb F is formed during translation of cord blood mRNA in the rabbit reticulocyte lysate system. The acetylation appeared to be enzymatic. This system contains an acetyltransferase capable of acetylating histones and hemoglobins. The enzyme, partially purified by histone-Sepharose affinity chromatography was capable of incorporating labeled acetyl- group from 1-[14C-acetyl]-CoA into both human Hb F0 and HB A0, but at a lower rate than for histones. Characterization of the labeled products indicated that the α-chains of both hemoglobins were being acetylated presumably at a lysyl-residue, but in the case of Hb F0 the amino-terminus of the γ-globin chains was acetylated as well. While histone-Sepharose bound more than 95% of the enzyme, Sepharose linked Hb F0, γ-globin chains, and Hb Bart's bound 14, 5, and 12% of the activity, respectively. Enzyme bound to these resins was not any more active on the hemoglobins than was the enzyme bound to the histone-Sepharose. The histone-Sepharose was also used to detect the enzyme in human cord blood red cells separated by dextran 40 density gradient centrifugation. Activity was found mostly in the young cells, and was directly related to the number of reticulocytes present in any one fraction

  18. Effect of elevated total CoA levels on metabolic pathways in cultured hepatocytes

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt2 cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 μM) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 μm/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of [14C]-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis

  19. Regulation of intermediary metabolism by protein acetylation

    Guan, Kun-Liang; Xiong, Yue

    2010-01-01

    Extensive studies during the past four decades have identified important roles for lysine acetylation in the regulation of nuclear transcription. Recent proteomic analyses on protein acetylation uncovered a large number of acetylated proteins in the cytoplasm and mitochondria, including most enzymes involved in intermediate metabolism. Acetylation regulates metabolic enzymes by multiple mechanisms, including via enzymatic activation or inhibition, and by influencing protein stability. Convers...

  20. Studies on biotin dependent carboxylases and the properties of carboxybiotin

    Biotin dependent carboxyl-transfer reactions have been studied using biotin and pyruvate carboxylases. The pH profile for the Mg2+ and MgATP dependent carboxylation of biotin by bicarbonate shows that an enzymic base with a pK of 6.5 must be unprotonated for catalysis to occur. The pH profiles for the carboxyl-transfer reaction of pyruvate carboxylase have been obtained by studying the decarboxylation of oxalacetate stimulated by the presence of oxamate. Similarly, 13C and 2H isotope effects have been measured for the decarboxylation of oxalacetate by both enzymic and nonenzymic means. From these studies the authors can conclude that carboxyl-transfer between biotin and oxalacetate is at least partially rate-limiting and is not concerted with proton-transfer. The lack of any apparent enzymic acid-base catalyst (the V/K profile for oxalacetate is pH independent) suggests that proton transfer may occur directly between biotin and the carbanion formed when oxalacetate is decarboxylated. The pH profile for the nonenzymatic decarboxylation of carboxybiotin shows a plateau below pH 4 (k = 0.012 min-1 at 20C), and a lower plateau above pH 8 (k = 0.005 min-1 at 250C). A proton inventory at low pH is linear, while at high pH it is curved. These data suggest that two different mechanisms operate at high and low pH

  1. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    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. PMID:27372278

  2. The COA360: a tool for assessing the cultural competency of healthcare organizations.

    LaVeist, Thomas A; Relosa, Rachel; Sawaya, Nadia

    2008-01-01

    The U.S. Census Bureau projects that by 2050, non-Hispanic whites will be in the numerical minority. This rapid diversification requires healthcare organizations to pay closer attention to cross-cultural issues if they are to meet the healthcare needs of the nation and continue to maintain a high standard of care. Although scorecards and benchmarking are widely used to gauge healthcare organizations' performance in various areas, these tools have been underused in relation to cultural preparedness or initiatives. The likely reason for this is the lack of a validated tool specifically designed to examine cultural competency. Existing validated cultural competency instruments evaluate individuals, not organizations. In this article, we discuss a study to validate the Cultural Competency Organizational Assessment--360 or the COA360, an instrument designed to appraise a healthcare organization's cultural competence. The Office of Minority Health and the Joint Commission have each developed standards for measuring the cultural competency of organizations. The COA360 is designed to assess adherence to both of these sets of standards. For this validation study, we enlisted a panel of national experts. The panel rated each dimension of the COA360, and the combination of items for each of the scale's 14 dimensions was rated above 4.13 (on 5-point scale). Our conclusion points to the validity of the COA360. As such, it is a valuable tool not only for assessing a healthcare organization's cultural readiness but also for benchmarking its progress in addressing cultural and diversity issues. PMID:18720687

  3. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea; Ruiz-Pérez, Luis M; González-Pacanowska, Dolores

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  4. Liver fatty acid binding protein (LFABP) transfers fatty acids and fatty acyl coas to membranes

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C.; Córsico, Betina

    2010-01-01

    The objective of this work was to analyze LFABP´s capacity to transfer acyl CoAs to artificial membranes and compare it to LCFA transfer employing natural ligands, in order to better understand the specific physiological role of LFABP in the cell.

  5. CoaSim: A Flexible Environment for Simulating Genetic Data under Coalescent Models

    Mailund; Schierup, Mikkel Heide; Pedersen, Christian Nørgaard Storm;

    2005-01-01

    get insight into these. Results We have created the CoaSim application as a flexible environment for Monte various types of genetic data under equilibrium and non-equilibrium coalescent variety of applications. Interaction with the tool is through the Guile version scripting language. Scheme scripts...

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

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

    2016-01-01

    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 ce...

  7. Fatal Intoxication with Acetyl Fentanyl.

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. PMID:26389815

  8. Biotin Responsive Multiple Carboxylase Deficiency Presenting as Diabetic Ketoacidosis.

    Jia-Woei Hou

    2004-02-01

    Full Text Available Multiple carboxylase deficiency (MCD is a rare inherited metabolic disease of biotindependency due to deficiency of holocarboxylase synthetase (HCS or biotinidase deficiency.A 30-month-old female patient who presented with the initial features of diabeticketoacidosis (severe metabolic acidosis, ketosis, and hyperglycemia, lactic acidemia, moderatehyperammonemia, and generalized organic aciduria is described. Associated symptomsand signs included erythematous skin rashes, alopecia and developmental delay. The patientresponded dramatically to treatment with biotin (10 mg/day showing normalization of clinicalsymptoms and most biochemical abnormalities. Based on the urine organic profile bygas chromatography/ mass spectrometry (GC/MS, the diagnosis of MCD was made. A plasmatandem mass study confirmed this diagnosis. The biotinase activity in serum was normal,indicating that this was a rare case of late-onset HCS deficiency.

  9. Intracellular signal transduction of PBAN action in the silkworm, Bombyx mori: involvement of acyl CoA reductase.

    Ozawa, R; Matsumoto, S

    1996-03-01

    In the silkworm, Bombyx mori, production of the sex pheromone bombykol is regulated by a neurohormone termed PBAN. We have detected the activity of acyl CoA reductase in the pheromone gland of B. mori by using palmitoyl CoA as a substrate. The acyl CoA reductase requires NADPH, but not NADH, as a proton dono. When the pheromone gland was incubated with the PBAN fragment peptide TKYFSPRLamide, palmitoyl CoA was incorporated and converted into the corresponding C16 alcohols. Radio HPLC analysis revealed that these C16 alcohols were hexadecan-1-ol (81.2%), (Z)-11-hexadecen-1-ol (12.3%), and (E, Z)-10, 12-hexadecadien-1-ol (= bombykol, 6.5%). The production of C16 alcohols in the pheromone gland was inhibited by the known bombykol biosynthesis inhibitors EDTA, LaCl3, W-7, trifluoperazine, p-nitrophenyl phosphate, NaF and compactin. By contrast, when the pheromone gland homogenate was incubated in the presence of palmitoyl CoA and NADPH, production of C16 alcohols was affected by compactin, W-7 and trifluoperazine, but not by EDTA, LaCl3, p-nitrophenyl phosphate and NaF. These results indicate that compactin, W-7 and trifluoperazine directly suppress the step catalyzed by acyl CoA reductase, whereas EDTA, LaCl3, pNPP, and NaF inhibit bombykol production by affecting other biochemical steps in the signal transduction of PBAN action. The present results also imply that PBAN regulates the step catalyzed by acyl CoA reductase and that palmitoyl CoA could be used as a substrate of the acyl CoA reductase that regulates bombykol biosynthesis. PMID:8900596

  10. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose

    Huan Huang; McIntosh, Avery L.; Martin, Gregory G.; Petrescu, Anca D.; Landrock, Kerstin K.; Danilo Landrock; Kier, Ann B.; Friedhelm Schroeder

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor-α (PPARα) in the nucleus, was found to bind TOFA and its activated CoA th...

  11. Protein Acetylation in Archaea, Bacteria, and Eukaryotes

    Jörg Soppa

    2010-01-01

    Full Text Available Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which—Alba—was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  12. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A;

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  13. Decreased renal vitamin K-dependent γ-glutamyl carboxylase activity in calcium oxalate calculi patients

    陈俊汇; 刘继红; 章咏裳; 叶章群; 王少刚

    2003-01-01

    Objective To study the activity of vitamin K-dependent γ-glutamyl carboxylase in patients with calcium oxalate (CaOx) urolithiasis compared with healthy individuals and to assess its relationship to the renal calcium oxalate urolithiasis. Methods Renal parenchymas were harvested from urolithic patients and renal tumor patients undergoing nephrectomy. The renal carboxylase activity was evaluated as the radioactivity of [14C] labeled sodium bicarbonate in carboxylic reactions in vitro using β-liquid scintillation counting. Results Significantly reduced activity of renal vitamin K-dependent γ-glutamyl carboxylase was observed in the urolithic group as compared with normal controls (P<0.01). Conclusion It suggests that the reduced carboxylase activity observed in the urolithic patients may play an important role in the course of renal calcium oxalate urolithiasis.

  14. Crystallization and structure of a recombinant ribulose-1,5-bisphosphate carboxylase

    Schneider, Gunter; Lindqvist, Ylva; Brändén, Carl-Ivar; Lorimer, George

    1988-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme in photosynthetic carbon dioxide fixation and photorespiration. The dimeric carboxylase from the photosynthetic bacterium Rhodospirillum rubrum has been cloned and expressed in E. coli. The recombinant enzyme has been crystallized in a number of different crystal forms. The three-dimensional structure of the enzyme has been determined by X-ray crystallographic methods to 2.9Åresolution.

  15. Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

    Koffas, Mattheos A. G.; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory

    2002-01-01

    Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overe...

  16. Sequence Classification: 182868 [

    Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|17987345|ref|NP_539979.1| BIOTIN CARBOX...YL CARRIER PROTEIN OF ACETYL-COA CARBOXYLASE || http://www.ncbi.nlm.nih.gov/protein/17987345 ...

  17. Sequence Classification: 565760 [

    Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|15965073|ref|NP_385426.1| PROBABLE BIOTIN CARB...OXYL CARRIER PROTEIN OF ACETYL-COA CARBOXYLASE (BCCP) || http://www.ncbi.nlm.nih.gov/protein/15965073 ...

  18. Sequence Classification: 512243 [

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17547505|ref|NP_520907.1| PROBABLE BIOTIN CARB...OXYL CARRIER PROTEIN OF ACETYL-COA CARBOXYLASE (BCCP) || http://www.ncbi.nlm.nih.gov/protein/17547505 ...

  19. Sequence Classification: 697789 [

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|34557455|ref|NP_907270.1| PUTATIVE BIOTIN CARB...OXYL CARRIER PROTEIN OF ACETYL-COA CARBOXYLASE || http://www.ncbi.nlm.nih.gov/protein/34557455 ...

  20. Unlocking the Barley Genome by Chromosomal and Comparative Genomics

    Mayer, K. F. X.; Martis, M.; Hedley, P. E.; Šimková, Hana; Liu, H.; Morris, J. A.; Steuernagel, B.; Taudien, S.; Kubaláková, Marie; Suchánková, Pavla; Doležel, Jaroslav; Stein, N.

    Roč. 23, č. 4 ( 2011 ), s. 1249-1263. ISSN 1040-4651 Institutional research plan: CEZ:AV0Z50380511 Keywords : PSEUDO-RESPONSE-REGULATOR * ACETYL-COA CARBOXYLASE * TRITICUM-AESTIVUM L. Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.987, year: 2011

  1. Unlocking the Barley Genome by Chromosomal and Comparative Genomics

    Mayer, K. F. X.; Martis, M.; Hedley, P. E.; Šimková, Hana; Liu, H.; Morris, J. A.; Steuernagel, B.; Taudien, S.; Kubaláková, Marie; Suchánková, Pavla; Doležel, Jaroslav; Stein, N.

    2011-01-01

    Roč. 23, č. 4 (2011), s. 1249-1263. ISSN 1040-4651 Institutional research plan: CEZ:AV0Z50380511 Keywords : PSEUDO-RESPONSE-REGULATOR * ACETYL-COA CARBOXYLASE * TRITICUM-AESTIVUM L. Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.987, year: 2011

  2. Swelling of acetylated wood in organic liquids

    Obataya, E; Obataya, Eiichi; Gril, Joseph

    2005-01-01

    To investigate the affinity of acetylated wood for organic liquids, Yezo spruce wood specimens were acetylated with acetic anhydride, and their swelling in various liquids were compared to those of untreated specimens. The acetylated wood was rapidly and remarkably swollen in aprotic organic liquids such as benzene and toluene in which the untreated wood was swollen only slightly and/or very slowly. On the other hand, the swelling of wood in water, ethylene glycol and alcohols remained unchanged or decreased by the acetylation. Consequently the maximum volume of wood swollen in organic liquids was always larger than that in water. The effect of acetylation on the maximum swollen volume of wood was greater in liquids having smaller solubility parameters. The easier penetration of aprotic organic liquids into the acetylated wood was considered to be due to the scission of hydrogen bonds among the amorphous wood constituents by the substitution of hydroxyl groups with hydrophobic acetyl groups.

  3. Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis

    F. Habarou

    2015-03-01

    Full Text Available Pyruvate carboxylase (PC is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A. Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1 and type B (patient 2. Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

  4. Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco.

    Toyota, Kentaro; Koizumi, Nozomu; Sato, Fumihiko

    2003-03-01

    Phosphoenolpyruvate carboxylase (PEPC), which catalyses the carboxylation of phosphoenolpyruvate using HCO(3)(-) to generate oxaloacetic acid, is an important enzyme in the primary metabolism of plants. Although the PEPC genes (ppc) comprise only a small gene family, the function of each gene is not clear, except for roles in C(4) photosynthesis and CAM. Three PEPC genes (Nsppc1-3) from the C(3) plant Nicotiana sylvestris were used to investigate their roles and regulation in a C(3) plant, and their regulation by phosphorus depletion in particular. First, the induction of PEPC by phosphorus depletion was confirmed. Next, Nsppc1 was determined to be mainly responsive to phosphorus deficiency at the transcriptional level. Further studies using transgenic tobacco harbouring a chimeric gene consisting of the 2.0 kb promoter region of Nsppc1 and the beta-glucuronidase (GUS) reporter showed that PEPC is transcriptionally induced. It was also found that sucrose had a synergistic effect on the induction of PEPC by phosphorus deficiency. A series of transgenic tobacco containing 5'-deletion mutants of Nsppc1 promoter::GUS fusion revealed that the -539 to -442 bp Nsppc1 promoter region, relative to the translation start site, was necessary for the response to phosphorus deficiency. Gain-of-function analysis using a construct containing three tandem repeats of the -539 to -442 bp region confirmed that this region was sufficient to induce the phosphorus-deficiency response in tobacco. PMID:12598567

  5. Histone Acetylation in Drug Addiction

    Renthal, William; Nestler, Eric J.

    2009-01-01

    Regulation of chromatin structure through post-translational modifications of histones (e.g. acetylation) has emerged as an important mechanism to translate a variety of environmental stimuli, including drugs of abuse, into specific changes in gene expression. Since alterations in gene expression are thought to contribute to the development and maintenance of the addicted state, recent efforts are aimed at identifying how drugs of abuse alter chromatin structure and the enzymes which regulate...

  6. The Crystal Structure of N-Acetyl-l-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation*†

    Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, and Mendel

    2008-01-01

    The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylgluta-mate have been determined at 2.5- and 2.6-Å resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-Å linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with ...

  7. Characterization of three protein components required for functional reconstitution of the epoxide carboxylase multienzyme complex from Xanthobacter strain Py2.

    Allen, J. R.; Ensign, S A

    1997-01-01

    Epoxide carboxylase from Xanthobacter strain Py2 catalyzes the reductant- and NAD+-dependent carboxylation of aliphatic epoxides to beta-keto acids. Epoxide carboxylase from Xanthobacter strain Py2 has been resolved from cell extracts by anion-exchange chromatography into three protein components, designated I, II, and III, that are obligately required for functional reconstitution of epoxide carboxylase activity. Component II has been purified to homogeneity on the basis of its ability to co...

  8. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions.

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas; Zechner, Rudolf; Choudhary, Chunaram

    2015-11-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3-targeted sites to chemical acetylation in vitro and fasting-induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low-level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues. PMID:26358839

  9. A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids.

    Lindenkamp, Nicole; Schürmann, Marc; Steinbüchel, Alexander

    2013-09-01

    In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoA-transferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64 kDa, respectively, and point to a quaternary structure of the native enzyme (α4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA-C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising - amongst others - the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16∆pct was generated. The growth and accumulation behaviour of this mutant on gluconate, gluconate plus 3,3'-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating

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

    Dominik M. Peter

    2016-04-01

    Full Text Available 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.

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

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

    2016-01-01

    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. PMID:27104508

  12. A Method to determine lysine acetylation stoichiometries

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  13. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas;

    2015-01-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or...... suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of...... fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation...

  14. Acetylation of woody lignocellulose: significance and regulation

    Prashant Mohan-Anupama Pawar

    2013-05-01

    Full Text Available Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1 summarize literature on lignocellulose acetylation in different tree species, 2 present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3 describe plant proteins involved in lignocellulose O-acetylation, 4 give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5 discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

  15. Acetylation-Mediated Suppression of Transcription-Independent Memory: Bidirectional Modulation of Memory by Acetylation

    Katja Merschbaecher; Jakob Haettig; Uli Mueller

    2012-01-01

    Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs), and the antagonistic histone deacetylases (HDACs) play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM). While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact...

  16. UNIQUE ACETYLATION OF OLIGOSACCHARIDES BY TRICHODERMA REESEI ACETYL ESTERASE IN WATER - VINYL ACETATE MIXTURE

    Purified T. reesei RUT C-30 acetyl esterase catalyzes acetyl transfer to a variety of carbohydrates in water in the presence of vinyl acetate as the acetyl group donor. The degree of conversion and the number of formed acetates depended on the acceptor used. With some acceptors, such as methyl or ...

  17. A Clinical Study to Validate the Pupil Rescaling Technique by using COAS Shack Hartmann Aberrometer.

    Kalikivayi, V; Kannan, K; Ganesan, A R

    2015-01-01

    In any optical system, optical aberrations of the imaging system affect the image quality. The human eye is also like an optical system which has optical aberrations influencing the quality of the retinal image. When pupil size exceeds 3 mm, ocular aberrations increase and play a major role on retinal image degradation. Pupil diameter is made constant in commercially available aberrometers by mathematically rescaling it. The aim of this study is to validate the pupil rescaling technique by using COAS (Complete Ophthalmic Analysis System)Shack Hartmann Aberrometer. Five subjects were recruited for this study. The measurements were taken over a moderately large pupil of 5mm in normal room illumination to allow for natural pupil dilation. The analyses diameter is fixed at 5 mm in COAS which means it rescales the aberration data to 5 mm if the pupil diameter recorded was more than 5 mm at the time of measurement. Ocular aberrations for natural and rescaled pupil sizes were analyzed. Estimation of ocular aberrations showed there was no statistical significance between natural pupil and rescaled pupil diameter. PMID:25996727

  18. Flexible DAQ card for detector systems utilizing the CoaXPress communication standard

    This work concerns the design and construction of a flexible FPGA based data acquisition system aimed for particle detectors. The interface card as presented was designed for large area detectors with millions of individual readout channels. Flexibility was achieved by partitioning the design into multiple PCBs, creating a set of modular blocks, allowing the creation of a wide variety of configurations by simply stacking functional PCBs together. This way the user can easily toggle the polarity of the high voltage bias supply or switch the downstream interface from CoaXPress to PCIe or stream directly HDMI. We addressed the issues of data throughput, data buffering, bias voltage generation, trigger timing and fine tuning of the whole readout chain enabling a smooth data transmission. On the current prototype, we have wire-bonded a MediPix2 MXR quad and connected it to a XILINX FPGA. For the downstream interface, we implemented the CoaXPress communication protocol, which enables us to stream data at 3.125 Gbps to a standard PC

  19. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM. PMID:27125317

  20. Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

    The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate

  1. Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog.

    Zhao, Gengxiang; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2013-01-25

    N-Acetyl-L-glutamate synthase catalyzes the conversion of AcCoA and glutamate to CoA and N-acetyl-L-glutamate (NAG), the first step of the arginine biosynthetic pathway in lower organisms. In mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase I in the urea cycle. We have previously reported the structures of NAGS from Neisseria gonorrhoeae (ngNAGS) with various substrates bound. Here we reported the preparation of the bisubstrate analog, CoA-S-acetyl-L-glutamate, the crystal structure of ngNAGS with CoA-NAG bound, and kinetic studies of several active site mutants. The results are consistent with a one-step nucleophilic addition-elimination mechanism with Glu353 as the catalytic base and Ser392 as the catalytic acid. The structure of the ngNAGS-bisubstrate complex together with the previous ngNAGS structures delineates the catalytic reaction path for ngNAGS. PMID:23261468

  2. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  3. Investigation of acetyl migrations in furanosides

    Migaud ME

    2006-07-01

    Full Text Available Abstract Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

  4. Histone Acetylation in Fungal Pathogens of Plants

    Junhyun Jeon

    2014-03-01

    Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

  5. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    Katja Merschbaecher

    Full Text Available Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs, and the antagonistic histone deacetylases (HDACs play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM. While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement.

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

    The X-ray structure of phosphopantetheine adenylyltransferase (PPAT) from M. tuberculosis in complex with its feedback regulator coenzyme A (CoA) was determined to 2.11 Å resolution. Unlike previous X-ray structures of PPAT–CoA complexes from other bacteria, which showed two distinct conformations of bound CoA, only one conformation of bound CoA is observed in the M. tuberculosis PPAT–CoA complex. 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 Å 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

  7. Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Leaves

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multi-functional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a...

  8. Characterization of phosphoenolpyruvate carboxylase from mature maize seeds: Properties of phosphorylated and dephosphorylated forms

    Černý, M.; Doubnerová, V.; Müller, Karel; Ryšlavá, H.

    2010-01-01

    Roč. 92, č. 10 (2010), s. 1362-1370. ISSN 0300-9084 R&D Projects: GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50380511 Keywords : Phosphoenolpyruvate carboxylase * Phosphorylation * Seed Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.787, year: 2010

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

    Wubben, T.; Mesecar, A.D. (Purdue); (UIC)

    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.

  10. COA2帮软件轻松换新家

    Sportsman

    2003-01-01

    Windows体积越来越大,而且在使用中还会不断膨胀自己,大部分软件也会默认安装在 文件夹中,时间一长,原来宽敞的C盘变得拥剂不堪,那么该如给C盘减肥呢?除到除临时文件等方法外,为什么不把原来装在C盘上的软件战略转移到其他分区?而COA2可以让你无需卸载和重装软件,直接将软件目录剪切到其他分区,从而完成快速搬家工程。

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

    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 [3H]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 μM 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

  12. Inhibition of neutral lipase from castor bean lipid bodies by coenzyme A (CoA) and Oleoyl-CoA

    The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [14C]trioleoylglycerol, releasing [14C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [14C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [14C]oleic acid produced from the lipase reaction to [14C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important in vivo in coordinating lipase activity with fatty acid oxidation

  13. Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulase-bisphosphate carboxylase activity

    The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P2) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P2 carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P2 carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P2 carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P2 carboxylase. The compound was analyzed by GC/MS, 13C NMR, and 1H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2-C-phosphohydroxymethyl)-D-ribonic acid]. The structure of the isolated compound differs from the Rbu-1,5-P2 carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate compared with the bisphosphate. The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P2 carboxylase activity in vivo

  14. Efficient acetylation of primary amines and amino acids in environmentally benign brine solution using acetyl chloride

    Kaushik Basu; Suchandra Chakraborty; Achintya Kumar Sarkar; Chandan Saha

    2013-05-01

    Acetyl chloride is one of the most commonly available and cheap acylating agent but its high reactivity and concomitant instability in water precludes its use to carry out acetylation in aqueous medium. The present methodology illustrates the efficient acetylation of primary amines and amino acids in brine solution by means of acetyl chloride under weakly basic condition in the presence of sodium acetate and/or triethyl amine followed by trituration with aqueous saturated bicarbonate solution. This effort represents the first efficient use of this most reactive but cheap acetylating agent to acetylate amines in excellent yields in aqueous medium. This is a potentially useful green chemical transformation where reaction takes place in environment-friendly brine solution leading to easy work-up and isolation of the amide derivatives. Mechanistic rationale of this methodology is also important.

  15. Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated.

    Lassila, Toni; Hokkanen, Juho; Aatsinki, Sanna-Mari; Mattila, Sampo; Turpeinen, Miia; Tolonen, Ari

    2015-12-21

    Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-β-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI). PMID:26558897

  16. Levels of histone acetylation in thyroid tumors.

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  17. p53 Acetylation: Regulation and Consequences

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer

  18. p53 Acetylation: Regulation and Consequences

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  19. p53 Acetylation: Regulation and Consequences

    Sara M. Reed

    2014-12-01

    Full Text Available Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  20. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2

    Schwer, Bjoern; Bunkenborg, Jakob; Verdin, Regis O; Andersen, Jens S; Verdin, Eric

    2006-01-01

    We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 b...

  1. Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.

    Suttie, J W; Hoskins, J A; Engelke, J; Hopfgartner, A; Ehrlich, H; Bang, N U; Belagaje, R M; Schoner, B; Long, G L

    1987-01-01

    The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme. PMID:3543932

  2. Reaction of phosphoenolpyruvate carboxylase with (Z)-3-bromophosphoenolpyruvate and (Z)-3-fluorophosphoenolpyruvate

    (Z)-3-Bromophosphoenolpyruvate inactivates phosphoenolpyruvate carboxylase from maize in the presence of HCO3- and either Mg2+ or Mn2+. The inactivation rate follows saturation kinetics. Inactivation is slower in the presence of phospholactate or epoxymaleate, both of which are inhibitors of the enzyme, or dithiothreitol. Inactivation is completely prevented by the presence of lactate dehydrogenase and NADH, and 3-bromolactate is formed during this treatment. If the reaction is conducted by using HC18O3-, the inorganic phosphate produced contains 18O. This and other evidence indicate that phosphoenolpyruvate carboxylase catalyzes conversion of bromophosphoenolpyruvate into bromopyruvate by way of the usual carboxyphosphate-enolate intermediate, and bromopyruvate is the species responsible for enzyme inactivation. (Z)-3-fluorophosphoenolpyruvate is transformed by the enzyme into a 6:1 mixture of 3-fluoropyruvate and 3-fluorooxalacetate, presumably by the same mechanism. The enzyme is not inactivated during this treatment

  3. Reaction of phosphoenolpyruvate carboxylase with (Z)-3-bromophosphoenolpyruvate and (Z)-3-fluorophosphoenolpyruvate

    Diaz, E.; O' Laughlin, J.T.; O' Leary, M.H.

    1988-02-23

    (Z)-3-Bromophosphoenolpyruvate inactivates phosphoenolpyruvate carboxylase from maize in the presence of HCO/sub 3//sup -/ and either Mg/sup 2 +/ or Mn/sup 2 +/. The inactivation rate follows saturation kinetics. Inactivation is slower in the presence of phospholactate or epoxymaleate, both of which are inhibitors of the enzyme, or dithiothreitol. Inactivation is completely prevented by the presence of lactate dehydrogenase and NADH, and 3-bromolactate is formed during this treatment. If the reaction is conducted by using HC/sup 18/O/sub 3//sup -/, the inorganic phosphate produced contains /sup 18/O. This and other evidence indicate that phosphoenolpyruvate carboxylase catalyzes conversion of bromophosphoenolpyruvate into bromopyruvate by way of the usual carboxyphosphate-enolate intermediate, and bromopyruvate is the species responsible for enzyme inactivation. (Z)-3-fluorophosphoenolpyruvate is transformed by the enzyme into a 6:1 mixture of 3-fluoropyruvate and 3-fluorooxalacetate, presumably by the same mechanism. The enzyme is not inactivated during this treatment.

  4. Expression, purification and crystallization of an archaeal-type phosphoenolpyruvate carboxylase

    The expression, purification, crystallization and preliminary diffraction analysis of an archaeal-type phosphoenolpyruvate carboxylase are described. Complete highly redundant X-ray data have been measured from a crystal diffracting to 3.13 Å resolution. An archaeal-type phosphoenolpyruvate carboxylase (PepcA) from Clostridium perfringens has been expressed in Escherichia coli in a soluble form with an amino-terminal His tag. The recombinant protein is enzymatically active and two crystal forms have been obtained. Complete diffraction data extending to 3.13 Å resolution have been measured from a crystal soaked in KAu(CN)2, using radiation at a wavelength just above the Au LIII edge. The asymmetric unit contains two tetramers of PepcA

  5. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  6. Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

    Gengxiang Zhao

    Full Text Available N-acetylglutamate synthase (NAGS catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG, an obligate cofactor for carbamyl phosphate synthetase I (CPSI in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.

  7. Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. PMID:23894642

  8. Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.

    Suttie, J W; Hoskins, J A; Engelke, J; Hopfgartner, A; Ehrlich, H.; Bang, N U; Belagaje, R M; Schoner, B; Long, G L

    1987-01-01

    The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the...

  9. Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency.

    Burri, B J; Sweetman, L.; Nyhan, W L

    1985-01-01

    Holocarboxylase synthetase activity has been determined in fibroblasts of seven patients with the neonatal form of biotin-responsive multiple carboxylase deficiency. The normal Km for biotin was 15 +/- 3 nmol/l, while in the patients the values ranged from 48 to 1,062 nmol/l. The mean maximum velocity was 27% of normal. Differences among the values obtained for the Km for biotin and the heat stability of holocarboxylase synthetase suggested that the patients studied represented at least four ...

  10. Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

    Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

    2014-01-01

    Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations ...

  11. Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

    Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B.; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison

    2014-01-01

    Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analys...

  12. Germline Deletion of Pantothenate Kinases 1 and 2 Reveals the Key Roles for CoA in Postnatal Metabolism

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

    2012-01-01

    Pantothenate kinase (PanK) phosphorylates pantothenic acid (vitamin B5) 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 ...

  13. The development of the 2, 4-dienoyl CoA reductase 1 gene (DECR 1) in pig

    2007-01-01

    2,4-dienoyl CoA reductase gene (DECR 1) is mapped on pig 4 q1.2, includes ten exons and nine introns of variable sizethat span 30 kb. DECR 1 gene participates in the β-oxidation pathway, affects the content of intramuscular fatty acid, especially thepercentage of linoleic acid. The expression of DECR 1 gene has important influence on IMF, the pH, and the meat colour of pork,further affects the meat quality.

  14. Molecular analysis of virulent genes (coa and spa) of staphylococcus aureus involved in natural cases of bovine mastitis

    The present study was undertaken to determine the distribution and genotypic characteristics of Staphylococcus aureus isolates recovered from naturally occurring mastitis in cattle and buffaloes. For this purpose a total of 1445 lactating cattle (653) and buffaloes (792) present at two experimental livestock farms Okara (Bahadarnagar) and Sahiwal (Qadiarabad), in and around district Faisalabad and slaughtered at an abattoir due to low milk yield and were screened for mastitis. California Mastitis Test (CMT) was used to detect sub clinical mastitis. The positive quarter milk samples were collected for culturing of S. aureus isolates. taphylococcus aureus isolates were identified on the basis of growth features, biochemical characteristics, coagulase test and as well as amplification of coagulase (coa) and spa (spa-X) genes specific to its virulence. S. aureus isolates (n=265) were characterized by Polymerase chain reaction to determine the frequency of coagulase (coa) and spa (spa-X) genes. From these isolates the amplification of the coagulase (coa) gene yielded three different PCR products approximately 204bp to 490bp while spa (spa-X) gene produced five different products ranging in size from 190bp to 320bp. PCR revealed that from all the coagulase positive S. aureus isolates 261(98.5%) had spa (spa-X) gene. The results of the present study indicated that S. aureus isolates recovered from bovine mastitis were genetically different within and among the various herds which may provide essential and valuable strategies to control staphylococcal infections in future. (author)

  15. On O(1S) and CO(a3Π) production from electron impact dissociation of CO2

    A novel method is employed to selectively detect O(1S) and CO(a 3Π) fragments following the dissociation of CO2 by electron impact. O(1S) atoms impinging on solid Xe produce a prominent emission feature at 725 nm, and less intense features at 375 and 550 nm. CO(a 3Π) molecules give a single emission feature spanning 250 to 350 nm. Time-of-flight spectra are presented for O(1S) and CO(a 3Π) from electron impact dissociation of CO2. The results for O(1S) production indicate contributions from pre-dissociation of excited states of CO2. Relative cross sections for the production of both species have been obtained. The cross section for O(1S) production has been made absolute by comparison with production of the same species from N2O. It has a threshold at 11.0 ± 0.5 eV and reaches a maximum of 1.69 x 10-17 cm2 at 50 eV. (author)

  16. Effect of Vitamin K-dependent Protein Precursor Propeptide, Vitamin K Hydroquinone, and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase*

    Higgins-Gruber, Shannon L.; Mutucumarana, Vasantha P.; Lin, Pen-Jen; Jorgenson, James W.; Stafford, Darrel W.; Straight, David L.

    2010-01-01

    The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates cause...

  17. Intracellular long-chain acyl CoAs activate TRPV1 channels.

    Yi Yu

    Full Text Available TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs, another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP2-binding residues in TRPV1. In contrast to PIP2, LC-CoA modulation of TRPV1 is independent of Ca2+i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca2+i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.

  18. Partitioning of Nitrogen among Ribulose-1,5-bisphosphate Carboxylase/Oxygenase, Phosphoenolpyruvate Carboxylase, and Pyruvate Orthophosphate Dikinase as Related to Biomass Productivity in Maize Seedlings.

    Sugiyama, T; Mizuno, M; Hayashi, M

    1984-07-01

    Maize (Zea mays L. cv Golden Cross Bantam T51) seedlings were grown under full sunlight or 50% sunlight in a temperature-controlled glasshouse at the temperatures of near optimum (30/25 degrees C) and suboptimum (17/13 degrees C) with seven levels of nitrate-N (0.4 to 12 millimolars). The contents of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPD), and ribulose-1,5-P(2) carboxylase/oxygenase (RuBisCO) were immunochemically determined for each treatment with rabbit antibodies raised against the respective maize leaf proteins (anti-PEPC and anti-PPD) or spinach leaf protein (anti-RuBisCO). The content of each enzymic protein increased with increasing N and raised under reduced temperature. The positive effect of light intensity on their contents was evident only at near optimal temperature. The relative increase in PEPC and PPD content with increasing N was significantly greater than that of RuBisCO irrespective of growth conditions. These enzymic proteins comprised about 8, 6, and 35% of total soluble protein, respectively, at near optimal growth condition. In contrast to significant increase in the proportion of soluble protein allocated to PEPC and PPD seen under certain conditions, the proportion allocated to RuBisCO decreased reciprocally with an increased biomass yield by N supply.These results indicated that the levels of PEPC and PPD parallel to maize biomass more tightly than that of RuBisCO at least under near optimal growth condition. PMID:16663684

  19. Oxidative Debenzylation and Acetylation of Hexabenzylhexaazaisowutzitane

    2002-01-01

    The oxidative reactivity of hexabenzylhexaazaisowutzitane(HBIW)under different conditions was studied. It was found that the N-benzyl groups were found to form benzoyl group after oxidation. They might also be first debenzylated and then acetylated by potassium permanganate in acetic anhydride/DMF.

  20. Property enhancement of optically transparent bionanofiber composites by acetylation

    Nogi, Masaya; Abe, Kentaro; Handa, Keishin; Nakatsubo, Fumiaki; Ifuku, Shinsuke; Yano, Hiroyuki

    2006-12-01

    The authors studied acetylation of bacterial cellulose (BC) nanofibers to widen the applications of BC nanocomposites in optoelectronic devices. The slight acetylation of BC nanofibers significantly reduces the hygroscopicity of BC nanocomposites, while maintaining their high optical transparency and thermal stability. Furthermore, the degradation in optical transparency at elevated temperature (200°C) was significantly reduced by acetylation treatment. Therefore, the acetylation of bionanofibers has an extraordinary potential as treatment for property enhancement of bionanofiber composites.

  1. aP2-Cre-mediated inactivation of acetyl-CoA carboxylase 1 causes growth retardation and reduced lipid accumulation in adipose tissues

    Adipose tissue is one of the major sites for fatty acid synthesis and lipid storage. We generated adipose (fat)-specific ACC1 knockout (FACC1KO) mice using the aP2-Cre/loxP system. FACC1KO mice showed prenatal growth retardation; after weaning, however, their weight gain was comparable to that of wi...

  2. Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior

    Bergauer, K.; Sintes, E.; van Bleijswijk, J.; Witte, H.; Herndl, G.J.

    2013-01-01

    Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating d

  3. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J;

    2011-01-01

    acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

  4. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves.

    Carmo-Silva, A Elizabete; Barta, Csengele; Salvucci, Michael E

    2011-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multifunctional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a viable strategy for increasing plant productivity. Advances in biotechnology have made this goal more attainable by making it possible to modify Rubisco in planta. To properly evaluate the properties of Rubisco, it is necessary to isolate the enzyme in pure form. This chapter describes procedures for rapid and efficient purification of Rubisco from leaves of several species. PMID:20960141

  5. The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

    Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel

    2008-03-14

    The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes. PMID:18184660

  6. The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

    Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel (Maryland); (GWU); (Georgia)

    2010-01-07

    The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.

  7. Software interface for high-speed readout of particle detectors based on the CoaXPress communication standard

    Hejtmánek, M.; Neue, G.; Voleš, P.

    2015-06-01

    This article is devoted to the software design and development of a high-speed readout application used for interfacing particle detectors via the CoaXPress communication standard. The CoaXPress provides an asymmetric high-speed serial connection over a single coaxial cable. It uses a widely available 75 Ω BNC standard and can operate in various modes with a data throughput ranging from 1.25 Gbps up to 25 Gbps. Moreover, it supports a low speed uplink with a fixed bit rate of 20.833 Mbps, which can be used to control and upload configuration data to the particle detector. The CoaXPress interface is an upcoming standard in medical imaging, therefore its usage promises long-term compatibility and versatility. This work presents an example of how to develop DAQ system for a pixel detector. For this purpose, a flexible DAQ card was developed using the XILINX Spartan 6 FPGA. The DAQ card is connected to the framegrabber FireBird CXP6 Quad, which is plugged in the PCI Express bus of the standard PC. The data transmission was performed between the FPGA and framegrabber card via the standard coaxial cable in communication mode with a bit rate of 3.125 Gbps. Using the Medipix2 Quad pixel detector, the framerate of 100 fps was achieved. The front-end application makes use of the FireBird framegrabber software development kit and is suitable for data acquisition as well as control of the detector through the registers implemented in the FPGA.

  8. Software interface for high-speed readout of particle detectors based on the CoaXPress communication standard

    This article is devoted to the software design and development of a high-speed readout application used for interfacing particle detectors via the CoaXPress communication standard. The CoaXPress provides an asymmetric high-speed serial connection over a single coaxial cable. It uses a widely available 75 Ω BNC standard and can operate in various modes with a data throughput ranging from 1.25 Gbps up to 25 Gbps. Moreover, it supports a low speed uplink with a fixed bit rate of 20.833 Mbps, which can be used to control and upload configuration data to the particle detector. The CoaXPress interface is an upcoming standard in medical imaging, therefore its usage promises long-term compatibility and versatility. This work presents an example of how to develop DAQ system for a pixel detector. For this purpose, a flexible DAQ card was developed using the XILINX Spartan 6 FPGA. The DAQ card is connected to the framegrabber FireBird CXP6 Quad, which is plugged in the PCI Express bus of the standard PC. The data transmission was performed between the FPGA and framegrabber card via the standard coaxial cable in communication mode with a bit rate of 3.125 Gbps. Using the Medipix2 Quad pixel detector, the framerate of 100 fps was achieved. The front-end application makes use of the FireBird framegrabber software development kit and is suitable for data acquisition as well as control of the detector through the registers implemented in the FPGA

  9. Partial genetic characterization of Stearoyl Coa-Desaturase´s structural region in Bubalus bubalis

    R.B. Thomazine

    2010-02-01

    Full Text Available Conjugated Linoleic Acids (CLAs comprise a family of positional and geometric isomers of linoleic acid. The main form of CLA, cis-9, trans-11-C18:2 show positive effects in cancer prevention and treatment. The major dietary sources of these fatty acids are derived from ruminant animals, in particular dairy products. In these animals, the endogenous synthesis mainly occurs in mammary gland by the action of enzyme Stearoyl CoA Desaturase (SCD. Different levels of expression and activity of SCD in mammary gland can explain partially the variation of CLA levels in fat milk. Considering a great fat concentration in bubaline milk and the benefit of a high and positive correlation between fat milk and CLA production, this study was carried on with the intention of sequencing and characterizing part of the gene that codifies SCD in buffaloes. Genomic DNA was extracted from blood samples of lactating bubaline which begins to the breed Murrah. After the (acho que nao precisa desse the extractions, PCR (Polymerase Chain Reaction reactions were made by using primers Z43D1 and E143F1. The fragments obtained in PCR were cloned into “T” vectors and transformed in competent cells DH10B line. After this, three samples of each fragment were sequenced from 5’ and 3’ extremities using a BigDye kit in an automatic sequencer. Sequences were edited in a consensus of each fragment and were submitted to BLAST-n / NCBI for similarity comparisions among other species. The sequence obtained with Z43D1 primers shows 938 bp enclosing exons 1 and 2 and intron 1. The primers E143F1 show 70 bp corresponding to exon 3 of bubaline SCD gene. Similarities were obtained between 85% and 97% among bubaline sequences and sequences of SCD gene described in human, mouse, rat, swine, bovine, caprine and ovine species. This study has permitted the identification and partial characterization of SCD codifing region in Bubalus bubalis specie.

  10. Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and co-immobilization with Azospirillum brasilense [corrected].

    Leyva, Luis A; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae. PMID:25129521

  11. Dynamic Protein Acetylation in Plant–Pathogen Interactions

    Song, Gaoyuan; Walley, Justin W.

    2016-01-01

    Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. PMID:27066055

  12. Attempts to apply affinity labeling techniques to ribulosebisphosphate carboxylase/oxygenase. [Comparison of spinach leaf and Rhodospirillum rubrum

    Hartman, F. C.; Norton, I. L.; Stringer, C. D.; Schloss, J. V.

    1978-01-01

    Studies on carboxylases/oxygenases from different species may be necessary to confirm that a residue implicated as essential is indeed an active-site component. To provide an especially stringent test case for the identification of species invariant structural features the enzymes from two phylogenetically distant species, spinach and Rhodospirillum rubrum, were compared. To date, the reactions of Br-butanone-P/sub 2/ and BrAcNHEtOP with the spinach enayme have been rather thoroughly characterized; only preliminary experiments have been completed with the R. rubrum enzyme. Both enzymes were isolated and assayed for carboxylase activity (spectrophotometrically or /sup 14/CO/sub 2/-fixation) and for oxygenase activity.

  13. Acetylation Is Indispensable for p53 Activation

    Tang, Yi; Zhao, Wenhui; Chen, Yue; Zhao, Yingming; Gu, Wei

    2008-01-01

    The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its...

  14. p53 Acetylation: Regulation and Consequences

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo ev...

  15. Dark/light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

    Vu, J.C.V.; Allen, L.H. Jr.; Bowes, G.

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from light-exposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO/sub 3//sup -/ and Mg/sup 2 +/ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C/sub 3/); P. maximum (C/sub 4/ phosphoenolpyruvate carboxykinase); P. milioides (C/sub 3//C/sub 4/); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C/sub 3/); P. miliaceum (C/sub 4/ NAD malic enzyme); Zea mays and Sorghum bicolor (C/sub 4/ NADP malic enzyme); Moricandia arvensis (C/sub 3//C/sub 4/); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C/sub 3/ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO/sub 2/ and Mg/sup 2 +/ activation, but which can be converted to an activatable state upon exposure of the leaf to light. 16 references, 2 tables.

  16. Dark/Light Modulation of Ribulose Bisphosphate Carboxylase Activity in Plants from Different Photosynthetic Categories 1

    Vu, J. Cu V.; Allen, Leon H.; Bowes, George

    1984-01-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO3− and Mg2+ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C3); P. maximum (C4 phosphoenolpyruvate carboxykinase); P. milioides (C3/C4); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C3); P. miliaceum (C4 NAD malic enzyme); Zea mays and Sorghum bicolor (C4 NADP malic enzyme); Moricandia arvensis (C3/C4); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C3 species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO2 and Mg2+ activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID:16663937

  17. Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories.

    Vu, J C; Allen, L H; Bowes, G

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO(3) (-) and Mg(2+) concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C(3)); P. maximum (C(4) phosphoenolpyruvate carboxykinase); P. milioides (C(3)/C(4)); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C(3)); P. miliaceum (C(4) NAD malic enzyme); Zea mays and Sorghum bicolor (C(4) NADP malic enzyme); Moricandia arvensis (C(3)/C(4)); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C(3) species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO(2) and Mg(2+) activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID:16663937

  18. Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

    [3H] Diethyl pyrocarbonate was synthesized from [3H] ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM Mg2+, and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with earlier experiments, suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources

  19. The neurobiology of acetyl-L-carnitine.

    Traina, Giovanna

    2016-01-01

    A large body of evidence points to the positive effects of dietary supplementation of acetyl-L-carnitine (ALC). Its use has shown health benefits in neuroinflammation, which is a common denominator in a host of neurodegenerative diseases. ALC is the principal acetyl ester of L-Carnitine (LC), and it plays an essential role in intermediary metabolism, acting as a donor of acetyl groups and facilitating the transfer of fatty acids from cytosol to mitochondria during beta-oxidation. Dietary supplementation of ALC exerts neuroprotective, neurotrophic, antidepressive and analgesic effects in painful neuropathies. ALC also has antioxidant and anti-apoptotic activity. Moreover, ALC exhibits positive effects on mitochondrial metabolism, and shows promise in the treatment of aging and neurodegenerative pathologies by slowing the progression of mental deterioration. In addition, ALC plays neuromodulatory effects on both synaptic morphology and synaptic transmission. These effects are likely due to affects of ALC through modulation of gene expression on several targets in the central nervous system. Here, we review the current state of knowledge on effects of ALC in the nervous system. PMID:27100509

  20. Fragrance material review on acetyl cedrene.

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. PMID:23907023

  1. Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

    We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position

  2. Acetylation phenotype variation in pediatric patients with atopic dermatitis

    Rafi A Majeed Al-Razzuqi

    2011-01-01

    Full Text Available Background: Few studies have been done on the relation between acetylator status and allergic diseases. Aim: To determine any possible association between acetylating phenotype in pediatric patients with atopic dermatitis (AD and the disease prognosis. Patients and Methods: Thirty-six pediatric patients and forty two healthy children as a control group were participated in the study. All participants received a single oral dose of dapsone of 1.54 mg/kg body weight, after an overnight fast. Using high performance liquid chromatography (HPLC, plasma concentrations of dapsone and its metabolite (monoacetyldapsone were estimated to phenotype the participants as slow and rapid acetylators according to their acetylation ratio (ratio of monoacetyldapsone to dapsone. Results: 72.2% of pediatric patients with AD showed slow acetylating status as compared to 69.4% of control individuals. Also, 73% of AD patients with slow acetylating phenotype had familial history of allergy. The severity of AD occurred only in slow acetylator patients. The eczematous lesions in slow acetylators presented mainly in the limbs, while in rapid acetylators, they were found mostly in face and neck. Conclusion: This study shows an association between the N-acetylation phenotype variation and clinical aspects of AD.

  3. New insights into structure-function relationships of oxalyl CoA decarboxylase from Escherichia coli.

    Werther, Tobias; Zimmer, Agnes; Wille, Georg; Golbik, Ralph; Weiss, Manfred S; König, Stephan

    2010-06-01

    The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5'-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5'-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites. PMID:20553497

  4. Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia : Mollusca)

    Cavanaugh, Colleen M.; Abbott, Marilyn S.; Veenhuis, Marten

    1988-01-01

    The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP2Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large

  5. Acetylation and characterization of spruce (Picea abies) galactoglucomannans.

    Xu, Chunlin; Leppänen, Ann-Sofie; Eklund, Patrik; Holmlund, Peter; Sjöholm, Rainer; Sundberg, Kenneth; Willför, Stefan

    2010-04-19

    Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, (1)H and (13)C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA. PMID:20144827

  6. Preparation, physicochemical characterization and application of acetylated lotus rhizome starches.

    Sun, Suling; Zhang, Ganwei; Ma, Chaoyang

    2016-01-01

    Acetylated lotus rhizome starches were prepared, physicochemically characterized and used as food additives in puddings. The percentage content of the acetyl groups and degree of substitution increased linearly with the amount of acetic anhydride used. The introduction of acetyl groups was confirmed via Fourier transform infrared (FT-IR) spectroscopy. The values of the pasting parameters were lower for acetylated starch than for native starch. Acetylation was found to increase the light transmittance (%), the freeze-thaw stability, the swelling power and the solubility of the starch. Sensorial scores for puddings prepared using native and acetylated lotus rhizome starches as food additives indicated that puddings produced from the modified starches with superior properties over those prepared from native starch. PMID:26453845

  7. Acetylation of Tau Inhibits Its Degradation and Contributes to Tauopathy

    Min, Sang-Won; Cho, Seo-Hyun; Zhou, Yungui; Schroeder, Sebastian; Haroutunian, Vahram; Seeley, William W.; Huang, Eric J.; Shen, Yong; Masliah, Eliezer; Mukherjee, Chandrani; Meyers, David; Cole, Philip A.; Ott, Melanie; Gan, Li

    2010-01-01

    Neurodegenerative tauopathies characterized by hyperphosphorylated tau include frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease (AD). Reducing tau levels improves cognitive function in mouse models of AD and FTDP-17, but the mechanisms regulating the turnover of pathogenic tau are unknown. We found that tau is acetylated and that tau acetylation prevents degradation of phosphorylated tau (p-tau). Using two antibodies specific for acetylated ta...

  8. Getting a Knack for NAC: N-Acetyl-Cysteine

    Sansone, Randy A.; Sansone, Lori A.

    2011-01-01

    N-acetyl-cysteine, N-acetylcysteine, N-acetyl cysteine, and N-acetyl-L-cysteine are all designations for the same compound, which is abbreviated as NAC. NAC is a precursor to the amino acid cysteine, which ultimately plays two key metabolic roles. Through its metabolic contribution to glutathione production, cysteine participates in the general antioxidant activities of the body. Through its role as a modulator of the glutamatergic system, cysteine influences the reward-reinforcement pathway....

  9. Obesity, cancer, and acetyl-CoA metabolism

    Lee, Joyce V.; Shah, Supriya A.; Wellen, Kathryn E.

    2013-01-01

    As rates of obesity soar in the Unites States and around the world, cancer attributed to obesity has emerged as major threat to public health. The link between obesity and cancer can be attributed in part to the state of chronic inflammation that develops in obesity. Acetyl-CoA production and protein acetylation patterns are highly sensitive to metabolic state and are significantly altered in obesity. In this article, we explore the potential role of nutrient-sensitive lysine acetylation in r...

  10. Determination of amphetamine by HPLC after acetylation.

    Veress, T

    2000-01-01

    An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market. PMID:10641931

  11. Resolving the Activation Site of PositiveRegulators in Plant PhosphoenolpyruvateCarboxylase

    2014-01-01

    Dear Editor, Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) islocated at an important branch point in the carbohydratemetabolism of plants. The enzyme is a homotetramer andcatalyzes the addition of bicarbonate to phosphoenolpyru-vate (PEP) to form oxaloacetate and phosphate. PEPC isregulated by metabolites and phosphorylation. AIIostericfeedback inhibition is mainly regulated by L-malate andL-aspartate which bind to a site separated from the activecenter (Kai et al., 1999; Paulus et al., 2013). Structure analy-sis of PEPC from Escherichia coli (Kai et al., 1999; Matsumuraet al., 2002), Zea rnays (Matsumura et al., 2002), Flaveria trin-ervia, and F. pringlei (Paulus et al., 2013) revealed that thesubstrate PEP and the feedback inhibitors bind to separatesites within each monomer.

  12. Is L-Carnitine Supplementation Beneficial in 3-Methylcrotonyl-CoA Carboxylase Deficiency?

    Thomsen, Jákup Andreas; Lund, Allan Meldgaard; Olesen, Jess Have; Mohr, Magni; Rasmussen, Jan

    2015-01-01

    Background: 3-Methylcrotonyl-CoA carboxylase deficiency (3-MCCd) is an autosomal recessive disorder in the catabolism of leucine. In the present study, we investigated the current and prior medical condition of patients with 3-MCCd in the Faroe Islands and their carnitine levels in blood, urine and...... muscle tissue with and without L-carnitine supplementation to evaluate the current treatment strategy of not recommending L-carnitine supplementation to Faroese 3-MCCd patients. Methods: Blood and urine samples and muscle biopsies were collected from patients at inclusion and at 3 months. Eight patients...... received L-carnitine supplementation when recruited; five did not. Included patients who received supplementation were asked to stop L-carnitine, the others were asked to initiate L-carnitine supplementation during the study. Symptoms were determined by review of hospital medical records and questionnaires...

  13. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk;

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  14. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  15. Protein lysine acetylation in bacteria: Current state of the art.

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  16. Differential patterns of histone acetylation in inflammatory bowel diseases

    Adcock Ian M

    2011-01-01

    Full Text Available Abstract Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD to study the expression of acetylated histones (H 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP in dextran sulfate sodium (DSS-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

  17. Probing the acetylation code of histone H4.

    Lang, Diana; Schümann, Michael; Gelato, Kathy; Fischle, Wolfgang; Schwarzer, Dirk; Krause, Eberhard

    2013-10-01

    Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein-protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-acetylated at K8/12/16 and fully tetra-acetylated. A set of 29 proteins was found enriched on the fully acetylated H4 tail while specific binders of the mono and bis-acetylated tails were barely detectable. These observations are in good agreement with earlier reports indicating that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than via site-specific recruitment of regulatory proteins. PMID:23970329

  18. Probing the acetylation code of histone H4.

    Lang, D; Schümann, M; Gelato, K.; Fischle, W; Schwarzer, D; Krause, E.

    2013-01-01

    Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein–protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-ace...

  19. Inhibition of E. coli P-enolpyruvate carboxylase by P-enol-3-bromopyruvate

    The generality of the mechanism based inhibition of P-enolpyruvate carboxylases (PEPCase) by P-enol-3-bromopyruvate (BrPEP) was tested by measuring its effects on the allosterically regulated enzyme from E. coli. In the presence of 1mM Mn2+, BrPEP appears to be a competitive inhibitor (K/sub i/ = 0.0087mM) of PEPCase. Incubation of 0.005mM PEPCase with 0.5mM (or 1.0mM)BrPEP along with H14CO3- and Mn2+, yielded, upon reduction with NaBH4, a protein containing radioactivity in an amount approximately proportional to that expected from the loss of catalytic activity. At both a 25- and a 50-fold excess (0.5mM and 1.0mM, respectively) of BrPEP to PEPCase subunits, first order loss of activity occurred with k values of 5.24 x 10-3 min-1 and 1.03 x 10-2 min-1, respectively. At the lower concentration of BrPEP the inactivation process appeared to be reversible after 40 min with no further inhibition occurring even up to two hours of incubation. At the higher concentration of BrPEP, the rate of inhibition slowed dramatically after 50 min and appeared insignificant over the next hour. These data suggest that BrPEP irreversibly inactivates the E. coli PEP carboxylase, but that there may be considerable dissociation of the product, Br-oxaloacetate, before irreversible binding occurs, and that the reduced rate of inactivation may be due to depletion of BrPEP

  20. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    Chou, Chi-Yuan; Tong, Liang (Columbia)

    2012-06-19

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  1. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha;

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double...... quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...... in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell...

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

    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).

  3. The production of CO+(A 2Π) from dissociative ionization of CO2: a fragment ion-photon coincidence spectroscopic investigation

    Fragment ion-photon coincidence (FIPCO) spectra by 120 eV electron impact on carbon dioxide (CO2) have been observed, in which optical emission in the 250-600 nm region has been detected. There are a dominant CO2+ peak and a weak, broad CO+ peak in the FIPCO spectra. The kinetic energy distribution of CO+ correlated with the CO+(A 2Π-X 2Σ+) emission has been estimated on the basis of the Monte Carlo simulation of the CO+ band shape. This CO+(A 2Π) ion is produced through the dissociation process, CO2+e-→CO2+*[MET I 2Πu] + 2e-→CO+(A 2Π) + O(3P) + 2e-, where MET refers to multiple electron transitions. The production of CO+(B 2Σ+) is negligible compared with that of CO+(A 2Π). The produced CO+(A 2Π) ion is in vibrationally excited levels, and there is little population in the vibrational levels, v'≤3. (author)

  4. Emerging Functions for N-Terminal Protein Acetylation in Plants

    Gibbs, Daniel J.

    2015-01-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour.

  5. Medial temporal N-acetyl-aspartate in pediatric major depression.

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  6. Medial temporal N-acetyl aspartate in pediatric major depression

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  7. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. PMID:26920270

  8. Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase: identification of the biotin carboxylase and biotin-carrier domains.

    Song, J.; Wurtele, E S; Nikolau, B J

    1994-01-01

    Soybean genomic clones were isolated based on hybridization to probes that code for the conserved biotinylation domain of biotin-containing enzymes. The corresponding cDNA was isolated and expressed in Escherichia coli through fusion to the bacterial trpE gene. The resulting chimeric protein was biotinylated in E. coli. Antibodies raised against the chimeric protein reacted specifically with an 85-kDa biotin-containing polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carboxylase (...

  9. Unchanged acetylation of isoniazid by alcohol intake

    Wilcke, J T R; Døssing, M; Angelo, H R;

    2004-01-01

    SETTING: In 10 healthy subjects, the influence of acute alcohol intake on the pharmacokinetics of isoniazid (INH) was studied. OBJECTIVE: To test the hypothesis that alcohol increases the conversion of INH by acetylation into its metabolite acetylisoniazid. DESIGN: In a crossover design, an oral...... dose of 300 mg INH was administered on 2 separate days, 14 days apart, with or without alcohol to a serum alcohol of about 21 mmol/l (1 g/l) maintained for 12 h. RESULTS: Neither the metabolism of INH nor that of acetylisoniazid was changed by acute alcohol intake. CONCLUSION: Acute alcohol intake has...... no impact on the conversion of INH to its metabolite acetylisoniazid, which is catalysed by the enzyme N-acetyltranferase. Accordingly, a metabolic effect of acute alcohol intake on INH metabolism probably contributes little to the therapeutic failure of anti-tuberculosis treatment among alcoholics....

  10. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  11. Electron spin resonance studies of ribulosebisphosphate carboxylase: identification of activator cation ligands.

    Miziorko, H M; Sealy, R C

    1984-01-31

    Ribulosebisphosphate carboxylase (RuBP carboxylase)forms a stable model complex containing stoichiometric amounts of enzyme sites, activator C0(2), divalent activator cation, and the transition-state analogue carboxyarabinitol bisphosphate (CABP). Incorporation of Mn(2+) in the model complex permits investigation of the environment of the activator cation by electron spin resonance (ESR)techniques. Measurements at 9 GHz on the Mn(2+)-containing complex prepared by using dimeric Rhodospirillum rubrum enzyme produce a spectrum which indicates that the cation is bound in an anisotropic environment. Measurements at 9 GHz on the spinach enzyme model complex produce a spectrum in which several of the fine structure transitions are obvious. In contrast, the spectrum produced from Mn(2+) bound to R. rubrum enzyme exhibits an intense powder pattern for the central fine structure transition; the other four fine structure transitions produce powder patterns that are in homogeneously broadened and therefore are not as apparent.Low-temperature measurements at high field (35 GHz) result in substantially simplified spectra. The spectrum of Mn(2+) bound to the R. rubrum enzyme shows less fine structure than the spectrum of Mn(2+) bound in the octameric spinach enzyme complex, where substantial hyperfine splitting is resolved in three of the five fine structure transitions. Measurements at 35 GHz on Mn (2+) bound in the dimeric R. rubrum enzyme complex produce spectra in which only the central fine structure transition produces a prominent signal. However, these samples are characterized by several narrow spectral features which permit investigation of the identity of Mn(2+)ligands by 170 perturbation techniques. Preparation of the R. rubrum RuBP carboxylase model complex in (17)O-enriched water results in a sample which exhibits an obviously broadened 35-GHz Mn(2+) spectrum in comparison to unenriched samples. Removal of H(2)(17)O by gel filtration abolished the spectral

  12. Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

    Biely, Peter; Cziszarava, Maria; Agger, Jane W.;

    2014-01-01

    Results The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most promin...

  13. Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

    Sugiura, Isamu; Furie, Bruce; Walsh, Christopher T.; Furie, Barbara C.

    1997-01-01

    The vitamin K-dependent γ-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to γ-carboxyglutamic acid in precursor proteins containing the γ-carboxylation recognition site (γ-CRS). During this reaction, glutamic acid is converted to γ-carboxyglutamic acid while vitamin KH2 is converted to vitamin K 2,3-epoxide. Recombinant bovine carboxylase was purified free of γ-CRS-containing propeptide and endogenous substrate in a single-step immunoaffinity procedure. We sh...

  14. Acetylation/deacetylation reactions of T-2, acetyl T-2, HT-2, and acetyl HT-2 toxins in bovine rumen fluid in vitro

    Munger, C.E.; Ivie, G.W.; Christopher, R.J.; Hammock, B.D.; Phillips, T.D.

    A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, underwent both acetylation and deacetylation reactions when incubated with bovine rumen fluid in vitro. Products from incubations of T-2 in rumen fluid included acetyl T-2, HT-2, and acetyl HT-2. Direct studies with tritiated samples of each of these metabolites confirmed their relatively facile interconversion in the rumen. Studies with (/sup 3/H)HT-2 under conditions of inhibited esterase activity (added diisopropyl fluorophosphate) showed that acetylation is preferred at C-3 vs. C-4. Studies with (/sup 3/H)acetyl T-2 indicated that deacetylation similarly occurs with greater rapidity at C-3. There were no indications that ester hydrolysis of these trichothecenes occurred at C-8 or C-15 or that they were subjected to epoxide reduction reactions. These data suggest that acetylation of T-2 and other trichothecenes in the rumen in situ may ultimately result in the absorption of more lipophilic metabolites whose toxicological and residual properties are at present unknown.

  15. Acetylation/deacetylation reactions of T-2, acetyl T-2, HT-2, and acetyl HT-2 toxins in bovine rumen fluid in vitro

    A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, underwent both acetylation and deacetylation reactions when incubated with bovine rumen fluid in vitro. Products from incubations of T-2 in rumen fluid included acetyl T-2, HT-2, and acetyl HT-2. Direct studies with tritiated samples of each of these metabolites confirmed their relatively facile interconversion in the rumen. Studies with [3H]HT-2 under conditions of inhibited esterase activity (added diisopropyl fluorophosphate) showed that acetylation is preferred at C-3 vs. C-4. Studies with [3H]acetyl T-2 indicated that deacetylation similarly occurs with greater rapidity at C-3. There were no indications that ester hydrolysis of these trichothecenes occurred at C-8 or C-15 or that they were subjected to epoxide reduction reactions. These data suggest that acetylation of T-2 and other trichothecenes in the rumen in situ may ultimately result in the absorption of more lipophilic metabolites whose toxicological and residual properties are at present unknown

  16. /sup 13/C nuclear magnetic resonance study of the CO/sub 2/ activation of ribulosebisphosphate carboxylase from Rhodospirillum rubrum

    O' Leary, M.H. (Univ. of Wisconsin, Madison); Joworski, R.J.; Hartman, F.C.

    1979-02-01

    Ribulosebisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) from Rhodospirillum rubrum is activated by CO/sub 2/ and Mg/sup 2 +/. /sup 13/C NMR spectra were determined for the unactivated enzyme and for enzyme that had been activated by /sup 13/CO/sub 2/ and Mg/sup 2 +/. In addition to the expected resonance for H/sup 13/CO/sub 3//sup -//CO/sub 3//sup 2 -/ at 161.8 ppM downfield from tetramethylsilane, the spectrum of the activated enzyme shows a broad resonance at 164.9 ppM. Analogy with previous NMR studies of /sup 13/CO/sub 2/ binding to hemoglobin suggests that the CO/sub 2/ activation of ribulosebisphosphate carboxylase involves formation of a carbamate between an enzyme amino group and CO/sub 2/.

  17. Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogues

    Lietzan, Adam D.; St. Maurice, Martin

    2013-01-01

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT doma...

  18. Biotin Uptake into Human Peripheral Blood Mononuclear Cells Increases Early in the Cell Cycle, Increasing Carboxylase Activities1,2

    Stanley, J. Steven; Mock, Donald M.; Griffin, Jacob B.; Zempleni, Janos

    2002-01-01

    Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.e., biotin uptake increased in the G1 phase of the cycle...

  19. Role of Histone Acetylation in Cell Cycle Regulation.

    Koprinarova, Miglena; Schnekenburger, Michael; Diederich, Marc

    2016-01-01

    Core histone acetylation is a key prerequisite for chromatin decondensation and plays a pivotal role in regulation of chromatin structure, function and dynamics. The addition of acetyl groups disturbs histone/DNA interactions in the nucleosome and alters histone/histone interactions in the same or adjacent nucleosomes. Acetyl groups can also provide binding sites for recruitment of bromodomain (BRD)-containing non-histone readers and regulatory complexes to chromatin allowing them to perform distinct downstream functions. The presence of a particular acetylation pattern influences appearance of other histone modifications in the immediate vicinity forming the "histone code". Although the roles of the acetylation of particular lysine residues for the ongoing chromatin functions is largely studied, the epigenetic inheritance of histone acetylation is a debated issue. The dynamics of local or global histone acetylation is associated with fundamental cellular processes such as gene transcription, DNA replication, DNA repair or chromatin condensation. Therefore, it is an essential part of the epigenetic cell response to processes related to internal and external signals. PMID:26303420

  20. Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation.

    Sellers, Katherine; Fox, Matthew P; Bousamra, Michael; Slone, Stephen P; Higashi, Richard M; Miller, Donald M; Wang, Yali; Yan, Jun; Yuneva, Mariia O; Deshpande, Rahul; Lane, Andrew N; Fan, Teresa W-M

    2015-02-01

    Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation. PMID:25607840

  1. Relationship between NH4+ assimilation rate and in vivo phosphoenolpyruvate carboxylase activity

    The rate of NH4+ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH4+ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H14CO3- into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C3 organism

  2. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    Meagher, R.B. [Georgia Univ., Athens, GA (United States). Dept. of Genetics

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  3. 3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

    Grünert Sarah C

    2012-05-01

    Full Text Available Abstract Background Isolated 3-methylcrotonyl-CoA carboxylase (MCC deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. Methods We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. Results Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. Conclusions Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.

  4. Ribulose-1,5-bisphosphate Carboxylase/Oxygenase content, assimilatory charge, and mesophyll conductance in leaves

    Eichelmann; Laisk

    1999-01-01

    The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 &mgr;mol active sites m-2. Mesophyll conductance (&mgr;) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of &mgr; on Et saturated at Et = 30 &mgr;mol active sites m-2 and &mgr; = 11 mm s-1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a &mgr; of only 6 to 8 mm s-1. &mgr; was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 &mgr;M-1 s-1. Our data show that the saturation of the relationship between Et and &mgr; is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites. PMID:9880359

  5. Functional metagenomic selection of ribulose 1, 5-bisphosphate carboxylase/oxygenase from uncultivated bacteria.

    Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A; Witte, Brian; Dourado, Manuella N; Anantharaman, Karthik; Arbing, Mark A; McCann, Shelley Hoeft; Oremland, Ronald S; Banfield, Jillian F; Wrighton, Kelly C; Tabita, F Robert

    2016-04-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized. PMID:26617072

  6. Pyruvate carboxylase as a sensitive protein biomarker for exogenous steroid chemicals

    Assessing protein responses to endocrine disrupting chemicals is critical for understanding the mechanisms of chemical action and for the assessment of hazards. In this study, the response of the liver proteome of male rare minnows (Gobiocypris rarus) treated with 17β-estradiol (E2) and females treated with 17α-methyltestosterone (MT) were analyzed. A total of 23 and 24 proteins were identified with differential expression in response to E2 and MT, respectively. Pyruvate carboxylase (PC) was the only common differentially expressed protein in both males and females after E2- and MT-treatments. The mRNA as well as the protein levels of PC were significantly down-regulated compared with that of the controls (p < 0.05). Our results suggest that endocrine disruptors interfere with genes and proteins of the TCA cycle and PC may be a sensitive biomarker of exposure to exogenous steroid chemicals in the liver of fish. - Highlights: • The hepatic proteomes of rare minnow (Gobiocypris rarus) exposed to E2 and MT were analyzed. • Differentially expressed proteins (23 and 24 respectively) were identified following E2 and MT exposure. • Four differentially expressed proteins associated with chemical stimulus were characterized. • PC was identified as a responsive biomarker for both estrogens and androgens. - Our results suggest PC may be a sensitive biomarker of exposure to exogenous steroid chemicals in the liver of fish

  7. Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

    Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

    2014-02-01

    Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products. PMID:24334667

  8. Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag.

    Bhaskaran, S; Smith, R H; Finer, J J

    1983-11-01

    Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. ;Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable. PMID:16663273

  9. A guarda Nacional Republicana no combate à sinistralidade rodoviária, educação ou coação?

    Lopes, Fábio

    2012-01-01

    O presente Trabalho de Investigação Aplicada (TIA) com o tema “A GNR no Combate à Sinistralidade Rodoviária, Educação ou Coação?” tem como objetivo esclarecer o papel da Guarda Nacional Republicana (GNR) no combate à sinistralidade rodoviária, assim como clarificar esse papel no domínio da fiscalização/coação e da educação. No desenvolver do presente estudo foram formuladas hipóteses e questões de investigação subordinadas à questão central: “Qual ...

  10. Intracellular signal transduction of PBAN action in lepidopteran insects: inhibition of sex pheromone production by compactin, an HMG CoA reductase inhibitor.

    Ozawa, R; Matsumoto, S; Kim, G H; Uchiumi, K; Kurihara, M; Shono, T; Mitsui, T

    1995-06-27

    Pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production in the pheromone glands of many species of female moths. In order to probe the biochemical steps as well as underlying mechanisms regulated by PBAN, we have tested the effect of chemicals on sex pheromone production by using an in vitro assay. Among the chemicals we tested here, compactin, a specific 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, clearly inhibited the pheromone biosynthesis in the silkworm, Bombyx mori, and the common cutworm, Spodoptera litura. Since the activation of HMG CoA reductase occurs by dephosphorylation mediated by a specific phosphatase and the biochemical step regulated by PBAN in bombykol biosynthesis is similar to the one catalyzed by HMG-CoA reductase in cholesterol biosynthesis, the present results support the idea that phosphoprotein phosphatase has a significant role to regulate bombykol production in the intracellular transduction of PBAN action in B. mori. PMID:7480881

  11. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-01-01

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. PMID:25742604

  12. Feedback regulation of cholesterol synthesis:sterol-accelerated ubiquitination and degradation of HMG CoA reductase

    Russell A DeBose-Boyd

    2008-01-01

    3Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate,an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids.The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism.Here,Ⅰwill discuss recent advances that shed light on one mechanism for control of reductase,which involves rapid degradation of the enzyme.Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2.Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78,which initiates ubiquitination of reductase.This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes.Thus,sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).

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

    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. PMID:26370185

  14. Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

    Ewing Andrew G

    2008-12-01

    Full Text Available Abstract Background Primordial germ cells (PGCs are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

  15. Study on the 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitory properties of Agaricus bisporus and extraction of bioactive fractions using pressurised solvent technologies

    Gil-Ramírez, Alicia; Clavijo, Cristina; Palanisamy, Marimuthu; Ruiz-Rodríguez, Alejandro; Navarro-Rubio, María; Pérez, Margarita; Marin, Francisco R.; Reglero, Guillermo; Soler-Rivas, Cristina

    2013-01-01

    [Background]: Agaricus bisporus mushrooms were able to lower cholesterol levels in hypercholesterolaemic rats and it was suggested that dietary fibre might inhibit cholesterol absorption. However, A. bisporus extracts were also able to inhibit the 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR, the key enzyme in the cholesterol biosynthetic pathway) and this might also contribute to the observed lowering of cholesterol levels in serum. [Results]: The methanol-water extracts obtained from A....

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

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie [Nankai; (Chinese Aca. Sci.)

    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. Acetylation of C/EBPα inhibits its granulopoietic function.

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S; Numata, Akihiko; Sárosi, Menyhárt B; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K; Gunaratne, Jayantha; Tenen, Daniel G

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  18. Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased ...

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

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

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

  20. Function-structure relationships of acetylated pea starches

    J. Huang

    2006-01-01

    Cowpea, chickpea and yellow pea starches were studied and the results showed that their properties were strongly related to the chemical fine structures of the starches. Furthermore, granular starches were modified using two types of chemical acetylation reagents and then separated into different size fractions. The amount of introduced acetyl groups was found to depend on the size of the granules for the reaction with rapidly reacting reagent acetic acid anhydride, whereas the amount of intr...

  1. Site-specific acetylation of ISWI by GCN5

    Chioda Mariacristina

    2007-08-01

    Full Text Available Abstract Background The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. Results We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. Conclusion Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(KacxPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

  2. Acetyl radical generation in cigarette smoke: Quantification and simulations

    Hu, Na; Green, Sarah A.

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high-performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commercial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass fiber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acetaldehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke.

  3. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  4. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    Yingmei Peng

    Full Text Available Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC while the other as bacteria-type pepcase (BTPC because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

  5. Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

    Ansaya Thonpho

    Full Text Available Pyruvate carboxylase (PC is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an activator element(s for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y, as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

  6. Epigenetic regulation of pyruvate carboxylase gene expression in the postpartum liver.

    Walker, C G; Crookenden, M A; Henty, K M; Handley, R R; Kuhn-Sherlock, B; White, H M; Donkin, S S; Snell, R G; Meier, S; Heiser, A; Loor, J J; Mitchell, M D; Roche, J R

    2016-07-01

    Hepatic gluconeogenesis is essential for maintenance of whole body glucose homeostasis and glucose supply for mammary lactose synthesis in the dairy cow. Upregulation of the gluconeogenic enzyme pyruvate carboxylase (PC) during the transition period is vital in the adaptation to the greater glucose demands associated with peripartum lactogenesis. The objective of this study was to determine if PC transcription in hepatocytes is regulated by DNA methylation and if treatment with a nonsteroidal anti-inflammatory drug (NSAID) alters methylation of an upstream DNA sequence defined as promoter 1. Dairy cows were left untreated (n=20), or treated with a NSAID during the first 5 d postcalving (n=20). Liver was biopsied at d 7 precalving and d 7, 14, and 28 postcalving. Total PC and transcript specific gene expression was quantified using quantitative PCR and DNA methylation of promoter 1 was quantified using bisulfite Sanger sequencing. Expression of PC changed over the transition period, with increased expression postcalving occurring concurrently with increased circulating concentration of nonesterified fatty acids. The DNA methylation percentage was variable at all sites quantified and ranged from 21 to 54% across the 15 CpG dinucleotides within promoter 1. The DNA methylation at wk 1 postcalving, however, was not correlated with gene expression of promoter 1-regulated transcripts and we did not detect an effect of NSAID treatment on DNA methylation or PC gene expression. Our results do not support a role for DNA methylation in regulating promoter 1-driven gene expression of PC at wk 1 postcalving. Further research is required to determine the mechanisms regulating increased PC expression over the transition period. PMID:27085418

  7. Photosynthetic Characteristics and Heterosis in Transgenic Hybrid Rice with Maize Phosphoenolpyruvate Carboxylase (pepc) Gene

    LI Ji-hang; XIANG Xun-chao; ZHOU Hua-qiang; HE Li-bin; ZHANG Kai-zheng; LI Ping

    2006-01-01

    Three F1 hybrids derived from the sterile rice lines Gang 46A, 776A and 2480A and the improved restorer line Shuhui 881 containing maize phosphoenolpyruvate carboxylase (pepc) gene were used to analyze the effect of pepc gene on the heterosis and photosynthetic characteristics, while the F1 obtained by crossing Shuhui 881 with the above three sterile lines served as controls. The dynamics of photosynthetic characteristics in leaves of three F1 with pepc gene and their controls were determined at the initial-tillering, maxium-tillering, elongation, initial-heading, heading, maturity stages, and other different times after flag leaf fully expanded. The PEPCase activities of the three F1 with pepc gene increased significantly as compared with control plants during the whole developmental stages. Moreover, the net photosynthesis rate (Pn) also increased to certain extent. The data showed that PEPCase activity was significantly correlated to Pn with a correlation coefficient of 0.6081**. The photosynthetic indexes of the three F1 with pepc gene were obviously superior with respective to controls in apparent quantum yield, light compensation point (LCP) and carboxylation efficiency (CE), while the CO2 compensation point (CCP) was lower than that of corresponding control. The Pn of the three F1 with pepc gene at light saturation point (LSP) and CO2 saturation point (CSP) was also higher than that of control plants. In addition, the three F1 with pepc gene had an average increase of 37.10% in grain yields per plant in comparison with control plants. The results indicated that the photosynthetic characteristics of hybrid rice containing pepc gene had been improved to some extent due to the introduction of pepc gene.

  8. Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase

    13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. /sup D/(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the βγ-bridge position to the β-nonbridge position in [18O4]ATP or washout of 18O from the γ-nonbridge positions. Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate is formed by nucleophilic attack of bicarbonate on ATP. Decarboxylation of carboxyphosphate in the active site generates CO2, which serves to carboxylate the isourea tautomer of biotin that is generated by the removal of the proton on N1' by the ionized sulfhydryl

  9. Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

    Marek M Galka

    Full Text Available Abscisic acid ((+-ABA is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC, x-ray crystallography and in silico modelling to identify putative (+-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP substrate. Functionally, (+-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM, but more potent inhibition of Rubisco activation (Ki of ~ 130 μM. Comparative structural analysis of Rubisco in the presence of (+-ABA with RuBP in the active site revealed only a putative low occupancy (+-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+-ABA binding site in the RuBP binding pocket. Overall we conclude that (+-ABA interacts with Rubisco. While the low occupancy (+-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

  10. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. PMID:26168906