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Sample records for acyl coa dehydrogenase

  1. Very long-chain acyl CoA dehydrogenase deficiency which was accepted as infanticide.

    PubMed

    Eminoglu, Tuba F; Tumer, Leyla; Okur, Ilyas; Ezgu, Fatih S; Biberoglu, Gursel; Hasanoglu, Alev

    2011-07-15

    Very-long-chain acyl-coenzyme A (CoA) dehydrogenase deficiency (VLCADD) (OMIM #201475) is an autosomal recessive disorder of fatty acid oxidation. Major phenotypic expressions are hypoketotic hypoglycemia, hepatomegaly, cardiomyopathy, myopathy, rhabdomyolysis, elevated creatinine kinase, and lipid infiltration of liver and muscle. At the same time, it is a rare cause of Sudden Infant Death Syndrome (SIDS) or unexplained death in the neonatal period [1-4]. We report a patient with VLCADD whose parents were investigated for infanticide because her three previous siblings had suddenly died after normal deliveries.

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

    PubMed

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

    2014-02-01

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

  3. Adult-onset multiple acyl CoA dehydrogenation deficiency associated with an abnormal isoenzyme pattern of serum lactate dehydrogenase.

    PubMed

    Sugai, Fuminobu; Baba, Kousuke; Toyooka, Keiko; Liang, Wen-Chen; Nishino, Ichizo; Yamadera, Misaki; Sumi, Hisae; Fujimura, Harutoshi; Nishikawa, Yoshiro

    2012-02-01

    We report a case of a 37 year-old male with multiple acyl-CoA dehydrogenation deficiency (MADD). The patient had suffered from exercise intolerance in his hip and thigh muscles for one year. Then, restriction of carbohydrates for a diet made his symptoms rapidly deteriorate. Blood test revealed compound heterozygosity for two novel missense mutations in the electron transfer flavoprotein dehydrogenase gene (ETFDH), and an abnormal LDH isoenzyme pattern: LDH-1 (60.0%) and LDH-2 (26.0%) predominated with abnormally elevated LDH-1/LDH-2 ratio (2.3), compared with muscle-derived LDH-5 (4.0%). Oral riboflavin treatment significantly improved his exercise intolerance and the LDH profile: LDH-1 (34.4%), LDH-2 (34.9%), LDH-5 (6.9%) and LDH-1/LDH-2 ratio (1.0). The abnormal LDH isoenzyme pattern may be one feature of adult-onset MADD selectively affecting type I muscle fibers with relatively high LDH-1 content. PMID:21907580

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

    PubMed Central

    2016-01-01

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

  5. Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

    PubMed Central

    Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

    2014-01-01

    Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

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

  8. Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.

    PubMed Central

    Yan, R T; Chen, J S

    1990-01-01

    Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay. Images PMID:2275527

  9. Purification and characterization of a novel pumpkin short-chain acyl-coenzyme A oxidase with structural similarity to acyl-coenzyme A dehydrogenases.

    PubMed

    De Bellis, L; Gonzali, S; Alpi, A; Hayashi, H; Hayashi, M; Nishimura, M

    2000-05-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

    PubMed Central

    Ruprecht, Amanda; Maddox, Jaymie; Stirling, Alexander J.; Visaggio, Nicole

    2015-01-01

    ABSTRACT The acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of Mycobacterium tuberculosis and Rhodococcus jostii RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the casC gene in R. jostii RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs. IMPORTANCE We report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from M. tuberculosis and Rhodococcus jostii RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of

  12. Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate

    PubMed Central

    Kormanik, Kaitlyn; Kang, Heejung; Cuebas, Dean; Vockley, Jerry; Mohsen, Al-Walid

    2012-01-01

    Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (KD app) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the Km for phenylbutyryl-CoA were 0.2 mM−1· sec−1 and 5.3 μM compared to 4.0 mM−1· sec−1 and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism. PMID:23141465

  13. Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds.

    PubMed

    Larson, Tony R; Edgell, Teresa; Byrne, James; Dehesh, Katayoon; Graham, Ian A

    2002-11-01

    Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.

  14. Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase.

    PubMed

    Satoh, Yasuharu; Murakami, Fumikazu; Tajima, Kenji; Munekata, Masanobu

    2005-05-01

    We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3 HB-co-4 HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3 HB-co-4 HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures. PMID:16233824

  15. 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.

    PubMed

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  18. Genetics Home Reference: short-chain acyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... Download PDF Open All Close All Description Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a condition that prevents the body from converting certain fats into energy, especially during periods without food (fasting). Signs and symptoms of SCAD deficiency may ...

  19. A covalent adduct of MbtN, an acyl-ACP dehydrogenase from Mycobacterium tuberculosis, reveals an unusual acyl-binding pocket.

    PubMed

    Chai, Ai-Fen; Bulloch, Esther M M; Evans, Genevieve L; Lott, J Shaun; Baker, Edward N; Johnston, Jodie M

    2015-04-01

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated with Mtb virulence. Here, the crystal structure of an Mtb enzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14-21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusual cis double bond in the mycobactin acyl chain.

  20. Cardiac Hypertrophy in Mice with Long-Chain Acyl-CoA Dehydrogenase (LCAD) or Very Long-Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency

    PubMed Central

    Cox, Keith B.; Liu, Jian; Tian, Liqun; Barnes, Stephen; Yang, Qinglin; Wood, Philip A.

    2009-01-01

    Cardiac hypertrophy is a common finding in human patients with inborn errors of long-chain fatty acid oxidation. Mice with either very long-chain acyl-CoA dehydrogenase deficiency (VLCAD−/−) or long-chain acyl-CoA dehydrogenase deficiency (LCAD−/−) develop cardiac hypertrophy. Cardiac hypertrophy, initially measured using heart/body weight ratios, was manifested most severely in LCAD−/− male mice. VLCAD−/− mice, as a group, showed a mild increase in normalized cardiac mass (8.8% hypertrophy compared to all wild-type [WT] mice). In contrast, LCAD−/− mice as a group showed more severe cardiac hypertrophy (32.2% increase compared to all WT mice). Based on a clear male predilection, we investigated the role of dietary plant estrogenic compounds commonly found in mouse diets due to soy or alfalfa components providing natural phytoestrogens or isoflavones in cardioprotection of LCAD−/− mice. Male LCAD−/− mice fed an isoflavone-free test diet had more severe cardiac hypertrophy (58.1% hypertrophy compared to WT mice fed the same diet. There were no significant differences in the female groups fed any of the diets. Echocardiography measurement performed on male LCAD deficient mice fed a standard diet at ~3 months of age confirmed the substantial cardiac hypertrophy in these mice compared with WT controls. Left ventricular wall thickness of interventricular septum and posterior wall was remarkably increased in LCAD−/− mice compared with that of WT controls. Accordingly, the calculated LV mass after normalization to body weight was increased about 40% in the LCAD−/− mice compared with WT mice. In summary, we found that metabolic cardiomyopathy, expressed as hypertrophy, developed in mice due to either VLCAD deficiency or LCAD deficiency; however, LCAD deficiency was the most profound and appeared to be attenuated either by endogenous estrogen in females or phytoestrogens in the diet as isoflavones in males. PMID:19736549

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

    PubMed Central

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

    2016-01-01

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

  2. Strategies for Correcting Very Long Chain Acyl-CoA Dehydrogenase Deficiency*

    PubMed Central

    Tenopoulou, Margarita; Chen, Jie; Bastin, Jean; Bennett, Michael J.; Ischiropoulos, Harry; Doulias, Paschalis-Thomas

    2015-01-01

    Very long acyl-CoA dehydrogenase (VLCAD) deficiency is a genetic pediatric disorder presenting with a spectrum of phenotypes that remains for the most part untreatable. Here, we present a novel strategy for the correction of VLCAD deficiency by increasing mutant VLCAD enzymatic activity. Treatment of VLCAD-deficient fibroblasts, which express distinct mutant VLCAD protein and exhibit deficient fatty acid β-oxidation, with S-nitroso-N-acetylcysteine induced site-specific S-nitrosylation of VLCAD mutants at cysteine residue 237. Cysteine 237 S-nitrosylation was associated with an 8–17-fold increase in VLCAD-specific activity and concomitant correction of acylcarnitine profile and β-oxidation capacity, two hallmarks of the disorder. Overall, this study provides biochemical evidence for a potential therapeutic modality to correct β-oxidation deficiencies. PMID:25737446

  3. Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism.

    PubMed

    Liu, Xiaojun; Wu, Long; Deng, Guisheng; Chen, Gong; Li, Nan; Chu, Xiusheng; Li, Ding

    2013-04-01

    Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors. PMID:23474214

  4. Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid.

    PubMed

    Bunik, Victoria I; Raddatz, Günter; Wanders, Ronald J A; Reiser, Georg

    2006-06-12

    Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.

  5. Equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in 14 horses associated with ingestion of Maple leaves (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum).

    PubMed

    van der Kolk, J H; Wijnberg, I D; Westermann, C M; Dorland, L; de Sain-van der Velden, M G M; Kranenburg, L C; Duran, M; Dijkstra, J A; van der Lugt, J J; Wanders, R J A; Gruys, E

    2010-01-01

    This case-series describes fourteen horses suspected of equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) also known as atypical myopathy of which seven cases were confirmed biochemically with all horses having had access to leaves of the Maple tree (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum). Assessment of organic acids, glycine conjugates, and acylcarnitines in urine was regarded as gold standard in the biochemical diagnosis of equine acquired multiple acyl-CoA dehydrogenase deficiency.

  6. Acquired multiple acyl-CoA dehydrogenase deficiency and marked selenium deficiency causing severe rhabdomyolysis in a horse

    PubMed Central

    Gomez, Diego E.; Valberg, Stephanie J.; Magdesian, K. Gary; Hanna, Paul E.; Lofstedt, Jeanne

    2015-01-01

    This report describes a case of severe rhabdomyolysis in a pregnant mare associated with histopathologic and biochemical features of both selenium deficiency and acquired multiple acyl-CoA dehydrogenase deficiency (MADD) due to seasonal pasture myopathy (SPM). This case highlights the importance of assessing plasma selenium levels in horses with clinical signs of pasture myopathy as this deficiency may be a contributing or exacerbating factor. PMID:26538673

  7. Effects of short-chain acyl-CoA dehydrogenase on cardiomyocyte apoptosis.

    PubMed

    Zeng, Zhenhua; Huang, Qiuju; Shu, Zhaohui; Liu, Peiqing; Chen, Shaorui; Pan, Xuediao; Zang, Linquan; Zhou, Sigui

    2016-07-01

    Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid β-oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert-butyl hydroperoxide (tBHP)-induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down-regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA-1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl-2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP-activated protein kinase α (p-AMPKα) and Peroxisome proliferator-activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways. PMID:26989860

  8. Acyl-CoA dehydrogenase drives heat adaptation by sequestering fatty acids

    PubMed Central

    Ma, Dengke K.; Li, Zhijie; Lu, Alice Y.; Sun, Fang; Chen, Sidi; Rothe, Michael; Menzel, Ralph; Sun, Fei; Horvitz, H. Robert

    2015-01-01

    Summary Cells adapt to temperature shifts by adjusting levels of lipid desaturation and membrane fluidity. This fundamental process occurs in nearly all forms of life, but its mechanism in eukaryotes is unknown. We discovered that the evolutionarily conserved C. elegans gene acdh-11 (acyl-CoA-dehydrogenase, ACDH) facilitates heat adaptation by regulating the lipid desaturase FAT-7. Human ACDH deficiency causes the most common inherited disorders of fatty acid oxidation, with syndromes that are exacerbated by hyperthermia. Heat up-regulates acdh-11 expression to decrease fat-7 expression. We solved the high-resolution crystal structure of ACDH-11 and established the molecular basis of its selective and high-affinity binding to C11/C12-chain fatty acids. ACDH-11 sequesters C11/C12-chain fatty acids and prevents these fatty acids from activating nuclear hormone receptors and driving fat-7 expression. Thus, the ACDH-11 pathway drives heat adaptation by linking temperature shifts to regulation of lipid desaturase levels and membrane fluidity via an unprecedented mode of fatty acid signaling. PMID:25981666

  9. A Korean patient with glutaric aciduria type 1 with a novel mutation in the glutaryl CoA dehydrogenase gene.

    PubMed

    Kim, Hee Su; Yu, Hee Joon; Lee, Jeehun; Park, Hyung-Doo; Kim, Ji Hye; Shin, Hyung-Jin; Jin, Dong Kyu; Lee, Munhyang

    2014-01-01

    Mutations in the glutaryl-CoA dehydrogenase gene can result in Glutaric aciduria type 1(GA 1) by accumulation of glutaric acid, 3-hydroxyglutaric acid (3-OH-GA), and glutarylcarnitine (C5DC). GA 1 is characterized by macrocephaly, subdural hemorrhage (SDH), and dystonic movement disorder after acute encephalopathic crisis. We report a Korean patient with GA1 and a novel mutation. A 16-month-old boy presented with SDH, macrocephaly, and developmental delay. In the neurologic examination, the patient had mild axial hypotonia, but otherwise normal neurologic functions. The brain MRI showed large amounts of bilateral SDH and high signal intensity in both basal ganglia and thalamus. Metabolic screening tests detected highly elevated urinary GA levels but 3-OH-glutaric acid was normal. C5DC was 0.94 μM/L (reference range < 0.3 μM/L). The patient had compound heterozygous mutations of the GCDH gene: p.Arg257Gln (c.770G>A) and p.Cys308Arg (c.922T>C). p.Cys308Arg is a novel mutation; reports of p.Arg257Gln were also rare both in Caucasians and Asian populations. In summary, we hereby report one Korean patient with GA1 with clinical, biochemical, and radiologic characteristics confirmed by genetic analysis. PMID:24795062

  10. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  11. Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site.

    PubMed

    Bharathi, Sivakama S; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E; Rardin, Matthew J; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W; Hirschey, Matthew D; Goetzman, Eric S

    2013-11-22

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

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

    PubMed

    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

  13. New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.

    PubMed

    Camões, Fátima; Islinger, Markus; Guimarães, Sofia C; Kilaru, Sreedhar; Schuster, Martin; Godinho, Luis F; Steinberg, Gero; Schrader, Michael

    2015-01-01

    Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid β-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid β-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient Δpex3 mutant revealed the existence of parallel and complex, cooperative β-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid β-oxidation is discussed.

  14. Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin.

    PubMed

    Van Calcar, Sandra C; Baker, Mei W; Williams, Phillip; Jones, Susan A; Xiong, Blia; Thao, Mai Choua; Lee, Sheng; Yang, Mai Khou; Rice, Greg M; Rhead, William; Vockley, Jerry; Hoffman, Gary; Durkin, Maureen S

    2013-01-01

    Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of l-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10years of expanded newborn screening using MS/MS in Wisconsin (2001-2011), 97 infants had elevated C5 values (≥0.44μmol/L), of whom five were Caucasian infants confirmed to have IVA. Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n=1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentration ≥0.44μmol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8-2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4-24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut-off would need to

  15. Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin

    PubMed Central

    Van Calcar, Sandra C.; Baker, Mei W.; Williams, Phillip; Jones, Susan A.; Xiong, Blia; Thao, Mai Choua; Lee, Sheng; Yang, Mai Khou; Rice, Greg M.; Rhead, William; Vockley, Jerry; Hoffman, Gary; Durkin, Maureen S.

    2015-01-01

    Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of L-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1 165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10 years of expanded newborn screening using MS/MS in Wisconsin (2001–2011), 97 infants had elevated C5 values (≥0.44 μmol/L), of whom five were Caucasian infants confirmed to have IVA Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n = 1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentrations ≥0.44 μmol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8–2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4–24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut

  16. Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

    PubMed

    Diekman, E F; Visser, G; Schmitz, J P J; Nievelstein, R A J; de Sain-van der Velden, M; Wardrop, M; Van der Pol, W L; Houten, S M; van Riel, N A W; Takken, T; Jeneson, J A L

    2016-01-01

    Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD. PMID:26881790

  17. Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency

    PubMed Central

    Diekman, E. F.; Visser, G.; Schmitz, J. P. J.; Nievelstein, R. A. J.; de Sain-van der Velden, M.; Wardrop, M.; Van der Pol, W. L.; Houten, S. M.; van Riel, N. A. W.; Takken, T.; Jeneson, J. A. L.

    2016-01-01

    Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD. PMID:26881790

  18. Development and pathomechanisms of cardiomyopathy in very long-chain acyl-CoA dehydrogenase deficient (VLCAD(-/-)) mice.

    PubMed

    Tucci, Sara; Flögel, Ulrich; Hermann, Sven; Sturm, Marga; Schäfers, Michael; Spiekerkoetter, Ute

    2014-05-01

    Hypertrophic cardiomyopathy is a typical manifestation of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), the most common long-chain β-oxidation defects in humans; however in some patients cardiac function is fully compensated. Cardiomyopathy may also be reversed by supplementation of medium-chain triglycerides (MCT). We here characterize cardiac function of VLCAD-deficient (VLCAD(-/-)) mice over one year. Furthermore, we investigate the long-term effect of a continuous MCT diet on the cardiac phenotype. We assessed cardiac morphology and function in VLCAD(-/-) mice by in vivo MRI. Cardiac energetics were measured by (31)P-MRS and myocardial glucose uptake was quantified by positron-emission-tomography (PET). Metabolic adaptations were identified by the expression of genes regulating glucose and lipid metabolism using real-time-PCR. VLCAD(-/-) mice showed a progressive decrease in heart function over 12 months accompanied by a reduced phosphocreatine-to-ATP-ratio indicative of chronic energy deficiency. Long-term MCT supplementation aggravated the cardiac phenotype into dilated cardiomyopathy with features similar to diabetic heart disease. Cardiac energy production and function in mice with a β-oxidation defect cannot be maintained with age. Compensatory mechanisms are insufficient to preserve the cardiac energy state over time. However, energy deficiency by impaired β-oxidation and long-term MCT induce cardiomyopathy by different mechanisms. Cardiac MRI and MRS may be excellent tools to assess minor changes in cardiac function and energetics in patients with β-oxidation defects for preventive therapy.

  19. COAs: Behind the Masks.

    ERIC Educational Resources Information Center

    Birke, Szifra

    1993-01-01

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

  20. Development and pathomechanisms of cardiomyopathy in very long-chain acyl-CoA dehydrogenase deficient (VLCAD(-/-)) mice.

    PubMed

    Tucci, Sara; Flögel, Ulrich; Hermann, Sven; Sturm, Marga; Schäfers, Michael; Spiekerkoetter, Ute

    2014-05-01

    Hypertrophic cardiomyopathy is a typical manifestation of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), the most common long-chain β-oxidation defects in humans; however in some patients cardiac function is fully compensated. Cardiomyopathy may also be reversed by supplementation of medium-chain triglycerides (MCT). We here characterize cardiac function of VLCAD-deficient (VLCAD(-/-)) mice over one year. Furthermore, we investigate the long-term effect of a continuous MCT diet on the cardiac phenotype. We assessed cardiac morphology and function in VLCAD(-/-) mice by in vivo MRI. Cardiac energetics were measured by (31)P-MRS and myocardial glucose uptake was quantified by positron-emission-tomography (PET). Metabolic adaptations were identified by the expression of genes regulating glucose and lipid metabolism using real-time-PCR. VLCAD(-/-) mice showed a progressive decrease in heart function over 12 months accompanied by a reduced phosphocreatine-to-ATP-ratio indicative of chronic energy deficiency. Long-term MCT supplementation aggravated the cardiac phenotype into dilated cardiomyopathy with features similar to diabetic heart disease. Cardiac energy production and function in mice with a β-oxidation defect cannot be maintained with age. Compensatory mechanisms are insufficient to preserve the cardiac energy state over time. However, energy deficiency by impaired β-oxidation and long-term MCT induce cardiomyopathy by different mechanisms. Cardiac MRI and MRS may be excellent tools to assess minor changes in cardiac function and energetics in patients with β-oxidation defects for preventive therapy. PMID:24530811

  1. Complex changes in the liver mitochondrial proteome of short chain acyl-CoA dehydrogenase deficient mice.

    PubMed

    Wang, Wei; Mohsen, Al-Walid; Uechi, Guy; Schreiber, Emanuel; Balasubramani, Manimalha; Day, Billy; Michael Barmada, M; Vockley, Jerry

    2014-05-01

    Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an autosomal recessive inborn error of metabolism that leads to the impaired mitochondrial fatty acid β-oxidation of short chain fatty acids. It is heterogeneous in clinical presentation including asymptomatic in most patients identified by newborn screening. Multiple mutations have been identified in patients; however, neither clear genotype-phenotype relationships nor a good correlation between genotype and current biochemical markers for diagnosis has been identified. The definition and pathophysiology of this deficiency remain unclear. To better understand this disorder at a global level, quantitative alterations in the mitochondrial proteome in SCAD deficient mice were examined using a combined proteomics approach: two-dimensional gel difference electrophoresis (2DIGE) followed by protein identification with MALDI-TOF/TOF and iTRAQ labeling followed by nano-LC/MALDI-TOF/TOF. We found broad mitochondrial dysfunction in SCAD deficiency. Changes in the levels of multiple energy metabolism related proteins were identified indicating that a more complex mechanism for development of symptoms may exist. Affected pathways converge on disorders with neurologic symptoms, suggesting that even asymptomatic individuals with SCAD deficiency may be at risk to develop more severe disease. Our results also identified a pattern associated with hepatotoxicity implicated in mitochondrial dysfunction, fatty acid metabolism, decrease of depolarization of mitochondria and mitochondrial membranes, and swelling of mitochondria, demonstrating that SCAD deficiency relates more directly to mitochondrial dysfunction and alteration of fatty acid metabolism. We propose several candidate molecules that may serve as markers for recognition of clinical risk associated with this disorder.

  2. Diet-Sensitive Sources of Reactive Oxygen Species in Liver Mitochondria: Role of Very Long Chain Acyl-CoA Dehydrogenases

    PubMed Central

    Cardoso, Ariel R.; Kakimoto, Pâmela A. H. B.; Kowaltowski, Alicia J.

    2013-01-01

    High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS. PMID:24116206

  3. Newborn screening for medium-chain acyl-CoA dehydrogenase deficiency: regional experience and high incidence of carnitine deficiency

    PubMed Central

    2013-01-01

    Background Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited defect in the mitochondrial fatty acid oxidation pathway, resulting in significant morbidity and mortality in undiagnosed patients. Newborn screening (NBS) has considerably improved MCADD outcome, but the risk of complication remains in some patients. The aim of this study was to evaluate the relationship between genotype, biochemical parameters and clinical data at diagnosis and during follow-up, in order to optimize monitoring of these patients. Methods We carried out a multicenter study in southwest Europe, of MCADD patients detected by NBS. Evaluated NBS data included free carnitine (C0) and the acylcarnitines C8, C10, C10:1 together with C8/C2 and C8/C10 ratios, clinical presentation parameters and genotype, in 45 patients. Follow-up data included C0 levels, duration of carnitine supplementation and occurrence of metabolic crises. Results C8/C2 ratio and C8 were the most accurate biomarkers of MCADD in NBS. We found a high number of patients homozygous for the prevalent c.985A > G mutation (75%). Moreover, in these patients C8, C8/C10 and C8/C2 were higher than in patients with other genotypes, while median value of C0 was significantly lower (23 μmol/L vs 36 μmol/L). The average follow-up period was 43 months. To keep carnitine levels within the normal range, carnitine supplementation was required in 82% of patients, and for a longer period in patients homozygotes for the c.985A>G mutation than in patients with other genotypes (average 31 vs 18 months). Even with treatment, median C0 levels remained lower in homozygous patients than in those with other genotypes (14 μmol/L vs 22 μmol/L). Two patients died and another three suffered a metabolic crisis, all of whom were homozygous for the c.985 A>G mutation. Conclusions Our data show a direct association between homozygosity for c.985A>G and lower carnitine values at diagnosis, and a higher dose of carnitine

  4. Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: acyl-CoA profiles in short-chain fatty acid oxidation defects

    PubMed Central

    Palladino, Andrew A.; Chen, Jie; Kallish, Staci; Stanley, Charles A.; Bennett, Michael J.

    2013-01-01

    The primary accumulating metabolites in fatty acid oxidation defects are intramitochondrial acyl-CoAs. Typically, secondary metabolites such as acylcarnitines, acylglycines and dicarboxylic acids are measured to study these disorders. Methods have not been adapted for tissue acyl-CoA measurement in defects with primarily acyl-CoA accumulation. Our objective was to develop a method to measure fatty acyl-CoA species that are present in tissues of mice with fatty acid oxidation defects using flow-injection tandem mass spectrometry. Following the addition of internal standards of [13C2] acetyl-CoA, [13C8] octanoyl-CoA, and [C17] heptadecanoic CoA, acyl-CoA’s are extracted from tissue samples and are injected directly into the mass spectrometer. Data is acquired using a 506.9 neutral loss scan and multiple reaction-monitoring (MRM). This method can identify all long, medium and short-chain acyl-CoA species in wild type mouse liver including predicted 3-hydroxyacyl-CoA species. We validated the method using liver of the short-chain-acyl-CoA dehydrogenase (SCAD) knock-out mice. As expected, there is a significant increase in [C4] butyryl-CoA species in the SCAD −/− mouse liver compared to wild type. We then tested the assay in liver from the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficient mice to determine the profile of acyl-CoA accumulation in this less predictable model. There was more modest accumulation of medium chain species including 3-hydroxyacyl-CoA’s consistent with the known chain-length specificity of the SCHAD enzyme. PMID:23117082

  5. Shrinking the FadE proteome of Mycobacterium tuberculosis: insights into cholesterol metabolism through identification of an α2β2 heterotetrameric acyl coenzyme A dehydrogenase family.

    PubMed

    Wipperman, Matthew F; Yang, Meng; Thomas, Suzanne T; Sampson, Nicole S

    2013-10-01

    The ability of the pathogen Mycobacterium tuberculosis to metabolize steroids like cholesterol and the roles that these compounds play in the virulence and pathogenesis of this organism are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. FadE26-FadE27 catalyzes the dehydrogenation of 3β-hydroxy-chol-5-en-24-oyl-CoA, an analog of the 5-carbon side chain cholesterol degradation intermediate. Genes encoding the α2β2 heterotetrameric ACAD structures are present in multiple regions of the M. tuberculosis genome, and subsets of these genes are regulated by four different transcriptional repressors or activators: KstR1 (also known as KstR), KstR2, Mce3R, and SigE. Homologous ACAD gene pairs are found in other Actinobacteria, as well as Proteobacteria. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four subpathways of cholesterol metabolism.

  6. SIRT3 and SIRT5 regulate the enzyme activity and cardiolipin binding of very long-chain acyl-CoA dehydrogenase.

    PubMed

    Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Uppala, Radha; Verdin, Eric; Gibson, Bradford W; Goetzman, Eric S

    2015-01-01

    SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane. PMID:25811481

  7. Shrinking the FadE Proteome of Mycobacterium tuberculosis: Insights into Cholesterol Metabolism through Identification of an α2β2 Heterotetrameric Acyl Coenzyme A Dehydrogenase Family

    PubMed Central

    Wipperman, Matthew F.; Yang, Meng; Thomas, Suzanne T.

    2013-01-01

    The ability of the pathogen Mycobacterium tuberculosis to metabolize steroids like cholesterol and the roles that these compounds play in the virulence and pathogenesis of this organism are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. FadE26-FadE27 catalyzes the dehydrogenation of 3β-hydroxy-chol-5-en-24-oyl-CoA, an analog of the 5-carbon side chain cholesterol degradation intermediate. Genes encoding the α2β2 heterotetrameric ACAD structures are present in multiple regions of the M. tuberculosis genome, and subsets of these genes are regulated by four different transcriptional repressors or activators: KstR1 (also known as KstR), KstR2, Mce3R, and SigE. Homologous ACAD gene pairs are found in other Actinobacteria, as well as Proteobacteria. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four subpathways of cholesterol metabolism. PMID:23836861

  8. Evolution of Acyl-Substrate Recognition by a Family of Acyl-Homoserine Lactone Synthases

    PubMed Central

    Christensen, Quin H.; Brecht, Ryan M.; Dudekula, Dastagiri; Greenberg, E. Peter; Nagarajan, Rajesh

    2014-01-01

    Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family. PMID:25401334

  9. Chlorsulfuron modifies biosynthesis of acyl Acid substituents of sucrose esters secreted by tobacco trichomes.

    PubMed

    Kandra, L; Wagner, G J

    1990-11-01

    Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID:16667871

  10. Chlorsulfuron Modifies Biosynthesis of Acyl Acid Substituents of Sucrose Esters Secreted by Tobacco Trichomes

    PubMed Central

    Kandra, Lili; Wagner, George J.

    1990-01-01

    Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID:16667871

  11. Anesthetic agents in patients with very long-chain acyl-coenzyme A dehydrogenase deficiency: a literature review.

    PubMed

    Redshaw, Charlotte; Stewart, Catherine

    2014-11-01

    Very long-chain acyl-coenzyme A dehydrongenase deficiency (VLCADD) is a rare disorder of fatty acid metabolism that renders sufferers susceptible to hypoglycemia, liver failure, cardiomyopathy, and rhabdomyolysis. The literature about the management of these patients is hugely conflicting, suggesting that both propofol and volatile anesthesia should be avoided. We have reviewed the literature and have concluded that the source papers do not support the statements that volatile anesthetic agents are unsafe. The reports on rhabdomyolysis secondary to anesthesia appear to be due to inadequate supply of carbohydrate not volatile agents. Catabolism must be avoided with minimal fasting, glucose infusions based on age and weight, and attenuation of emotional and physical stress. General anesthesia appears to be protective of stress-induced catabolism and may offer benefits in children and anxious patients over regional anesthesia. Propofol has not been demonstrated to be harmful in VLCADD but is presented in an emulsion containing very long-chain fatty acids which can cause organ lipidosis and itself can inhibit mitochondrial fatty acid metabolism. It is therefore not recommended. Suxamethonium-induced myalgia may mimic symptoms of rhabdomyolysis and cause raised CK therefore should be avoided. Opioids, NSAIDS, regional anesthesia, and local anesthetic techniques have all been used without complication. PMID:25069536

  12. Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency.

    PubMed

    Kim, Seok-Hyung; Scott, Sarah A; Bennett, Michael J; Carson, Robert P; Fessel, Joshua; Brown, H Alex; Ess, Kevin C

    2013-06-01

    Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463) ) that has an inactivating mutation in the etfa gene. dxa(vu463) recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463) zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463) zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

  13. Multi-organ Abnormalities and mTORC1 Activation in Zebrafish Model of Multiple Acyl-CoA Dehydrogenase Deficiency

    PubMed Central

    Kim, Seok-Hyung; Scott, Sarah A.; Bennett, Michael J.; Carson, Robert P.; Fessel, Joshua; Brown, H. Alex; Ess, Kevin C.

    2013-01-01

    Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxavu463) that has an inactivating mutation in the etfa gene. dxavu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxavu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxavu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity. PMID:23785301

  14. Multiple Acyl-CoA Dehydrogenation Deficiency (Glutaric Aciduria Type II) with a Novel Mutation of Electron Transfer Flavoprotein-Dehydrogenase in a Cat.

    PubMed

    Wakitani, Shoichi; Torisu, Shidow; Yoshino, Taiki; Hattanda, Kazuhisa; Yamato, Osamu; Tasaki, Ryuji; Fujita, Haruo; Nishino, Koichiro

    2014-01-01

    Multiple acyl-CoA dehydrogenation deficiency (MADD; also known as glutaric aciduria type II) is a human autosomal recessive disease classified as one of the mitochondrial fatty-acid oxidation disorders. MADD is caused by a defect in the electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH) molecule, but as yet, inherited MADD has not been reported in animals. Here we present the first report of MADD in a cat. The affected animal presented with symptoms characteristic of MADD including hypoglycemia, hyperammonemia, vomiting, diagnostic organic aciduria, and accumulation of medium- and long-chain fatty acids in plasma. Treatment with riboflavin and L-carnitine ameliorated the symptoms. To detect the gene mutation responsible for MADD in this case, we determined the complete cDNA sequences of feline ETFα, ETFβ, and ETFDH. Finally, we identified the feline patient-specific mutation, c.692T>G (p.F231C) in ETFDH. The affected animal only carries mutant alleles of ETFDH. p.F231 in feline ETFDH is completely conserved in eukaryotes, and is located on the apical surface of ETFDH, receiving electrons from ETF. This study thus identified the mutation strongly suspected to have been the cause of MADD in this cat. PMID:24142280

  15. Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

    PubMed Central

    Iolascon, A; Parrella, T; Perrotta, S; Guardamagna, O; Coates, P M; Sartore, M; Surrey, S; Fortina, P

    1994-01-01

    Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders. Images PMID:7966191

  16. Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

    PubMed Central

    Weichler, Maria-Teresa; Kurteva-Yaneva, Nadya; Przybylski, Denise; Schuster, Judith; Müller, Roland H.; Harms, Hauke

    2015-01-01

    The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502–15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of

  17. Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA.

    PubMed

    Weichler, Maria-Teresa; Kurteva-Yaneva, Nadya; Przybylski, Denise; Schuster, Judith; Müller, Roland H; Harms, Hauke; Rohwerder, Thore

    2015-07-01

    The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of

  18. Sexual dimorphism of lipid metabolism in very long-chain acyl-CoA dehydrogenase deficient (VLCAD-/-) mice in response to medium-chain triglycerides (MCT).

    PubMed

    Tucci, Sara; Flögel, Ulrich; Spiekerkoetter, Ute

    2015-07-01

    Medium-chain triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders. Previously it was shown that long-term MCT supplementation strongly affects lipid metabolism in mice. We here investigate sex-specific effects in mice with very-long-chain-acyl-CoA dehydrogenase (VLCAD) deficiency in response to a long-term MCT modified diet. We quantified blood lipids, acylcarnitines, glucose, insulin and free fatty acids, as well as tissue triglycerides in the liver and skeletal muscle under a control and an MCT diet over 1 year. In addition, visceral and hepatic fat content and muscular intramyocellular lipids (IMCL) were assessed by in vivo(1)H magnetic resonance spectroscopy (MRS) techniques. The long-term application of an MCT diet induced a marked alteration of glucose homeostasis. However, only VLCAD-/- female mice developed a severe metabolic syndrome characterized by marked insulin resistance, dyslipidemia, severe hepatic and visceral steatosis, whereas VLCAD-/- males seemed to be protected and only presented with milder insulin resistance. Moreover, the highly saturated MCT diet is associated with a decreased hepatic stearoyl-CoA desaturase 1 (SCD1) activity in females aggravating the harmful effects of a saturated MCT diet. Long-term MCT supplementation deeply affects lipid metabolism in a sexual dimorphic manner resulting in a severe metabolic syndrome only in female mice. These findings are striking since the first signs of insulin resistance already occur in female VLCAD-/- mice during their reproductive period. How these metabolic adaptations are finally regulated needs to be determined. More important, the relevance of these findings for humans under these dietary modifications needs to be investigated. PMID:25887160

  19. Three RFLPs defining a haplotype associated with the common mutation in a human medium-chain acyl-CoA dehydrogenase (MCAD) deficiency occur in Alu repeats

    SciTech Connect

    Zhifang Zhang; Yeqing Zhou; Kelly, D.P.; Strauss, A.W. St. Louis Children's Hospital, MO ); Kolvraa, S.; Gregersen, N. )

    1993-06-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of fatty-acid oxidation and may cause sudden infant death. Previous studies revealed that (i) homozygosity for an A-to-G mutation at nucleotide 985 of the mRNA coding region (A985G) is an extremely common cause of MCAD deficiency and (ii) MCAD deficiency is strongly associated with a particular haplotype for RFLPs for BanII, PstI, and TaqI. TaqI allele 2 is always associated with the A985G mutation in human MCAD deficiency. In this study, the authors have delineated the molecular basis of the RFLPs for PstI, BamHI, and TaqI in the human MCAD gene. Their results prove that the three RFLPs are caused by point mutations in the 8 kb of DNA encompassing exons 8--10 of the human MCAD gene. The TaqI polymorphism is caused by a C-to-A substitution 392 bp upstream of the exon 8, and the PstI and BamHI polymorphisms are due to T-to-C and G-to-A substitutions, respectively, which are 727 and 931 bp downstream of exon 10, respectively. All three RFLPs lie within Alu repetitive sequences. Comparison of intronic sequences immediately following exon 10 from two normal individuals with different haplotypes showed that this region contains densely packed Alu repeats and is highly polymorphic. The results are consistent both with a founder effect as the cause of the high prevalence of a single (A985G) mutation in MCAD deficiency and with its association with a particular haplotype for these intragenic RFLPs. 27 refs., 6 figs., 1 tab.

  20. Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

    PubMed

    Olsen, Rikke K J; Koňaříková, Eliška; Giancaspero, Teresa A; Mosegaard, Signe; Boczonadi, Veronika; Mataković, Lavinija; Veauville-Merllié, Alice; Terrile, Caterina; Schwarzmayr, Thomas; Haack, Tobias B; Auranen, Mari; Leone, Piero; Galluccio, Michele; Imbard, Apolline; Gutierrez-Rios, Purificacion; Palmfeldt, Johan; Graf, Elisabeth; Vianey-Saban, Christine; Oppenheim, Marcus; Schiff, Manuel; Pichard, Samia; Rigal, Odile; Pyle, Angela; Chinnery, Patrick F; Konstantopoulou, Vassiliki; Möslinger, Dorothea; Feichtinger, René G; Talim, Beril; Topaloglu, Haluk; Coskun, Turgay; Gucer, Safak; Botta, Annalisa; Pegoraro, Elena; Malena, Adriana; Vergani, Lodovica; Mazzà, Daniela; Zollino, Marcella; Ghezzi, Daniele; Acquaviva, Cecile; Tyni, Tiina; Boneh, Avihu; Meitinger, Thomas; Strom, Tim M; Gregersen, Niels; Mayr, Johannes A; Horvath, Rita; Barile, Maria; Prokisch, Holger

    2016-06-01

    Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis. PMID:27259049

  1. Sexual dimorphism of lipid metabolism in very long-chain acyl-CoA dehydrogenase deficient (VLCAD-/-) mice in response to medium-chain triglycerides (MCT).

    PubMed

    Tucci, Sara; Flögel, Ulrich; Spiekerkoetter, Ute

    2015-07-01

    Medium-chain triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders. Previously it was shown that long-term MCT supplementation strongly affects lipid metabolism in mice. We here investigate sex-specific effects in mice with very-long-chain-acyl-CoA dehydrogenase (VLCAD) deficiency in response to a long-term MCT modified diet. We quantified blood lipids, acylcarnitines, glucose, insulin and free fatty acids, as well as tissue triglycerides in the liver and skeletal muscle under a control and an MCT diet over 1 year. In addition, visceral and hepatic fat content and muscular intramyocellular lipids (IMCL) were assessed by in vivo(1)H magnetic resonance spectroscopy (MRS) techniques. The long-term application of an MCT diet induced a marked alteration of glucose homeostasis. However, only VLCAD-/- female mice developed a severe metabolic syndrome characterized by marked insulin resistance, dyslipidemia, severe hepatic and visceral steatosis, whereas VLCAD-/- males seemed to be protected and only presented with milder insulin resistance. Moreover, the highly saturated MCT diet is associated with a decreased hepatic stearoyl-CoA desaturase 1 (SCD1) activity in females aggravating the harmful effects of a saturated MCT diet. Long-term MCT supplementation deeply affects lipid metabolism in a sexual dimorphic manner resulting in a severe metabolic syndrome only in female mice. These findings are striking since the first signs of insulin resistance already occur in female VLCAD-/- mice during their reproductive period. How these metabolic adaptations are finally regulated needs to be determined. More important, the relevance of these findings for humans under these dietary modifications needs to be investigated.

  2. A Historical Cohort Study on the Efficacy of Glucocorticoids and Riboflavin Among Patients with Late-onset Multiple Acyl-CoA Dehydrogenase Deficiency

    PubMed Central

    Liu, Xin-Yi; Wang, Zhi-Qiang; Wang, Dan-Ni; Lin, Min-Ting; Wang, Ning

    2016-01-01

    Background: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common type of lipid storage myopathies in China. Most patients with late-onset MADD are well responsive to riboflavin. Up to now, these patients are often treated with glucocorticoids as the first-line drug because they are misdiagnosed as polymyositis without muscle biopsy or gene analysis. Although glucocorticoids seem to improve the fatty acid metabolism of late-onset MADD, the objective evaluation of their rationalization on this disorder and comparison with riboflavin treatment are unknown. Methods: We performed a historical cohort study on the efficacy of the two drugs among 45 patients with late-onset MADD, who were divided into glucocorticoids group and riboflavin group. Detailed clinical information of baseline and 1-month follow-up were collected. Results: After 1-month treatment, a dramatic improvement of muscle strength was found in riboflavin group (P < 0.05). There was no significant difference in muscle enzymes between the two groups. Significantly, the number of patients with full recovery in glucocorticoids group was less than the number in riboflavin group (P < 0.05). On the other hand, almost half of the patients in riboflavin group still presented high-level muscle enzymes and weak muscle strength after 1-month riboflavin treatment, meaning that 1-month treatment duration maybe insufficient and patients should keep on riboflavin supplement for a longer time. Conclusions: Our results provide credible evidences that the overall efficacy of riboflavin is superior to glucocorticoids, and a longer duration of riboflavin treatment is necessary for patients with late-onset MADD. PMID:26830983

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

    ERIC Educational Resources Information Center

    Thomas, Jacob

    2009-01-01

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

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

    SciTech Connect

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

    2006-03-24

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

  5. Role of CoA and acetyl-CoA in regulating cardiac fatty acid and glucose oxidation.

    PubMed

    Abo Alrob, Osama; Lopaschuk, Gary D

    2014-08-01

    CoA (coenzyme A) and its derivatives have a critical role in regulating cardiac energy metabolism. This includes a key role as a substrate and product in the energy metabolic pathways, as well as serving as an allosteric regulator of cardiac energy metabolism. In addition, the CoA ester malonyl-CoA has an important role in regulating fatty acid oxidation, secondary to inhibiting CPT (carnitine palmitoyltransferase) 1, a key enzyme involved in mitochondrial fatty acid uptake. Alterations in malonyl-CoA synthesis by ACC (acetyl-CoA carboxylase) and degradation by MCD (malonyl-CoA decarboxylase) are important contributors to the high cardiac fatty acid oxidation rates seen in ischaemic heart disease, heart failure, obesity and diabetes. Additional control of fatty acid oxidation may also occur at the level of acetyl-CoA involvement in acetylation of mitochondrial fatty acid β-oxidative enzymes. We find that acetylation of the fatty acid β-oxidative enzymes, LCAD (long-chain acyl-CoA dehydrogenase) and β-HAD (β-hydroxyacyl-CoA dehydrogenase) is associated with an increase in activity and fatty acid oxidation in heart from obese mice with heart failure. This is associated with decreased SIRT3 (sirtuin 3) activity, an important mitochondrial deacetylase. In support of this, cardiac SIRT3 deletion increases acetylation of LCAD and β-HAD, and increases cardiac fatty acid oxidation. Acetylation of MCD is also associated with increased activity, decreases malonyl-CoA levels and an increase in fatty acid oxidation. Combined, these data suggest that malonyl-CoA and acetyl-CoA have an important role in mediating the alterations in fatty acid oxidation seen in heart failure. PMID:25110000

  6. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase activities in plant mitochondria: interaction via a common coenzyme a pool.

    PubMed

    Dry, I B; Wiskich, J T

    1987-08-15

    2-Oxoglutarate (2-OG)-dependent O2 uptake by washed or purified turnip (Brassica rapa L.) and pea (Pisum sativum L. cv. Massey Gem) leaf mitochondria, in the presence of malonate, was inhibited between 65 and 90% by micromolar levels of pyruvate. The inhibition was not observed in the absence of malonate and was reversed by alpha-cyano-4-hydroxycinnamic acid. The inhibition was also reversed by oxaloacetate or by malate, but not by any other tricarboxylic acid cycle intermediates. The stimulation of O2 uptake by oxaloacetate was half maximal at 8-9 microM and was transient, indicating its action was not mediated through the complete metabolic removal of pyruvate. Pyruvate had not effect on 2-OG oxidation under conditions in which pyruvate dehydrogenase was not active, indicating that pyruvate metabolism, rather than pyruvate itself, was responsible for producing the inhibition of 2-OG oxidation. Similar results were obtained with detergent-treated mitochondrial extracts with the exception that the inhibition of 2-OG oxidation by pyruvate could also be reversed by coenzyme A. The results suggest that pyruvate inhibits 2-oxoglutarate oxidation, in intact plant mitochondria, by sequestering intramitochondrial CoA as acetyl-CoA and, in the absence of citrate synthase activity, reduces the amount of free coenzyme A available for 2-oxoglutarate dehydrogenase. These results indicate that pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase share a common CoA pool within plant mitochondria and that the turnover of the acyl-CoA product of one enzyme will dramatically influence the activity of the other.

  7. Recent NASA Dryden COA Experience

    NASA Technical Reports Server (NTRS)

    Cobleigh, Brent

    2008-01-01

    This viewgraph presentation concerns the experience that Dryden has had with Certificate of Authorization (COA) in reference to unmanned aerial systems (UAS). It reviews recent Certificate of Authorization UAS's i.e., 2005 Altair NOAA Mission, 2006 Altair Western States Fire Mission, and 2007 Ikhana. The priorities for the safety process is reviewed, as are typical UAS hazards. Slides also review the common COA provisions, best practices and lessons learned, the 2005 NOAA/NASA Science Demonstration Flights and the use of the UAS systems during fire emergencies.

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

    SciTech Connect

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

    2005-01-01

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

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

    PubMed

    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

  10. [Phosphoprotein phosphatase nonspecifically hydrolyzes CoA].

    PubMed

    Reziapkin, V I; Moiseenok, A G

    1988-01-01

    CoA hydrolysis was studied by a homogenous phosphoprotein phosphatase (EC 3.1 3.16) preparation from bovine spleen nuclei at pH 5.8. Phosphoprotein phosphatase catalyzed hydrolysis of the CoA 3'-phosphoester bond to form dephospho-CoA and Pi. The Km value for phosphoprotein phosphatase with CoA as substrate was 3.7 mM, the specific activity - 0.26 mmol Pi.min-1.mg-1. Phosphoprotein phosphatase did not essentially catalyze the calcium pantothenate hydrolysis (not more than 2% as compared with the CoA hydrolysis rate). PMID:2849829

  11. Proteomics and gene expression analyses of mitochondria from squalene-treated apoE-deficient mice identify short-chain specific acyl-CoA dehydrogenase changes associated with fatty liver amelioration.

    PubMed

    Ramírez-Torres, Adela; Barceló-Batllori, Sílvia; Fernández-Vizarra, Erika; Navarro, María A; Arnal, Carmen; Guillén, Natalia; Acín, Sergio; Osada, Jesús

    2012-05-17

    Squalene, a hydrocarbon involved in cholesterol biosynthesis, is an abundant component in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE knock-out mice. To study the effect of squalene on mitochondrial proteins in fatty liver, 1 g/kg/day of this isoprenoid was administered to those mice. After 10 weeks, hepatic fat was assessed and protein extracts from mitochondria enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by MS analysis. Squalene administration modified the expression of eighteen proteins involved in different metabolic processes, 12 associated with hepatic fat content. Methionine adenosyltransferase I alpha (Mat1a) and short-chain specific acyl-CoA dehydrogenase (Acads) showed significant increased and decreased transcripts, respectively, consistent with their protein changes. These mRNAs were also studied in wild-type mice receiving squalene, where Mat1a was found increased and Acads decreased. However, this mRNA was significantly increased in the absence of apolipoprotein E. These results suggest that squalene action may be executed through a complex regulation of mitochondrial protein expression, including changes in Mat1a and Acads levels. Indeed, Mat1a is a target of squalene administration while Acads reflects the anti-steatotic properties of squalene.

  12. A Genetically Amenable Platensimycin- and Platencin- Overproducer as a Platform for Biosynthetic Explorations: a Showcase of PtmO4, a Long-Chain Acyl-CoA Dehydrogenase

    PubMed Central

    Rudolf, Jeffrey D.; Dong, Liao-Bin; Huang, Tingting; Shen, Ben

    2015-01-01

    Platensimycin (PTM) and platencin (PTN) are members of a new class of promising drug leads that target bacterial and mammalian fatty acid synthases. We previously cloned and sequenced the PTM and PTN gene clusters, discovered six additional PTM-PTN dual producing strains, and demonstrated the dramatic overproduction of PTM and PTN by inactivating the pathway-specific regulators ptmR1 or ptnR1 in four different strains. Our ability to utilize these PTM-PTN dual overproducing strains was limited by their lack of genetic amenability. Here we report the construction of Streptomyces platensis SB12029, a genetically amenable, in-frame ΔptmR1 dual PTM-PTN overproducing strain. To highlight the potential of this strain for future PTM and PTN biosynthetic studies, we created the ΔptmR1 ΔptmO4 double mutant S. platensis SB12030. Fourteen PTM and PTN congeners, ten of which were new, were isolated from SB12030, shedding new insights into PTM and PTN biosynthesis. PtmO4, a long-chain acyl-CoA dehydrogenase, is strongly implicated to catalyze β-oxidation of the diterpenoid intermediates in to the PTM and PTN scaffolds. SB12029 sets the stage for future biosynthetic and bioengineering studies of the PTM and PTN family of natural products. PMID:26055255

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

    SciTech Connect

    Steffen, C.A.; Smith, C.M.

    1987-05-01

    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 Bt/sub 2/ cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 ..mu..M) + 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 ..mu..m/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 (/sup 14/C)-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.

  14. De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

    An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing.

  15. De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

    An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing. PMID:26284828

  16. Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase.

    PubMed

    Pellett, J D; Becker, D F; Saenger, A K; Fuchs, J A; Stankovich, M T

    2001-06-26

    The effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme A dehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160 and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemical properties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engage in favorable pi-sigma interactions with the isoalloxazine ring of the flavin to help stabilize formation of the anionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine (F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (E(ox/hq)) to shift negative by 44-54 mV. The E(ox/hq) value was also found to decrease when aromatic residues containing electron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mV for the F160Y and F160W mutants, respectively, are attributed to increased pi-pi repulsive interactions between the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potential for the butyryl-CoA/crotonyl-CoA couple (E(BCoA/CCoA)) to shift 14 mV negative, stabilizing the oxidized product. Formation of product was found to be even more favorable in complexes with the F160Y and F160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.

  17. A novel 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis strain DPN7T acting as a key enzyme during catabolism of 3,3'-dithiodipropionic acid is a member of the acyl-CoA dehydrogenase superfamily.

    PubMed

    Schürmann, Marc; Deters, Anika; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2013-04-01

    3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7(T). During investigation of a Tn5::mob-induced mutant defective in growth on 3,3'-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO3(2-)). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min(-1) mg(-1), an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s(-1) mM(-1) for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for the

  18. A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

    PubMed Central

    Schürmann, Marc; Deters, Anika; Wübbeler, Jan Hendrik

    2013-01-01

    3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7T. During investigation of a Tn5::mob-induced mutant defective in growth on 3,3′-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO32−). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min−1 mg−1, an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s−1 mM−1 for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for

  19. Aberrant protein acylation is a common observation in inborn errors of acyl-CoA metabolism.

    PubMed

    Pougovkina, Olga; Te Brinke, Heleen; Wanders, Ronald J A; Houten, Sander M; de Boer, Vincent C J

    2014-09-01

    Inherited disorders of acyl-CoA metabolism, such as defects in amino acid metabolism and fatty acid oxidation can present with severe clinical symptoms either neonatally or later in life, but the pathophysiological mechanisms are often incompletely understood. We now report the discovery of a novel biochemical mechanism that could contribute to the pathophysiology of these disorders. We identified increased protein lysine butyrylation in short-chain acyl-CoA dehydrogenase (SCAD) deficient mice as a result of the accumulation of butyryl-CoA. Similarly, in SCAD deficient fibroblasts, lysine butyrylation was increased. Furthermore, malonyl-CoA decarboxylase (MCD) deficient patient cells had increased levels of malonylated lysines and propionyl-CoA carboxylase (PCC) deficient patient cells had increased propionylation of lysines. Since lysine acylation can greatly impact protein function, aberrant lysine acylation in inherited disorders associated with acyl-CoA accumulation may well play a role in their disease pathophysiology. PMID:24531926

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

    DOEpatents

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

    2009-04-28

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

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

    DOEpatents

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

    2004-07-20

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

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

    DOEpatents

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

    2005-09-13

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

  3. Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

    PubMed

    Miklaszewska, Magdalena; Banaś, Antoni

    2016-08-01

    Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols.

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

    ERIC Educational Resources Information Center

    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…

  5. Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes[S

    PubMed Central

    Soupene, Eric; Kuypers, Frans A.

    2015-01-01

    The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes. PMID:26290611

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

    SciTech Connect

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

    2011-12-31

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

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

    SciTech Connect

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

    2011-02-18

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

  8. Structural basis for a bispecific NADP+ and CoA binding site in an archaeal malonyl-coenzyme A reductase.

    PubMed

    Demmer, Ulrike; Warkentin, Eberhard; Srivastava, Ankita; Kockelkorn, Daniel; Pötter, Markus; Marx, Achim; Fuchs, Georg; Ermler, Ulrich

    2013-03-01

    Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP(+) and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP(+) and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3'- and 2'-ribose phosphate group of CoA and NADP(+), respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP(+). Evolution from an NADP(+) to a bispecific NADP(+) and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP(+) binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.

  9. Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

    PubMed

    Jasieniecka-Gazarkiewicz, Katarzyna; Demski, Kamil; Lager, Ida; Stymne, Sten; Banaś, Antoni

    2016-01-01

    Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme. PMID:26643989

  10. Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.

    PubMed

    Thornburg, Chelsea K; Wortas-Strom, Susan; Nosrati, Meisam; Geiger, James H; Walker, Kevin D

    2015-10-13

    A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of

  11. Oxidative acylation using thioacids

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.

    1997-01-01

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

  12. Genetics Home Reference: succinyl-CoA:3-ketoacid CoA transferase deficiency

    MedlinePlus

    ... CoA:3-ketoacid CoA transferase deficiency succinyl-CoA:3-ketoacid CoA transferase deficiency Enable Javascript to view ... PDF Open All Close All Description Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inherited ...

  13. Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells*s⃞

    PubMed Central

    Soupene, Eric; Serikov, Vladimir; Kuypers, Frans A.

    2008-01-01

    Human acyl-coenzyme A binding domain-containing member 6 (ACBD6) is a modular protein that carries an acyl-CoA binding domain at its N terminus and two ankyrin motifs at its C terminus. ACBD6 binds long-chain acyl-CoAs with a strong preference for unsaturated, C18:1-CoA and C20:4-CoA, over saturated, C16:0-CoA, acyl species. Deletion of the C terminus, which is not conserved among the members of this family, did not affect the binding capacity or the substrate specificity of the protein. ACBD6 is not a ubiquitous protein, and its expression is restricted to tissues and progenitor cells with functions in blood and vessel development. ACBD6 was detected in bone marrow, spleen, placenta, cord blood, circulating CD34+ progenitors, and embryonic-like stem cells derived from placenta. In placenta, the protein was only detected in CD34+ progenitor cells present in blood and in CD31+ endothelial cells surrounding the blood vessels. These cells were also positive for the marker CD133, and they probably constitute hemangiogenic stem cells, precursors of both blood and vessels. We propose that human ACBD6 represents a cellular marker for primitive progenitor cells with functions in hematopoiesis and vascular endothelium development. PMID:18268358

  14. The presence of acyl-CoA hydrolase in rat brown-adipose-tissue peroxisomes.

    PubMed

    Alexson, S E; Osmundsen, H; Berge, R K

    1989-08-15

    The subcellular distribution of acyl-CoA hydrolase was studied in rat brown adipose tissue, with special emphasis on possible peroxisomal localization. Subcellular fractionation by sucrose-density-gradient centrifugation, followed by measurement of short-chain (propionyl-CoA) acyl-CoA hydrolase in the presence of NADH, resulted in two peaks of activity in the gradient: one peak corresponded to the distribution of cytochrome oxidase (mitochondrial marker enzyme), and another peak of activity coincided with the peroxisomal marker enzyme catalase. The distribution of the NADH-inhibited short-chain hydrolase activity fully resembled that of cytochrome oxidase. The substrate-specificity curve of the peroxisomal acyl-CoA hydrolase activity indicated the presence of a single enzyme exhibiting a broad substrate specificity, with maximal activity towards fatty acids with chain lengths of 3-12 carbon atoms. The mitochondrial acyl-CoA hydrolase substrate specificity, in contrast, indicated the presence of at least two acyl-CoA hydrolases (of short- and medium-chain-length specificity). The peroxisomal acyl-CoA hydrolase activity was inhibited by CoA at low (microM) concentrations and by ATP at high concentrations (greater than 0.8 mM). In contrast with the mitochondrial short-chain hydrolase, the peroxisomal acyl-CoA hydrolase activity was not inhibited by NADH. PMID:2573347

  15. Fatty acylation of proteins: The long and the short of it.

    PubMed

    Resh, Marilyn D

    2016-07-01

    Long, short and medium chain fatty acids are covalently attached to hundreds of proteins. Each fatty acid confers distinct biochemical properties, enabling fatty acylation to regulate intracellular trafficking, subcellular localization, protein-protein and protein-lipid interactions. Myristate and palmitate represent the most common fatty acid modifying groups. New insights into how fatty acylation reactions are catalyzed, and how fatty acylation regulates protein structure and function continue to emerge. Myristate is typically linked to an N-terminal glycine, but recent studies reveal that lysines can also be myristoylated. Enzymes that remove N-terminal myristoyl-glycine or myristate from lysines have now been identified. DHHC proteins catalyze S-palmitoylation, but the mechanisms that regulate substrate recognition by individual DHHC family members remain to be determined. New studies continue to reveal thioesterases that remove palmitate from S-acylated proteins. Another area of rapid expansion is fatty acylation of the secreted proteins hedgehog, Wnt and Ghrelin, by Hhat, Porcupine and GOAT, respectively. Understanding how these membrane bound O-acyl transferases recognize their protein and fatty acyl CoA substrates is an active area of investigation, and is punctuated by the finding that these enzymes are potential drug targets in human diseases. PMID:27233110

  16. Discovery of amide (peptide) bond synthetic activity in Acyl-CoA synthetase.

    PubMed

    Abe, Tomoko; Hashimoto, Yoshiteru; Hosaka, Hideaki; Tomita-Yokotani, Kaori; Kobayashi, Michihiko

    2008-04-25

    Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.

  17. A Liver-Specific Defect of Acyl-CoA Degradation Produces Hyperammonemia, Hypoglycemia and a Distinct Hepatic Acyl-CoA Pattern

    PubMed Central

    Gauthier, Nicolas; Wu, Jiang Wei; Wang, Shu Pei; Allard, Pierre; Mamer, Orval A.; Sweetman, Lawrence; Moser, Ann B.; Kratz, Lisa; Alvarez, Fernando; Robitaille, Yves; Lépine, François; Mitchell, Grant A.

    2013-01-01

    Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-14C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication. PMID:23861731

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

    SciTech Connect

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

    2008-04-02

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

  19. Coagglutination (COA) test for the rapid diagnosis of cryptococcal meningitis.

    PubMed

    Koshi, G; Anandi, V; Shastry, J C; Cheriyan, A M; Abraham, J

    1989-07-01

    Cryptococcus coagglutination (COA) test reagent was prepared locally and showed no cross reactions with different species of bacteria or yeasts or with 75 control sera including 25 that gave positive results for RA factor. We used the COA test to detect cryptococcus antigen in the CSF and we could confirm the diagnosis of 11 out of 115 suspected cases of fungal meningitis; the titre varied from 4 to 128. A four-fold rise in titre confirmed the diagnostic value and a steady fall in titre in three patients on therapy indicated the prognostic value of the test. The earliest confirmation was in a renal transplant patient on the eighth day after onset of symptoms. The COA test was negative with the CSF of 118 patients with chronic meningitis. Cryptococcal colony forming units (cfu) in CSF varied from 100 to greater than 100,000/ml and correlated well with microscopy and with the COA antigen titre in CSF. Four out of the 11 patients who had cryptococcaemia, had 50,000-100,000 cfu/ml in the CSF. Cryptococcus antigen was detected by COA in the serum of all 11 patients, even in those with only 100 cfu/ml in CSF. In the three post-renal transplant patients, who were being monitored regularly, the diagnosis was made early and all three recovered on antifungal therapy with no relapse to date (1-2 years). All the others, including the two primary CNS infections, succumbed to the disease because they presented late for diagnosis and therapy. The cryptococcus COA test is a simple and specific test that can be used as a rapid test to confirm early diagnosis and permit prompt therapy, which should improve the prognosis in CNS and other forms of systemic cryptococcosis. Moreover, it is reproducible and cost-effective, particularly in countries where the latex and other expensive test reagents are not generally available. PMID:2664182

  20. Mammalian long-chain acyl-CoA synthetases.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2008-05-01

    Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

  1. Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development

    PubMed Central

    Hsiao, An-Shan; Haslam, Richard P.; Michaelson, Louise V.; Liao, Pan; Chen, Qin-Fang; Sooriyaarachchi, Sanjeewani; Mowbray, Sherry L.; Napier, Johnathan A.; Tanner, Julian A.; Chye, Mee-Len

    2014-01-01

    Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein–lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (β-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds. PMID:25423293

  2. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.

    PubMed

    Snyder, Nathaniel W; Tombline, Gregory; Worth, Andrew J; Parry, Robert C; Silvers, Jacob A; Gillespie, Kevin P; Basu, Sankha S; Millen, Jonathan; Goldfarb, David S; Blair, Ian A

    2015-04-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.

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

    PubMed

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

    2015-01-01

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

  4. Acyl-Lipid Metabolism

    PubMed Central

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

    2013-01-01

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

  5. Acyl-Lipid Metabolism

    PubMed Central

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

    2010-01-01

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

  6. Acyl-lipid metabolism.

    PubMed

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

    2013-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Dillon, Mark; Hall, Philip

    2010-01-01

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

  8. Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

    PubMed Central

    McAllister, Kelly A.; Peery, Robert B.; Zhao, Genshi

    2006-01-01

    Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology. PMID:16788183

  9. Acyl-acyl carrier protein: Lysomonogalactosyldiacylglycerol acyl transferase in Anabaena variabilis

    SciTech Connect

    Chen, H.H.

    1989-01-01

    Monogalactosyldiacylglycerol was produced when membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were incubated with ({sup 14}C)acyl-acyl carrier protein. This enzymatic synthesis of monogalactosyldiacylglycerol localized in the membranes was not dependent on any added cofactors, such as ATP, coenzyme A, and dithiothreitol. Palmitoyl-, stearoyl-, and oleoyl-acyl carrier proteins were approximately equally active as substrates with Km of 0.37, 0.36, and 0.23 {mu}M, respectively. The ({sup 14}C)acyl group was exclusively transferred to the sn-1 hydroxyl of the glycerol backbone of monogalactosyldiacylglycerol as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. Using a double labelled ({sup 14}C)acyl-({sup 14}C)acyl carrier protein, this enzyme catalyzed the direct transfer of the acyl group from acyl-acyl carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by the increased activity with the addition of the lysomonogalactosyldiacylglycerol suspension. A specific galactolipid acyl hydrolase activity was released into the soluble protein fraction when the membranes of Anabaena variabilis were treated with 2% Triton X-100. The positional specificity of this acyl hydrolase was demonstrated to be similar to that of Rhizopus lipase, i.e. only the acyl group at the sn-1 position was hydrolyzed. The acyl hydrolase which was also localized in the membrane fraction of Anabaena variabilis was presumably responsible for producing endogenous lysomonogalactosyldiacylglycerol used by the acyltransferase.

  10. Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

    The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases. Images PMID:2170335

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

    PubMed Central

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

    2014-01-01

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

  12. Inherited disorders of 3-methylcrotonyl CoA carboxylation.

    PubMed

    Leonard, J V; Seakins, J W; Bartlett, K; Hyde, J; Wilson, J; Clayton, B

    1981-01-01

    The clinical course of 4 patients who had reduced activities of 3-methylcrotonyl CoA carboxylase (also called 3-methylcrotonylglycinuria) is described. Two children presented with a metabolic acidosis, one in the neonatal period and the other with episodes of acidosis that started in the second year of life. In the other 2 children neurological symptoms were prominent, one having infantile spasms and the other developmental regression with a skin rash and alopecia. Three of the children responded well to oral biotin and dietary protein restriction but the fourth, despite a biochemical response to biotin, has a severe neurological handicap. The clinical presentation of inborn errors of 3-methylcrotonyl CoA carboxylase is variable. Metabolic acidosis may not be conspicuous and instead neurological features may predominate.

  13. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  14. Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

    PubMed

    Tsuchiya, Yugo; Pham, Uyen; Hu, Wanzhou; Ohnuma, Shin-Ichi; Gout, Ivan

    2014-01-01

    Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis. PMID:24831956

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

    SciTech Connect

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

    2012-10-15

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

  16. Characterization of a Bifunctional Archaeal Acyl Coenzyme A Carboxylase

    PubMed Central

    Chuakrut, Songkran; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2003-01-01

    Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an α4β4γ4 subunit structure. The optimum temperature for the enzyme was 60 to 70°C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent Km for acetyl-CoA was 0.17 ± 0.03 mM, with a Vmax of 43.3 ± 2.8 U mg−1, and the Km for propionyl-CoA was 0.10 ± 0.008 mM, with a Vmax of 40.8 ± 1.0 U mg−1. This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit. PMID:12533469

  17. The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis.

    PubMed

    Keating, D H; Zhang, Y; Cronan, J E

    1996-05-01

    Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis. Jackowski and Rock (J. Biol. Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected. We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates.

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

    SciTech Connect

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

    2015-09-18

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

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

    DOE PAGES

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

    2015-09-18

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

  20. Effects of long-chain fatty-acyl esters of coenzyme A and carnitine on cell-free rat heart preparations.

    PubMed

    Varela, A; Savino, E A

    1987-06-01

    The purpose of this study was to investigate the effects of fatty acyl CoA and carnitine esters on the glycolytic system of the rat heart. Using a respiring incubation mixture containing a whole-heart homogenate it was observed that oleoyl-CoA slowed down the glucose disappearance whereas lactate accumulation did not change. Experiments were also performed by means of an incubation mixture prepared with a soluble heart extract, considered to contain all glycolytic enzymes present in heart fibres. Palmitoyl-CoA or oleoyl-CoA as well as palmitoyl carnitine, added separately or together, were unable to alter the glucose disappearance and lactate accumulation in this mixture. These data suggest that long chain acyl-esters have not direct inhibitory actions on the heart glycolytic activity. However, CoA esters seem to exert indirect inhibitory effects which may be relevant to the myocardium under oxygen restriction situations.

  1. Acylation of Ferrocene: A Greener Approach

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  2. Acylation of Streptomyces type II polyketide synthase acyl carrier proteins.

    PubMed

    Crosby, J; Byrom, K J; Hitchman, T S; Cox, R J; Crump, M P; Findlow, I S; Bibb, M J; Simpson, T J

    1998-08-14

    Acyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis. The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP. Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine. The presence of this intramolecular disulphide was confirmed by tryptic digestion of the ACP followed by ESMS analysis of the fragments. An act Cys17Ser ACP was engineered by site-directed mutagenesis. S-Acyl adducts of act C17S, oxytetracycline and griseusin holo ACPs were rapidly formed by reaction with hexanoyl, 5-ketohexanoyl and protected acetoacetyl imidazolides. Comparisons with type 11 FAS ACPs were made.

  3. The essential function of genes for a hydratase and an aldehyde dehydrogenase for growth of Pseudomonas sp. strain Chol1 with the steroid compound cholate indicates an aldolytic reaction step for deacetylation of the side chain.

    PubMed

    Holert, Johannes; Jagmann, Nina; Philipp, Bodo

    2013-08-01

    In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD(+) as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

  4. The Natural Mentors of Adolescent Children of Alcoholics (COAs): Implications for Preventive Practices.

    ERIC Educational Resources Information Center

    Cavell, Timothy A.; Meehan, Barbara T.; Heffer, Robert W.; Holladay, Janice J.

    2002-01-01

    Late adolescent children of alcoholics (COAs) were interviewed about their relationship with a natural mentor. Results showed that a typical mentor was a same-sex relative who had been responsible for initiating the mentor-like relationship. Differences in the reported adjustment of COAs with and without natural mentors are considered in light of…

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

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

    PubMed

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

    2012-07-01

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

  9. Modified branched-chain amino acid pathways give rise to acyl acids of sucrose esters exuded from tobacco leaf trichomes.

    PubMed

    Kandra, G; Severson, R; Wagner, G J

    1990-03-10

    A major diversion of carbon from branched-chain amino acid biosynthesis/catabolism to form acyl moieties of sucrose esters (6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-beta-D- fructofuranosides) was observed to be associated with specialized trichome head cells which secrete large amounts of sucrose esters. Surface chemistry and acetyl and acyl substituent groups of tobacco (T.I. 1068) sucrose esters were identified and quantified by gas chromatography/mass spectrometry. Sucrose esters were prominent surface constituents and 3-methylvaleric acid, 2- and 3-methylbutyric acid, and methylpropionic acid accounted for 60%, 25% and 9%, respectively, of total C3--C7 acyl substituents. Radiolabeled Thr, Ile, Val, Leu, pyruvate and Asp, metabolites of branched-chain amino acid pathways, were compared with radioactively labeled acetate and sucrose as donors of carbon to sucrose, acetyl and acyl components of sucrose esters using epidermal peels with undisturbed trichomes. Preparations of biosynthetically competent trichome heads (site of sucrose ester formation) were also examined. Results indicate that 3-methylvaleryl and 2-methylbutyryl groups are derived from the Thr pathway of branched-chain amino acid metabolism, 3-methylbutyryl and methylpropionyl groups are formed via the pyruvate pathway, and that acetyl groups are principally formed directly via acetyl-CoA. Arguments are presented which rule out participation of fatty acid synthase in the formation of prominent acyl acids. Results suggest that the shunting of carbon away from the biosynthesis of Val, Leu and Ile may be due to a low level of amino acid utilization in protein synthesis in specialized glandular head cells of trichomes. This would result in the availability of corresponding oxo acids for CoA activation and esterification to form sucrose esters. Preliminary evidence was found for the involvement of cycling reactions in oxo-acid-chain lengthening and for utilization of pyruvate-derived 2

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

    SciTech Connect

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

    2013-11-01

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

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

    PubMed

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

    2013-11-01

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

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

    SciTech Connect

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

    2012-08-10

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

  13. Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide

    SciTech Connect

    Towler, D.; Glaser, L.

    1986-05-01

    Incubation of Saccharomyces cerevisiae strain JR153 with either (/sup 3/H)myristate or (/sup 3/H)palmitate demonstrates the synthesis of proteins that contain covalently bound fatty acids. A unique set of proteins is labeled by each fatty acid. Detailed analysis of a 20-kDa protein labeled with myristic acid demonstrates that myristate is linked to the amino-terminal glycine. We describe an enzymatic activity in yeast that will transfer myristic acid to the amino terminus of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg, whose sequence was derived from a known N-myristoylated acyl protein, the catalytic subunit of cAMP-dependent protein kinase of bovine cardiac muscle. The acylation reaction is dependent on ATP and CoA, is enriched in a crude membrane fraction, and will use myristate but not palmitate as the acyl donor. Specificity of the glycyl peptide substrate is demonstrated by the observation that other glycyl peptides do not competitively inhibit myristoylation of Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg.

  14. Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.

    PubMed

    Drozak, Jakub; Veiga-da-Cunha, Maria; Kadziolka, Beata; Van Schaftingen, Emile

    2014-03-01

    Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

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

    SciTech Connect

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

    2012-10-01

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

  16. Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase.

    PubMed

    Van Hellemond, J J; Opperdoes, F R; Tielens, A G

    1998-03-17

    Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same alpha-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes of Leishmania mexicana mexicana, L. infantum and Phytomonas sp., and procyclics of Trypanosoma brucei were analyzed as well as the parasitic helminth Fasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT of L. mexicana mexicana promastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km 0.26 mM) and a low affinity for succinate (Km 6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles.

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

    PubMed

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

    1988-05-01

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

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

    PubMed

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

    2016-09-13

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

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

    PubMed

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

    2016-09-13

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

  20. Modulation of FadR binding capacity for acyl-CoA fatty acids through structure-guided mutagenesis.

    PubMed

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

    2015-10-01

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is thus of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl-CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology. PMID:26385696

  1. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

    1998-01-06

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

  2. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

    1999-03-30

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

  3. Stearoyl-Acyl Carrier Protein and Unusual Acyl-Acyl Carrier Protein Desaturase Activities Are Differentially Influenced by Ferredoxin1

    PubMed Central

    Schultz, David J.; Suh, Mi Chung; Ohlrogge, John B.

    2000-01-01

    Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Δ9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [14C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium × hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Δ9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction. PMID:11027717

  4. The Physiology of Protein S-acylation

    PubMed Central

    Chamberlain, Luke H.; Shipston, Michael J.

    2015-01-01

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

  5. Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

    PubMed

    Choosangtong, Kamonman; Sirithanakorn, Chaiyos; Adina-Zada, Abdul; Wallace, John C; Jitrapakdee, Sarawut; Attwood, Paul V

    2015-07-22

    We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity. PMID:26149215

  6. Structure of succinyl-CoA:3-ketoacid CoA transferase from Drosophila melanogaster

    PubMed Central

    Zhang, Min; Xu, Han-Yang; Wang, Yi-Cui; Shi, Zhu-Bing; Zhang, Nan-Nan

    2013-01-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT) plays a crucial role in ketone-body metabolism. SCOT from Drosophila melanogaster (DmSCOT) was purified and crystallized. The crystal structure of DmSCOT was determined at 2.64 Å resolution and belonged to space group P212121, with unit-cell parameters a = 76.638, b = 101.921, c = 122.457 Å, α = β = γ = 90°. Sequence alignment and structural analysis identified DmSCOT as a class I CoA transferase. Compared with Acetobacter aceti succinyl-CoA:acetate CoA transferase, DmSCOT has a different substrate-binding pocket, which may explain the difference in their substrate specificities. PMID:24100554

  7. Topology and acylation of spiralin.

    PubMed Central

    Wróblewski, H; Nyström, S; Blanchard, A; Wieslander, A

    1989-01-01

    Of the 51 polypeptides detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the plasma membrane of the helical mollicute Spiroplasma melliferum, 21 are acylated, predominantly with myristic (14:0) and palmitic (16:0) chains. This is notably the case for spiralin, the major membrane protein of this bacterium, which contains an average of 0.7 acyl chains per polypeptide, attached very probably by ester bonds to alcohol amino acids. The amphiphilicity of spiralin was demonstrated by the behavior of the protein in charge-shift electrophoresis, its incorporation into liposomes, and its ability to form in the absence of lipids and detergents, globular protein micelles (diameter, approximately 15 nm). The presence of epitopes on the two faces of the cell membrane, as probed by antibody adsorption and crossed immunoelectrophoresis, and the strong interaction between spiralin and the intracytoplasmic fibrils show that spiralin is a transmembrane protein. The mean hydropathy of the amino acid composition of spiralin (-0.30) is on the hydrophilic side of the scale. Surprisingly, the water-insoluble core of spiralin micelles, which is the putative membrane anchor, has a still more hydrophilic amino acid composition (mean hydropathy, -0.70) and is enriched in glycine and serine residues. Taking into account all these properties, we propose a topological model for spiralin featuring a transbilayer localization with hydrophilic domains protruding on the two faces of the membrane and connected by a small domain embedded within the apolar region of the lipid bilayer. In this model, the membrane anchoring of the protein is strengthened by a covalently bound acyl chain. Images PMID:2768198

  8. Versatility of acyl-acyl carrier protein synthetases.

    PubMed

    Beld, Joris; Finzel, Kara; Burkart, Michael D

    2014-10-23

    The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms. PMID:25308274

  9. Modified Acyl-ACP desaturase

    DOEpatents

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

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

  10. Modified acyl-ACP desaturase

    DOEpatents

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

    1998-01-06

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

  11. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: kinetics.

    PubMed

    Thompson, P; Reid, E E; Lyttle, C R; Dennis, D T

    1977-05-01

    A steady-state kinetic analysis has been performed on the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria and castor bean (Ricinus communis L.) proplastids. Substrate interaction kinetics for all substrates gave parallel lines consistent with a multisite ping-pong mechanism. Product inhibition studies showed uncompetitive inhibition between acetyl-CoA and pyruvate and competitive inhibition between NADH and NAD(+), both of which are also consistent with this mechanism. In the mitochondrial complex, acetyl-CoA showed noncompetitive inhibition versus CoA which suggests that the intermediate complex is kinetically important in the lipoamide transacetylase component of this complex. In contrast, the proplastid complex showed competitive inhibition in this interaction. NADH is a noncompetitive inhibitor versus CoA in both complexes indicating that these complexes, like the mammalian complex, may have protein-protein interactions between the second and third enzymes of the complex. Since NADH also shows noncompetitive inhibition versus pyruvate, this interaction may extend to all components of the complex. Acetyl-CoA shows noncompetitive inhibition versus NAD(+) which may also be a result of interaction between the second and third enzymes of the complex. The limiting Michaelis constants for substrates and the inhibitor constants for both complexes were determined.

  12. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  13. Lipid Acyl Chain Remodeling in Yeast

    PubMed Central

    Renne, Mike F.; Bao, Xue; De Smet, Cedric H.; de Kroon, Anton I. P. M.

    2015-01-01

    Membrane lipid homeostasis is maintained by de novo synthesis, intracellular transport, remodeling, and degradation of lipid molecules. Glycerophospholipids, the most abundant structural component of eukaryotic membranes, are subject to acyl chain remodeling, which is defined as the post-synthetic process in which one or both acyl chains are exchanged. Here, we review studies addressing acyl chain remodeling of membrane glycerophospholipids in Saccharomyces cerevisiae, a model organism that has been successfully used to investigate lipid synthesis and its regulation. Experimental evidence for the occurrence of phospholipid acyl chain exchange in cardiolipin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine is summarized, including methods and tools that have been used for detecting remodeling. Progress in the identification of the enzymes involved is reported, and putative functions of acyl chain remodeling in yeast are discussed. PMID:26819558

  14. Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum

    SciTech Connect

    Ragsdale, S.W.

    1992-01-01

    The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H[sub 4]folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe[sup 3-4]S[sub 4

  15. Chemical reactivity of the naproxen acyl glucuronide and the naproxen coenzyme A thioester towards bionucleophiles.

    PubMed

    Olsen, Jørgen; Bjørnsdottir, Inga; Tjørnelund, Jette; Honoré Hansen, Steen

    2002-06-20

    Drugs may be metabolised to reactive electrophilic species that spontaneously react with proteins. The presence of such drug-protein adducts has been associated with drug toxicity. In this study, the reactivity of the major metabolite of naproxen--the 1-beta-O-glucuronide (Nap-GlcU)--was compared to the corresponding naproxen coenzyme A (Nap-CoA) thioester. The reactivity of the two metabolites was assessed in vitro in a phosphate buffer (pH 7.4; 0.1 M) at 37 degrees C towards the model bionucleophiles glutathione and human serum albumin (HSA). The reaction between the electrophilic species (Nap-GlcU and Nap-CoA) and glutathione forming the Nap-glutathione conjugate was monitored using LC-MS-MS and LC-UV, respectively. It was shown that Nap-CoA resulted in an approximate 100-fold higher formation of Nap-glutathione conjugate than Nap-GlcU. The presence of Nap-CoA also resulted in acylated HSA with a rate and a yield that was significantly higher than reported for Nap-GlcU. In summary, the data suggest that CoA metabolites may be more reactive species than acyl glucuronides that previously have been associated with severe drug related side effects in vivo.

  16. Stability-increasing effects of anthocyanin glycosyl acylation.

    PubMed

    Zhao, Chang-Ling; Yu, Yu-Qi; Chen, Zhong-Jian; Wen, Guo-Song; Wei, Fu-Gang; Zheng, Quan; Wang, Chong-De; Xiao, Xing-Lei

    2017-01-01

    This review comprehensively summarizes the existing knowledge regarding the chemical implications of anthocyanin glycosyl acylation, the effects of acylation on the stability of acylated anthocyanins and the corresponding mechanisms. Anthocyanin glycosyl acylation commonly refers to the phenomenon in which the hydroxyl groups of anthocyanin glycosyls are esterified by aliphatic or aromatic acids, which is synthetically represented by the acylation sites as well as the types and numbers of acyl groups. Generally, glycosyl acylation increases the in vitro and in vivo chemical stability of acylated anthocyanins, and the mechanisms primarily involve physicochemical, stereochemical, photochemical, biochemical or environmental aspects under specific conditions. Additionally, the acylation sites as well as the types and numbers of acyl groups influence the stability of acylated anthocyanins to different degrees. This review could provide insight into the optimization of the stability of anthocyanins as well as the application of suitable anthocyanins in food, pharmaceutical and cosmetic industries. PMID:27507456

  17. LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.

    PubMed

    Frey, Alexander J; Feldman, Daniel R; Trefely, Sophie; Worth, Andrew J; Basu, Sankha S; Snyder, Nathaniel W

    2016-05-01

    Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters. PMID:26968563

  18. Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation

    PubMed Central

    Jump, Donald B.; Torres-Gonzalez, Moises; Olson, L. Karl

    2010-01-01

    Acetyl CoA carboxylase (ACC1 & ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid β-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL & palmitate (16:0) and linoleate (18:2,n-6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 & 18:2,n-6; IC50 ~ 5 nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2,n-6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids. PMID:21184748

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

    PubMed

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

    2014-04-01

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

  20. Acyl-acyl-carrier protein: lysomonogalactosyldiacylglycerol acyltransferase from the cyanobacterium Anabaena variabilis.

    PubMed

    Chen, H H; Wickrema, A; Jaworski, J G

    1988-12-16

    Membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were capable of catalyzing the direct transfer of the acyl group from acyl-acyl-carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. Other glycolipids including monoglucosyldiacylglycerol and digalactosyldiacylglycerol were not products of this reaction. The transfer was not dependent on any added cofactors. Palmitoyl-, stearoyl- and oleoyl-acyl-carrier protein were approximately equally active as substrates. Transfer was exclusively to the C-1 of the glycerol, as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. In addition to the single galactolipid, a second minor reaction product was free fatty acid, presumably due to hydrolysis of the acyl-acyl-carrier protein. Using a double-labelled [14C]acyl-[14C]acyl-carrier protein, the reaction was demonstrated to be a transfer reaction, rather than a simple exchange of acyl groups with endogenous monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by increasing activity with the addition of liposomes of lysomonogalactosyldiacylglycerol.

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

    PubMed Central

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

    2014-01-01

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

  2. Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli.

    PubMed

    Heath, R J; Rock, C O

    1996-01-26

    Long chain acyl-acyl carrier protein (acyl-ACP) has been implicated as a physiological inhibitor of fatty acid biosynthesis since acyl-ACP degradation by thioesterase overexpression leads to constitutive, unregulated fatty acid production. The biochemical targets for acyl-ACP inhibition were unknown, and this work identified two biosynthetic enzymes that were sensitive to acyl-ACP feedback inhibition. Palmitoyl-ACP inhibited the incorporation of [14C]malonyl-CoA into long chain fatty acids in cell-free extracts of Escherichia coli. A short chain acyl-ACP species with the electrophoretic properties of beta-hydroxybutyryl-ACP accumulated concomitant with the overall decrease in the amount of [14C]malonyl-CoA incorporation, indicating that the first elongation cycle was targeted by acyl-ACP. All of the proteins required to catalyze the first round of fatty acid synthesis from acetyl-CoA plus malonyl-CoA in vitro were isolated, and the first fatty acid elongation cycle was reconstituted with these purified components. Analysis of the individual enzymes and the pattern of intermediate accumulation in the reconstituted system identified initiation of fatty acid synthesis by beta-ketoacyl-ACP synthase III (fabH) and enoyl-ACP reductase (fabI) in the elongation cycle as two steps attenuated by long chain acyl-ACP.

  3. Antifibrotic Activity of Acylated and Unacylated Ghrelin

    PubMed Central

    Angelino, Elia; Reano, Simone; Ferrara, Michele; Agosti, Emanuela; Graziani, Andrea; Filigheddu, Nicoletta

    2015-01-01

    Fibrosis can affect almost all tissues and organs, it often represents the terminal stage of chronic diseases, and it is regarded as a major health issue for which efficient therapies are needed. Tissue injury, by inducing necrosis/apoptosis, triggers inflammatory response that, in turn, promotes fibroblast activation and pathological deposition of extracellular matrix. Acylated and unacylated ghrelin are the main products of the ghrelin gene. The acylated form, through its receptor GHSR-1a, stimulates appetite and growth hormone (GH) release. Although unacylated ghrelin does not bind or activate GHSR-1a, it shares with the acylated form several biological activities. Ghrelin peptides exhibit anti-inflammatory, antioxidative, and antiapoptotic activities, suggesting that they might represent an efficient approach to prevent or reduce fibrosis. The aim of this review is to summarize the available evidence regarding the effects of acylated and unacylated ghrelin on different pathologies and experimental models in which fibrosis is a predominant characteristic. PMID:25960743

  4. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

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

  5. Dynamics of the Heat Stress Response of Ceramides with Different Fatty-Acyl Chain Lengths in Baker's Yeast.

    PubMed

    Chen, Po-Wei; Fonseca, Luis L; Hannun, Yusuf A; Voit, Eberhard O

    2015-08-01

    The article demonstrates that computational modeling has the capacity to convert metabolic snapshots, taken sequentially over time, into a description of cellular, dynamic strategies. The specific application is a detailed analysis of a set of actions with which Saccharomyces cerevisiae responds to heat stress. Using time dependent metabolic concentration data, we use a combination of mathematical modeling, reverse engineering, and optimization to infer dynamic changes in enzyme activities within the sphingolipid pathway. The details of the sphingolipid responses to heat stress are important, because they guide some of the longer-term alterations in gene expression, with which the cells adapt to the increased temperature. The analysis indicates that all enzyme activities in the system are affected and that the shapes of the time trends in activities depend on the fatty-acyl CoA chain lengths of the different ceramide species in the system.

  6. Dynamics of the Heat Stress Response of Ceramides with Different Fatty-Acyl Chain Lengths in Baker's Yeast.

    PubMed

    Chen, Po-Wei; Fonseca, Luis L; Hannun, Yusuf A; Voit, Eberhard O

    2015-08-01

    The article demonstrates that computational modeling has the capacity to convert metabolic snapshots, taken sequentially over time, into a description of cellular, dynamic strategies. The specific application is a detailed analysis of a set of actions with which Saccharomyces cerevisiae responds to heat stress. Using time dependent metabolic concentration data, we use a combination of mathematical modeling, reverse engineering, and optimization to infer dynamic changes in enzyme activities within the sphingolipid pathway. The details of the sphingolipid responses to heat stress are important, because they guide some of the longer-term alterations in gene expression, with which the cells adapt to the increased temperature. The analysis indicates that all enzyme activities in the system are affected and that the shapes of the time trends in activities depend on the fatty-acyl CoA chain lengths of the different ceramide species in the system. PMID:26241868

  7. LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters.

    PubMed

    Neubauer, Stefan; Chu, Dinh Binh; Marx, Hans; Sauer, Michael; Hann, Stephan; Koellensperger, Gunda

    2015-09-01

    Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U(13)C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.

  8. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaene variabilis

    SciTech Connect

    Chen, H.H.; Wickrema, A.; Jaworski, J.

    1987-04-01

    The authors previously reported the direct acylation of monogalactosyldiacylglycerol (MGDG) by an enzyme in the membranes of the cyanobacterium Anabaena variabilis. The enzyme requires acyl-acyl carrier protein (acyl-ACP) as substrate, but had no other additional cofactor requirements. Palmitoyl-, stearoyl- and oleoyl-ACP were all effective substrates. The A. variabilis membranes also had a hydrolase activity which metabolized the acyl-ACP to yield free fatty acid and ACP. Possible mechanisms for the acylation reaction include either acyl exchange with existing MGDG or direct acyl transfer to a lyso-MGDG, with concomitant release of free ACP. The mechanism of this reaction has been resolved using a double labelled (/sup 14/C)acyl-(/sup 14/)ACP substrate prepared with E. coli acyl-ACP synthetase. Following incubation with the enzyme, the unreacted (/sup 14/)acyl-(/sup 14/)ACP was isolated and the (/sup 14/)acyl/(/sup 14/)ACP ratio determined. Comparison of this ratio to that of the original substrate indicated no change and eliminated acyl exchange as a possible mechanism. Therefore, the direct acylation of lyso-MGDG is the proposed mechanism for this enzyme.

  9. Microbial Tailoring of Acyl Peptidic Siderophores

    PubMed Central

    2015-01-01

    Marine bacteria produce an abundance of suites of acylated siderophores characterized by a unique, species-dependent headgroup that binds iron(III) and one of a series of fatty acid appendages. Marinobacter sp. DS40M6 produces a suite of seven acylated marinobactins, with fatty acids ranging from saturated and unsaturated C12–C18 fatty acids. In the present study, we report that in the late log phase of growth, the fatty acids are hydrolyzed by an amide hydrolase producing the peptidic marinobactin headgroup. Halomonas aquamarina str. DS40M3, another marine bacterium isolated originally from the same sample of open ocean water as Marinobacter sp. DS40M6, produces the acyl aquachelins, also as a suite composed of a peptidic headgroup distinct from that of the marinobactins. In contrast to the acyl marinobactins, hydrolysis of the suite of acyl aquachelins is not detected, even when H. aquamarina str. DS40M3 is grown into the stationary phase. The Marinobacter cell-free extract containing the acyl amide hydrolase is active toward exogenous acyl-peptidic siderophores (e.g., aquachelin C, loihichelin C, as well as octanoyl homoserine lactone used in quorum sensing). Further, when H. aquamarina str. DS40M3 is cultured together with Marinobacter sp. DS40M6, the fatty acids of both suites of siderophores are hydrolyzed, and the aquachelin headgroup is also produced. The present study demonstrates that coculturing bacteria leads to metabolically tailored metabolites compared to growth in a single pure culture, which is interesting given the importance of siderophore-mediated iron acquisition for bacterial growth and that Marinobacter sp. DS40M6 and H. aquamarina str. DS40M3 were isolated from the same sample of seawater. PMID:24735218

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

    PubMed Central

    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 or brain iron accumulation that accompany the human disease. Pank1/Pank2 double knockout (dKO) mice were derived to determine if the mild phenotypes of the single knockout mice are due to the ability of the two isoforms to compensate for each other in CoA biosynthesis. Postnatal development was severely affected in the dKO mice. The dKO pups developed progressively severe hypoglycemia and hyperketonemia by postnatal day 10 leading to death by day 17. Hyperketonemia arose from impaired whole-body ketone utilization illustrating the requirement for CoA in energy generation from ketones. dKO pups had reduced CoA and decreased fatty acid oxidation coupled with triglyceride accumulation in liver. dKO hepatocytes could not maintain the NADH levels compared to wild-type hepatocytes. These results revealed an important link between CoA and NADH levels, which was reflected by deficiencies in hepatic oleate synthesis and gluconeogenesis. The data indicate that PanK1 and PanK2 can compensate for each other to supply tissue CoA, but PanK1 is more important to CoA levels in liver whereas PanK2 contributes more to CoA synthesis in the brain. PMID:22815849

  11. Ratio of active to inactive forms of acyl carrier protein in Escherichia coli.

    PubMed

    Jackowski, S; Rock, C O

    1983-12-25

    Acyl carrier protein (ACP) functions as a cofactor in fatty acid biosynthesis due to the covalent linkage of an acyl moiety to its 4'-phosphopantetheine prosthetic group. This prosthetic group undergoes turnover in vivo and since the apoprotein is functionally inactive, the interconversion between ACP and apo-ACP has been considered as a possible regulatory point in lipid biosynthesis. To investigate this possibility, the ratio of ACP to apo-ACP was measured in Escherichia coli. An apo-ACP standard was synthesized using [ACP] phosphodiesterase (EC 3.1.4.14) and could be clearly separated from ACP by conformationally sensitive gel electrophoresis, thus providing a reliable assay for the presence of these two species. Antibodies specific for ACP were purified from rabbit serum on an ACP-Sepharose column and subsequently used to synthesize an immunoaffinity column. Chromatography of leucine-labeled cell extracts on this support resulted in the specific binding of ACP, but apo-ACP was not detected in either logarithmically growing or stationary phase cells, although both ACP species bound to the purified anti-ACP IgG. Apo-ACP was not detected as an intermediate in ACP biosynthesis, suggesting that apo-ACP is rapidly converted to ACP following translation. CoA is the biosynthetic precursor to the ACP prosthetic group, but apo-ACP did not accumulate when the intracellular CoA concentration was severely depressed in strain SJ16 (panD), a beta-alanine auxotroph. Strain MP4 (acpS) is conditionally defective in [ACP]synthase (EC 2.7.8.7) and apo-ACP was the predominant form of ACP synthesized in this strain under nonpermissive conditions. Even under conditions that permitted growth, apo-ACP comprised 70% of the total ACP pool in strain MP4. Strain MP4 possessed a phospholipid to protein ratio within the normal range, suggesting that the ratio of ACP to apo-ACP can be significantly altered without affecting total lipid content. Thus, it appears that the prosthetic group

  12. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

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

    2009-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

  14. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  15. Identification of N-Acyl Phosphatidylserine Molecules in Eukaryotic Cells

    PubMed Central

    Guan, Ziqiang; Li, Shengrong; Smith, Dale C.; Shaw, Walter A.; Raetz, Christian R. H.

    2008-01-01

    While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acyl phosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1 % of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, mono-unsaturated and poly-unsaturated species. N-acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells and yeast, but not E. coli. N-acyl-PSs may be biosynthetic precursors of N-acyl serine molecules, such as the recently reported signaling lipid N-arachidonoyl serine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acyl serine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoyl ethanolamine (anadamide) from N-arachidonoyl phosphatidylethanolamine. PMID:18031065

  16. Testosterone induction of microsomal acyl-CoA reductase and a cytosolic regulatory protein in mouse preputial glands.

    PubMed

    Lee, T C; Kirk, P; Snyder, F

    1986-01-01

    Alkyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids are prominent components of many mammalian cells; moreover, an acetylated form of an alkyl phospholipid was recently found to possess potent hypotensive, inflammatory and allergic properties. In our studies, preputial glands of mice were selected as a model to investigate the regulation of factors involved in the biosynthesis of ether-linked lipids, since these glands contain high concentrations of ether-linked neutral lipids that are under the influence of hormonal control. We found that a key enzyme in the ether-lipid metabolic pathway, microsomal acyl-CoA reductase that catalyzes the formation of long-chain fatty alcohols (precursor of the O-alkyl chain), was increased 16-fold after injecting testosterone into male, castrated mice. This induction was highly specific, since testosterone did not affect another microsomal enzyme, NADPH-cytochrome c reductase. Based on kinetics of enzyme activity changes, the half-life of acyl-CoA reductase was calculated to be 61-70 h. In addition, the activity of a cytosolic stimulatory protein for the acyl-CoA reductase (but not for a different cytosolic protein, lactate dehydrogenase) was also enhanced in the testosterone-treated, male, castrated mice. These findings indicate that acyl-CoA reductase is an important regulatory enzyme in the reactions that lead to the formation of the ether bond in glycerolipids and that it is modulated through hormonal control. PMID:3940533

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

    PubMed

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

    2016-08-01

    KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain. PMID:27477280

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

    DOE PAGES

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

    2014-01-01

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

  19. GOAT induced ghrelin acylation regulates hedonic feeding.

    PubMed

    Davis, J F; Perello, M; Choi, D L; Magrisso, I J; Kirchner, H; Pfluger, P T; Tschoep, M; Zigman, J M; Benoit, S C

    2012-11-01

    Ghrelin is an orexigenic hormone that regulates homeostatic and reward-related feeding behavior. Recent evidence indicates that acylation of ghrelin by the gut enzyme ghrelin O-acyl transferase (GOAT) is necessary to render ghrelin maximally active within its target tissues. Here we tested the hypothesis that GOAT activity modulates food motivation and food hedonics using behavioral pharmacology and mutant mice deficient for GOAT and the ghrelin receptor (GHSR). We evaluated operant responding following pharmacological administration of acyl-ghrelin and assessed the necessity of endogenous GOAT activity for operant responding in GOAT and GHSR-null mice. Hedonic-based feeding behavior also was examined in GOAT-KO and GHSR-null mice using a "Dessert Effect" protocol in which the intake of a palatable high fat diet "dessert" was assessed in calorically-sated mice. Pharmacological administration of acyl-ghrelin augmented operant responding; notably, this effect was dependent on intact GHSR signaling. GOAT-KO mice displayed attenuated operant responding and decreased hedonic feeding relative to controls. These behavioral results correlated with decreased expression of the orexin-1 receptor in reward-related brain regions in GOAT-KO mice. In summary, the ability of ghrelin to stimulate food motivation is dependent on intact GHSR signaling and modified by endogenous GOAT activity. Furthermore, GOAT activity is required for hedonic feeding behavior, an effect potentially mediated by forebrain orexin signaling. These data highlight the significance of the GOAT-ghrelin system for the mediation of food motivation and hedonic feeding.

  20. Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols.

    PubMed Central

    Gerold, P; Jung, N; Azzouz, N; Freiberg, N; Kobe, S; Schwarz, R T

    1999-01-01

    The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy. PMID:10585859

  1. Acylation of lysophosphatidylcholine and glycerolphosphate and fatty acid pattern in phosphatidylcholine and -ethanolamine in microsomes of normal and dystrophic human muscle.

    PubMed

    Kunze, D; Rüstow, B; Kuksis, A; Myher, J J

    1986-02-01

    Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were isolated from microsomes obtained from normal and dystrophic human muscle and the fatty acid (FA) pattern estimated by GLC. In PC a decrease of the fatty acids of 16:0 and 18:2 and an increase of 18:0 and 18:1 was observed. In PE the decrease measured 18:2 and the increase 18:0 and 18:1. The acylation of lysophosphatidylcholine (LPC) and glycerol-3-phosphate (G3P) was measured in a microsomal system containing exogenously added LPC or G3P and labelled palmitic and oleic acid CoA esters. The incorporation of both labelled fatty acids in LPC-forming PC is reduced in dystrophic microsomes. On the other hand the acylation of glycerolphosphate and the formation of phosphatidic acid (PA) is greater in dystrophic microsomes when compared with normal controls. Possible correlations between the shifted FA pattern and the acylation rate by dystrophic microsomes measured in vitro in the two systems are discussed.

  2. Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21.

    PubMed

    Barrientos, Álvaro; Merino, Estefanía; Casabon, Israël; Rodríguez, Joaquín; Crowe, Adam M; Holert, Johannes; Philipp, Bodo; Eltis, Lindsay D; Olivera, Elías R; Luengo, José M

    2015-01-01

    Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

  3. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

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

    SciTech Connect

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

    2006-05-26

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

  5. Inhibition of Long Chain Acyl Coenzyme A Synthetases during Fatty Acid Loading Induces Lipotoxicity in Macrophages

    PubMed Central

    Saraswathi, Viswanathan; Hasty, Alyssa H.

    2009-01-01

    OBJECTIVES Obesity is often associated with hypertriglyceridemia and elevated free fatty acids (FFAs) which are independent risk factors for cardiovascular disease and diabetes. While impairment of cholesterol homeostasis is known to induce toxicity in macrophages, the consequence of altered fatty acid homeostasis is not clear. METHODS AND RESULTS Long chain acyl CoA synthetases (ACSLs) play a critical role in fatty acid homeostasis by channeling fatty acids to diverse metabolic pools. We treated mouse peritoneal macrophages (MPMs) with VLDL or FFAs in the presence of triacsin C, an inhibitor of the three ACSL isoforms present in macrophages. Treatment of macrophages with VLDL and triacsin C resulted in reduced TG accumulation but increased intracellular FFA levels which induced lipotoxicity characterized by induction of apoptosis. Treatment of MPMs with the saturated fatty acid stearic acid in the presence of triacsin C increased intracellular stearic acid and induced apoptosis. Stromal vascular cells collected from high fat diet-fed mice displayed foam cell morphology and exhibited increased mRNA levels of macrophage markers and ACSL1. Importantly, all of these changes were associated with increased FFA level in AT. CONCLUSIONS Inhibition of ACSLs during fatty acid loading results in apoptosis via accumulation of FFAs. Our data have implications in understanding the consequences of dysregulated fatty acid metabolism in macrophages. PMID:19679826

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

    PubMed

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

    1988-02-01

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

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

    SciTech Connect

    Hayashi, H.; Miwa, A. )

    1989-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    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.

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

    PubMed

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

    2014-04-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

  10. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz.

    PubMed

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S S; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended.

  11. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

    PubMed Central

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S. S.; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended. PMID:27175208

  12. cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

    SciTech Connect

    Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.; Tanaka, K.

    1986-05-01

    MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the ..beta..-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonal monospecific antibody. Single-stranded (/sup 32/P)labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.

  13. Genetics Home Reference: medium-chain acyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... down (metabolize) a group of fats called medium-chain fatty acids. These fatty acids are found in foods and the body's fat tissues. Fatty acids are a major source of energy for the heart and muscles. During periods of fasting, ... of this enzyme, medium-chain fatty acids are not metabolized properly. As a ...

  14. Genetics Home Reference: very long-chain acyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... metabolize) a group of fats called very long-chain fatty acids. These fatty acids are found in foods and the body's fat tissues. Fatty acids are a major source of energy for the heart and muscles. During periods of fasting, ... of this enzyme, very long-chain fatty acids are not metabolized properly. As a ...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2010-11-01

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

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

    PubMed

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

    2016-03-01

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

  19. Acylated but not des-acyl ghrelin is neuroprotective in an MPTP mouse model of Parkinson's disease.

    PubMed

    Bayliss, Jacqueline A; Lemus, Moyra; Santos, Vanessa V; Deo, Minh; Elsworth, John D; Andrews, Zane B

    2016-05-01

    The gut hormone ghrelin is widely beneficial in many disease states. However, ghrelin exists in two distinctive isoforms, each with its own metabolic profile. In Parkinson's Disease (PD) acylated ghrelin administration is neuroprotective, however, the role of des-acylated ghrelin remains unknown. In this study, we wanted to identify the relative contribution each isoform plays using the MPTP model of PD. Chronic administration of acylated ghrelin in mice lacking both isoforms of ghrelin (Ghrelin KO) attenuated the MPTP-induced loss on tyrosine hydroxylase (TH) neuronal number and volume and TH protein expression in the nigrostriatal pathway. Moreover, acylated ghrelin reduced the increase in glial fibrillary acidic protein and Ionized calcium binding adaptor molecule 1 microglia in the substantia nigra. However, injection of acylated ghrelin also elevated plasma des-acylated ghrelin, indicating in vivo deacetylation. Next, we chronically administered des-acylated ghrelin to Ghrelin KO mice and observed no neuroprotective effects in terms of TH cell number, TH protein expression, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 cell number. The lack of a protective effect was mirrored in ghrelin-O-acyltransferase KO mice, which lack the ability to acylate ghrelin and consequently these mice have chronically increased plasma des-acyl ghrelin. Plasma corticosterone was elevated in ghrelin-O-acyltransferase KO mice and with des-acylated ghrelin administration. Overall, our studies suggest that acylated ghrelin is the isoform responsible for in vivo neuroprotection and that pharmacological approaches preventing plasma conversion from acyl ghrelin to des-acyl ghrelin may have clinical efficacy to help slow or prevent the debilitating effects of PD. Ghrelin exists in the plasma as acyl and des-acyl ghrelin. We determined the form responsible for in vivo neuroprotection in a mouse model of Parkinson's disease. Although exogenous acyl ghrelin

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

    PubMed

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

    2010-10-01

    Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response. PMID:20830700

  1. Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Abbanat, D R; Ferry, J G

    1990-01-01

    The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide. PMID:2123865

  2. The pyruvate dehydrogenase E1 alpha gene is testosterone and prolactin regulated in prostate epithelial cells.

    PubMed

    Costello, L C; Liu, Y; Zou, J; Franklin, R B

    2000-02-01

    The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells. PMID:10711720

  3. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  4. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

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

    PubMed Central

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

    2014-01-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

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

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

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

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

    PubMed Central

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

    2016-01-01

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

  8. The role of acyl-glucose in anthocyanin modifications.

    PubMed

    Sasaki, Nobuhiro; Nishizaki, Yuzo; Ozeki, Yoshihiro; Miyahara, Taira

    2014-11-14

    Higher plants can produce a wide variety of anthocyanin molecules through modification of the six common anthocyanin aglycons that they present. Thus, hydrophilic anthocyanin molecules can be formed and stabilized by glycosylation and acylation. Two types of glycosyltransferase (GT) and acyltransferase (AT) have been identified, namely cytoplasmic GT and AT and vacuolar GT and AT. Cytoplasmic GT and AT utilize UDP-sugar and acyl-CoA as donor molecules, respectively, whereas both vacuolar GT and AT use acyl-glucoses as donor molecules. In carnation plants, vacuolar GT uses aromatic acyl-glucoses as the glucose donor in vivo; independently, vacuolar AT uses malylglucose, an aliphatic acyl-glucose, as the acyl-donor. In delphinium and Arabidopsis, p-hydroxybenzoylglucose and sinapoylglucose are used in vivo as bi-functional donor molecules by vacuolar GT and AT, respectively. The evolution of these enzymes has allowed delphinium and Arabidopsis to utilize unique donor molecules for production of highly modified anthocyanins.

  9. Physiological Consequences of Compartmentalized Acyl-CoA Metabolism*

    PubMed Central

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

    2015-01-01

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

  10. 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.

    PubMed Central

    Raybuck, S A; Ramer, S E; Abbanat, D R; Peters, J W; Orme-Johnson, W H; Ferry, J G; Walsh, C T

    1991-01-01

    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. PMID:1987173

  11. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

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

    EPA Science Inventory

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

  13. Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape

    PubMed Central

    Weselake, Randall J; Madhavji, Milan; Szarka, Steve J; Patterson, Nii A; Wiehler, William B; Nykiforuk, Cory L; Burton, Tracy L; Boora, Parveen S; Mosimann, Steven C; Foroud, Nora A; Thibault, Benjamin J; Moloney, Maurice M; Laroche, André; Furukawa-Stoffer, Tara L

    2006-01-01

    Background Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1–116)His6, with calculated molecular mass of 13,278 Da. Results BnDGAT1(1–116)His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1–116)His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisΔ13)-CoA over oleoyl (18:1cisΔ9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1–116)His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1–116)His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. Conclusion Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER. PMID:17192193

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

    SciTech Connect

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

    1986-05-01

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

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

    SciTech Connect

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

    1985-09-01

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

  16. Acylated iridoids with cytotoxicity from Valeriana jatamansi.

    PubMed

    Lin, Sheng; Shen, Yun-Heng; Li, Hui-Liang; Yang, Xian-Wen; Chen, Tao; Lu, Long-Hai; Huang, Zheng-Sheng; Liu, Run-Hui; Xu, Xi-Ke; Zhang, Wei-Dong; Wang, Hui

    2009-04-01

    Thirteen new acylated iridoids, jatamanvaltrates A-M (1-13), together with nine known valepotriates (14-22), were isolated from the whole plants of Valeriana jatamansi (syn. Valeriana wallichii). The structures of these new compounds were assigned by detailed interpretation of spectroscopic data. Jatamanvaltrates D (4) and H (9) are the first examples of naturally occurring valepotriates containing an o-hydroxybenzoyloxy moiety at C-10. All isolated compounds were tested for their cytotoxicity against lung adenocarcinoma (A549), metastatic prostate cancer (PC-3M), colon cancer (HCT-8), and hepatoma (Bel7402) cell lines.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  20. Transcriptomic and Reverse Genetic Analysesof Branched-Chain Fatty Acid and Acyl Sugar Production in Solanum pennellii and Nicotiana benthamiana1[W][OA

    PubMed Central

    Slocombe, Stephen P.; Schauvinhold, Ines; McQuinn, Ryan P.; Besser, Katrin; Welsby, Nicholas A.; Harper, Andrea; Aziz, Naveed; Li, Yi; Larson, Tony R.; Giovannoni, James; Dixon, Richard A.; Broun, Pierre

    2008-01-01

    Acyl sugars containing branched-chain fatty acids (BCFAs) are exuded by glandular trichomes of many species in Solanaceae, having an important defensive role against insects. From isotope-feeding studies, two modes of BCFA elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species Solanum pennellii and Datura metel; and (2) α-keto acid elongation-mediated one-carbon increments in several tobacco (Nicotiana) species and a Petunia species. To investigate the molecular mechanisms underlying BCFAs and acyl sugar production in trichomes, we have taken a comparative genomic approach to identify critical enzymatic steps followed by gene silencing and metabolite analysis in S. pennellii and Nicotiana benthamiana. Our study verified the existence of distinct mechanisms of acyl sugar synthesis in Solanaceae. From microarray analyses, genes associated with α-keto acid elongation were found to be among the most strongly expressed in N. benthamiana trichomes only, supporting this model in tobacco species. Genes encoding components of the branched-chain keto-acid dehydrogenase complex were expressed at particularly high levels in trichomes of both species, and we show using virus-induced gene silencing that they are required for BCFA production in both cases and for acyl sugar synthesis in N. benthamiana. Functional analysis by down-regulation of specific KAS I genes and cerulenin inhibition indicated the involvement of the fatty acid synthase complex in BCFA production in S. pennellii. In summary, our study highlights both conserved and divergent mechanisms in the production of important defense compounds in Solanaceae and defines potential targets for engineering acyl sugar production in plants for improved pest tolerance. PMID:18931142

  1. Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

    PubMed

    Sirithanakorn, Chaiyos; Jitrapakdee, Sarawut; Attwood, Paul V

    2016-08-01

    The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis. PMID:27379711

  2. Acyl-ACP Substrate Recognition in Burkholderia mallei BmaI1 Acyl-Homoserine Lactone Synthase

    PubMed Central

    2015-01-01

    The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme. PMID:25215658

  3. Emulsifying properties of acylated rapeseed (Brassica napus L.) peptides.

    PubMed

    Sánchez-Vioque, Raúl; Bagger, Christian L; Larré, Colette; Guéguen, Jacques

    2004-03-01

    A peptide fraction having an average size of 5.6 amino acids has been purified from a rapeseed hydrolyzate, acylated using C(10)-C(14) acyl chlorides, and the surface tension values at the air-water interface and emulsifying properties studied. As compared with standard surface-active proteins, such as bovine serum albumin (BSA), and with detergents such as sodium dodecyl sulfate (SDS), acylated peptides exhibited particular surface characteristics. The surface tension at air-water interface of acylated peptides ranged from 29.1 to 37.8 mN/m at equilibrium; these values were considerably lower than those for BSA and closer those for SDS, suggesting that acylated peptides pack at the air-water interface more like detergents than like proteins. The adsorption of acylated peptides to the oil-water interface was slower than for SDS or BSA, as deduced from the rather large size of oil droplets in emulsions (31-17 microm). Consequently, these emulsions creamed extensively during aging. Nevertheless, emulsions generated from acylated peptides were in general more stable to phase separation than those prepared from SDS. The C(14) acylated peptides were more effective for generating emulsions than the C(10) and C(12) derivatives, especially concerning the stability of emulsions against coalescence and phase separation, which was better than SDS and close to BSA.

  4. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    SciTech Connect

    Ohlrogge, John B.

    2013-11-05

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

  5. Are There Acyl-Homoserine Lactones within Mammalian Intestines?

    PubMed Central

    Swearingen, Matthew C.; Sabag-Daigle, Anice

    2013-01-01

    Many Proteobacteria are capable of quorum sensing using N-acyl-homoserine lactone (acyl-HSL) signaling molecules that are synthesized by LuxI or LuxM homologs and detected by transcription factors of the LuxR family. Most quorum-sensing species have at least one LuxR and one LuxI homolog. However, members of the Escherichia, Salmonella, Klebsiella, and Enterobacter genera possess only a single LuxR homolog, SdiA, and no acyl-HSL synthase. The most obvious hypothesis is that these organisms are eavesdropping on acyl-HSL production within the complex microbial communities of the mammalian intestinal tract. However, there is currently no evidence of acyl-HSLs being produced within normal intestinal communities. A few intestinal pathogens, including Yersinia enterocolitica, do produce acyl-HSLs, and Salmonella can detect them during infection. Therefore, a more refined hypothesis is that SdiA orthologs are used for eavesdropping on other quorum-sensing pathogens in the host. However, the lack of acyl-HSL signaling among the normal intestinal residents is a surprising finding given the complexity of intestinal communities. In this review, we examine the evidence for and against the possibility of acyl-HSL signaling molecules in the mammalian intestine and discuss the possibility that related signaling molecules might be present and awaiting discovery. PMID:23144246

  6. Lysine fatty acylation promotes lysosomal targeting of TNF-α

    PubMed Central

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-01-01

    Tumor necrosis factor-α (TNF-α) is a proinflammation cytokine secreted by various cells. Understanding its secretive pathway is important to understand the biological functions of TNF-α and diseases associated with TNF-α. TNF-α is one of the first proteins known be modified by lysine fatty acylation (e.g. myristoylation). We previously demonstrated that SIRT6, a member of the mammalian sirtuin family of enzymes, can remove the fatty acyl modification on TNF-α and promote its secretion. However, the mechanistic details about how lysine fatty acylation regulates TNF-α secretion have been unknown. Here we present experimental data supporting that lysine fatty acylation promotes lysosomal targeting of TNF-α. The result is an important first step toward understanding the biological functions of lysine fatty acylation. PMID:27079798

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

    PubMed

    Kem, Michelle P; Butler, Alison

    2015-06-01

    Acyl peptidic siderophores are produced by a variety of bacteria and possess unique amphiphilic properties. Amphiphilic siderophores are generally produced in a suite where the iron(III)-binding headgroup remains constant while the fatty acid appendage varies by length and functionality. Acyl peptidic siderophores are commonly synthesized by non-ribosomal peptide synthetases; however, the method of peptide acylation during biosynthesis can vary between siderophores. Following biosynthesis, acyl siderophores can be further modified enzymatically to produce a more hydrophilic compound, which retains its ferric chelating abilities as demonstrated by pyoverdine from Pseudomonas aeruginosa and the marinobactins from certain Marinobacter species. Siderophore hydrophobicity can also be altered through photolysis of the ferric complex of certain β-hydroxyaspartic acid-containing acyl peptidic siderophores. PMID:25677460

  8. Evolution of multicomponent pheromone signals in small ermine moths involves a single fatty-acyl reductase gene

    PubMed Central

    Liénard, Marjorie A.; Hagström, Åsa K.; Lassance, Jean-Marc; Löfstedt, Christer

    2010-01-01

    Fatty-acyl CoA reductases (FAR) convert fatty acids into fatty alcohols in pro- and eukaryotic organisms. In the Lepidoptera, members of the FAR gene family serve in the biosynthesis of sex pheromones involved in mate communication. We used a group of closely related species, the small ermine moths (Lepidoptera: Yponomeutidae) as a model to investigate the role of FARs in the biosynthesis of complex pheromone blends. Homology-based molecular cloning in three Yponomeuta species led to the identification of multiple putative FAR transcripts homologous to FAR genes from the Bombyx mori genome. The expression of one transcript was restricted to the female pheromone-gland tissue, suggesting a role in pheromone biosynthesis, and the encoded protein belonged to a recently identified Lepidoptera-specific pgFAR gene subfamily. The Yponomeuta evonymellus pgFAR mRNA was up-regulated in sexually mature females and exhibited a 24-h cyclic fluctuation pattern peaking in the pheromone production period. Heterologous expression confirmed that the Yponomeuta pgFAR orthologs in all three species investigated [Y. evonymellus (L.), Yponomeuta padellus (L.), and Yponomeuta rorellus (Hübner)] encode a functional FAR with a broad substrate range that efficiently promoted accumulation of primary alcohols in recombinant yeast supplied with a series of biologically relevant C14- or C16-acyl precursors. Taken together, our data evidence that a single alcohol-producing pgFAR played a critical function in the production of the multicomponent pheromones of yponomeutids and support the hypothesis of moth pheromone-biosynthetic FARs belonging to a FAR gene subfamily unique to Lepidoptera. PMID:20534481

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

    PubMed

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

    2015-10-27

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

  10. K2CO3-promoted formation of aryl esters from primary aryl amides by the acyl-acyl exchange process.

    PubMed

    Bian, Yongjun; Qu, Xingyu

    2016-04-28

    A new acyl-acyl exchange reaction has been developed for the formation of aryl esters from primary aryl amides. The reaction could occur under mild reaction conditions with catalytic quantities of K2CO3, and could afford moderate to good yields of the desired products. PMID:27035611

  11. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  12. Friedel-Craft acylation of ar-himachalene: synthesis of acyl-ar-himachalene and a new acyl-hydroperoxide.

    PubMed

    Hossini, Issam; Harrad, Mohamed Anoir; Ait Ali, Mustapha; El Firdoussi, Larbi; Karim, Abdallah; Valerga, Pedro; Puerta, M Carmen

    2011-01-01

    Friedel-Craft acylation at 100 °C of 2,5,9,9-tetramethyl-6,7,8,9-tetrahydro-5H-benzocycloheptene [ar-himachalene], a sesquiterpenic hydrocarbon obtained by catalytic dehydrogenation of α-, β- and γ-himachalenes, produces a mixture of two compounds: (3,5,5,9-tetramethyl-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl)-ethanone (2, in 69% yield), with a conserved reactant backbone, and 3, with a different skeleton, in 21% yield. The crystal structure of 3 reveals it to be 1-(8-ethyl-8-hydroperoxy-3,5,5-trimethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-ethanone. In this compound O-H…O bonds form dimers. These hydrogen-bonds, in conjunction with weaker C-H…O interactions, form a more extended supramolecular arrangement in the crystal. PMID:21760570

  13. A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency.

    PubMed

    Shafqat, Naeem; Kavanagh, Kate L; Sass, Jörn Oliver; Christensen, Ernst; Fukao, Toshiyuki; Lee, Wen Hwa; Oppermann, Udo; Yue, Wyatt W

    2013-11-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT . PMID:23420214

  14. Biotin deficiency in the cat and the effect on hepatic propionyl CoA carboxylase.

    PubMed

    Carey, C J; Morris, J G

    1977-02-01

    Biotin deficiency was produced in growing kittens by feeding a diet containing dried, raw egg white. After receiving either an 18.5% egg white diet for 25 weeks, or a 32% egg white diet for 12 weeks, they exhibited dermal lesions characterized by alopecia, scaly dermatitis and achromotrichia, which increased in severity with the deficiency. Females developed accumulations of dried salivary, nasal and lacrymal secretions in the facial region although a male did not. There was a loss of body weight in all cats as the deficiency progressed. Hepatic propionyl CoA carboxylase activities were measured on biopsy samples of liver during biotin deficiency and after biotin supplementation. In the deficient state, activities were 4% and 24% of that following biotin supplementation. Propionyl carboxylase activity in the liver of the cat was comparable to that reported in the rat and chick in the deficient and normal states. Subcutaneous injection of 0.25 mg biotin every other day while continuing to receive the egg white diet caused remission of clinical signs, a body weight gain and increased food intake.

  15. Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers[W

    PubMed Central

    Klempien, Antje; Kaminaga, Yasuhisa; Qualley, Anthony; Nagegowda, Dinesh A.; Widhalm, Joshua R.; Orlova, Irina; Shasany, Ajit Kumar; Taguchi, Goro; Kish, Christine M.; Cooper, Bruce R.; D’Auria, John C.; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2012-01-01

    Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway. PMID:22649270

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

    PubMed

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

    2014-03-01

    Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2M GdnHCl (as compared to drastic loss of native structure in 2M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein. PMID:24309513

  17. Clustering of mutations in methylmalonyl CoA mutase associated with mut- methylmalonic acidemia.

    PubMed Central

    Crane, A. M.; Ledley, F. D.

    1994-01-01

    Mutations have been described in human methylmalonyl CoA mutase (MCM) that exhibit partial defects in enzyme activity, including cobalamin-dependent (i.e., mut-) or interallelic complementation. This work describes mutations in cells from four patients, three of whom exhibit a cobalamin-dependent phenotype and all four of whom exhibit interallelic complementation. Four novel mutations (R694W, G648D, G630E, and G626C) are identified that cluster near the carboxyl terminus of the protein, a region close to another mut- mutation (G717V). Each of these mutations was shown to express a phenotype congruent with that of the parental cell line, after transfection into mut0 fibroblasts, and each exhibits interallelic complementation in cotransfection assays with clones bearing a R93H mutation. The activity of mutant enzymes expressed in Saccharomyces cerevisiae parallels the residual activity of the parental cell lines and exhibits novel sensitivities to pH and salt. The clustering of these mutations identifies a region of MCM that most likely represents the cobalamin-binding domain. The location of this domain, as well as the pattern of sequence preservation between the homologous human and Probiono-bacterium shermanii enzymes, suggests a mechanism for interallelic complementation in which the cobalamin-binding defect is complemented in trans from the heterologous subunits of the dimer. Images Figure 6 PMID:7912889

  18. Enhanced activity of acetyl CoA synthetase adsorbed on smart microgel: an implication for precursor biosynthesis.

    PubMed

    Dubey, Nidhi Chandrama; Tripathi, Bijay Prakash; Müller, Martin; Stamm, Manfred; Ionov, Leonid

    2015-01-28

    Acetyl coenzyme A (acetyl CoA) is an essential precursor molecule for synthesis of metabolites such as the polyketide-based drugs (tetracycline, mitharamycin, Zocor, etc.) fats, lipids, and cholesterol. Acetyl CoA synthetase (Acs) is one of the enzymes that catalyzes acetyl CoA synthesis, and this enzyme is essentially employed for continuous supply of the acetyl CoA for the production of these metabolites. To achieve reusable and a more robust entity of the enzyme, we carried out the immobilization of Acs on poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgels via adsorption. Cationic PNIPAm-PEI microgel was synthesized by one-step graft copolymerization of NIPAm and N,N-methylene bis-acrylamide (MBA) from PEI. Adsorption studies of Acs on microgel indicated high binding of enzymes, with a maximum binding capacity of 286 μg/mg of microgel for Acs was achieved. The immobilized enzymes showed improved biocatalytic efficiency over free enzymes, beside this, the reaction parameters and circular dichroism (CD) spectroscopy studies indicated no significant changes in the enzyme structure after immobilization. This thoroughly characterized enzyme bioconjugate was further immobilized on an ultrathin membrane to assess the same reaction in flow through condition. Bioconjugate was covalently immobilized on a thin layer of preformed microgel support upon polyethylene terephthalate (PET) track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of cross-linked bioconjugate. The membrane reactor showed consistent operational stability and maintained >70% of initial activity after 7 consecutive operation cycles. PMID:25561344

  19. Genetic Diversity of Staphylocoagulase Genes (coa): Insight into the Evolution of Variable Chromosomal Virulence Factors in Staphylococcus aureus

    PubMed Central

    Watanabe, Shinya; Ito, Teruyo; Sasaki, Takashi; Li, Shanshuang; Uchiyama, Ikuo; Kishii, Kozue; Kikuchi, Ken; Skov, Robert Leo; Hiramatsu, Keiichi

    2009-01-01

    Background The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. Methodology/Principal Findings We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. Conclusion The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome. PMID:19492076

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

    EPA Science Inventory

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

  1. A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

    SciTech Connect

    McIntire, W.S. Univ. of California, San Francisco ); Wemmer, D.E. ); Chistoserdov, A.; Lidstrom, M.E. )

    1991-05-10

    Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

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

    PubMed

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

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. PMID:23541561

  3. Acyl-acyl carrier protein thioesterase activity from sunflower (Helianthus annuus L.) seeds.

    PubMed

    Martínez-Force, E; Cantisán, S; Serrano-Vega, M J; Garcés, R

    2000-10-01

    During sunflower (Helianthus annuus L.) seed formation there was an active period of lipid biosynthesis between 12 and 28 days after flowering (DAF). The maximum in-vitro acyl-acyl carrier protein (ACP) thioesterase activities (EC 3.1.2.14) were found at 15 DAF, preceding the largest accumulation of lipid in the seed. Data from the apparent kinetic parameters, Vmax and Km, from seeds of 15 and 30 DAF, showed that changes in acyl-ACP thioesterase activity are not only quantitative, but also qualitative, since, although the preferred substrate was always oleoyl-ACP, the affinity for palmitoyl-ACP decreased, whereas that for stearoyl-ACP increased with seed maturation. Bisubstrate assays carried out at 30 DAF seemed to indicate that the total activity found in mature seeds is due to a single enzyme with 100/75/15 affinity for oleoyl-ACP/stearoyl-ACP/ palmitoyl-ACP. In contrast, at 15 DAF, enzymatic data together with partial sequences from cDNAs indicated the presence of at least two enzymes with different properties, a FatA-like thioesterase, with a high affinity for oleoyl-ACP, plus a FatB-like enzyme, with preference for long-chain saturated fatty acids, both being expressed during the active lipid biosynthesis period. Competition assays carried out with CAS-5, a mutant with a higher content of palmitic acid in the seed oil, indicated that a modified FatA-type thioesterase is involved in the mutant phenotype.

  4. Palladium-Catalyzed Environmentally Benign Acylation.

    PubMed

    Suchand, Basuli; Satyanarayana, Gedu

    2016-08-01

    Recent trends in research have gained an orientation toward developing efficient strategies using innocuous reagents. The earlier reported transition-metal-catalyzed carbonylations involved either toxic carbon monoxide (CO) gas as carbonylating agent or functional-group-assisted ortho sp(2) C-H activation (i.e., ortho acylation) or carbonylation by activation of the carbonyl group (i.e., via the formation of enamines). Contradicting these methods, here we describe an environmentally benign process, [Pd]-catalyzed direct carbonylation starting from simple and commercially available iodo arenes and aldehydes, for the synthesis of a wide variety of ketones. Moreover, this method comprises direct coupling of iodoarenes with aldehydes without activation of the carbonyl and also without directing group assistance. Significantly, the strategy was successfully applied to the synthesis n-butylphthalide and pitofenone. PMID:27377566

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

    SciTech Connect

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

    2011-06-01

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

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

    PubMed Central

    2016-01-01

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

  7. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

    PubMed Central

    Wu, Gang; Fiser, András; ter Kuile, Benno; Šali, Andrej; Müller, Miklós

    1999-01-01

    Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. PMID:10339579

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

    SciTech Connect

    Not Available

    1990-01-01

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

  9. Sorbitol dehydrogenase is a zinc enzyme.

    PubMed Central

    Jeffery, J; Chesters, J; Mills, C; Sadler, P J; Jörnvall, H

    1984-01-01

    Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins. PMID:6370679

  10. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

    PubMed Central

    Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

    2015-01-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

  11. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: regulation.

    PubMed

    Thompson, P; Reid, E E; Lyttle, C R; Dennis, D T

    1977-05-01

    The activity of the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria is inhibited when MgATP is added to the reaction mixture; 50% inhibition occurs at 4 mm ATP. The inhibition does not increase with time and is higher in the more highly purified preparations. Crude preparations of the complex show a time-dependent inactivation when incubated with 7.5 mm MgATP alone but this is not found with the more highly purified complex. This inactivation does not occur at 0 C. The complex could not be reactivated by high concentrations of Mg(2+). It is suggested that a phosphorylation-dephosphorylation mechanism may occur in plants, but the phosphatase and kinase are not tightly bound to the complex and are lost on isolation. The complex does not respond in a significant manner to energy charge. The NAD(+) to NADH ratio is the principal means of regulation of the complex, NADH being competitive with NAD(+) for the dihydrolipoamide component. The CoA to acetyl-CoA ratio is not important in regulation.The castor bean (Ricinus communis L.) proplastid complex is inhibited by the addition of 2 mm MgATP to the assay mixture. The inhibition is immediate, suggesting that phosphorylation of the enzyme is not involved or must be very rapid. Incubation of the complex with 20 mm MgCl(2) causes an activation of the complex. Maximum activity is not expressed in this case for 30 minutes. A similar activation can be achieved by preincubating the complex with 1 mm pyruvate. These data suggest that the complex is not fully activated on isolation.

  12. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

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

    2013-08-01

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

  13. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

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

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway. PMID:23677922

  14. Amine, Alcohol and Phosphine Catalysts for Acyl Transfer Reactions

    NASA Astrophysics Data System (ADS)

    Spivey, Alan C.; Arseniyadis, Stellios

    An overview of the area of organocatalytic asymmetric acyl transfer processes is presented including O- and N-acylation. The material has been ordered according to the structural class of catalyst employed rather than reaction type with the intention to draw mechanistic parallels between the manner in which the various reactions are accelerated by the catalysts and the concepts employed to control transfer of chiral information from the catalyst to the substrates.

  15. Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum. Progress report, August 15, 1993--March 24, 1995

    SciTech Connect

    Ragsdale, S.W.

    1992-11-01

    The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H{sub 4}folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe{sup 3-4}S{sub 4}]-CO species which has been characterized by magnetic resonance methods can be described as [Ni-X-Fe{sub 3-4}S{sub 4}]-C{equivalent_to}O. We showed that this species is the catalytically competent precursor of the carbonyl group of acetyl-CoA. We have made progress in the synthesis of seleno-coenzyme A, which we will use to probe the binding of CoA to CODH. We also have compared the CODH from Methanosarcina thermophila with the C. thermoaceticum enzyme by EPR and electrochemical methods and found that the metal sites of these enzymes are remarkably similar given the evolutionary separation between archaea and bacteria domains.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    Cancer.gov

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

  18. Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius.

    PubMed

    Extance, Jonathan; Crennell, Susan J; Eley, Kirstin; Cripps, Roger; Hough, David W; Danson, Michael J

    2013-10-01

    Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

  19. Purification of Recombinant Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (ACAT1) from H293 Cells and Binding Studies Between the Enzyme and Substrates Using Difference Intrinsic Fluorescence Spectroscopy†

    PubMed Central

    Chang, Catherine CY; Miyazaki, Akira; Dong, Ruhong; Kheirollah, Alireza; Yu, Chunjiang; Geng, Yong; Higgs, Henry N; Chang, Ta-Yuan

    2010-01-01

    Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl CoA binds to ACAT1 with Kd=1.9 μM, and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl CoA elicits a similar spectrum change with much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3beta-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1. PMID:20964445

  20. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaena variabilis. [Anabaena variabilis

    SciTech Connect

    Chen, H.H.; Wickrema, A.; Jaworski, J.

    1987-05-01

    The authors previously reported the direct acylation of monogalactosyldiacylglycerol (MGDG) by an enzyme in the membranes of the cyanobacterium (Anabaena variabilis. The enzyme requires acyl-acyl carrier protein (acyl-ACP) as substrate, but had no other additional cofactor requirements. Palmitoyl-, stearoyl- and oleoyl-ACP were all effective substrates. The A. variabilis membranes also had a hydrolase activity which metabolized the acyl-ACP to yield free fatty acid and ACP. Possible mechanisms for the acylation reaction include either acyl exchange with existing MGDG or direct acyl transfer to a lyso-MGDG, with concomitant release of free ACP. The mechanism of this reaction has been resolved using a double labelled (/sup 14/C)acyl-(/sup 14/C)ACP substrate prepared with E. coli acyl-ACP synthetase. Following incubation with the enzyme, the unreacted (/sup 14/C)acyl-(/sup 14/C)ACP was isolated and the (/sup 14/C)acyl/(/sup 14/C)ACP ratio determined. Comparison of this ratio to that of the original substrate indicated no change and eliminated acyl exchange as a possible mechanism. Therefore, the direct acylation of lyso-MGDG is the proposed mechanism for this enzyme. The reaction is apparently specific for MGDG synthesis, as other glycolipids and phospholipids were not labelled during incubations.

  1. Regioselective Acylation of Diols and Triols: The Cyanide Effect.

    PubMed

    Peng, Peng; Linseis, Michael; Winter, Rainer F; Schmidt, Richard R

    2016-05-11

    Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective O-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-cis-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at -78 °C the thermodynamically unfavorable axial O-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-O-unproteced galacto- and fucopyranosides and 2,3-O-unprotected mannopyranosides. Even for 3,4,6-O-unprotected galactopyranosides as triols, axial 4-O-acylation is appreciably faster than O-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the "cyanide effect" is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial O-acylation. PMID:27104625

  2. Site-specific S-Acylation of Influenza Virus Hemagglutinin

    PubMed Central

    Brett, Katharina; Kordyukova, Larisa V.; Serebryakova, Marina V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2014-01-01

    S-Acylation of hemagglutinin (HA), the main glycoprotein of influenza viruses, is an essential modification required for virus replication. Using mass spectrometry, we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus also be suitable to analyze cellular proteins. We observed cell type-specific differences in the fatty acid pattern of HA: more stearate was attached if human viruses were grown in mammalian compared with avian cells. No underacylated peptides were detected in the mass spectra, and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with [3H]palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern. PMID:25349209

  3. Medium-chain acyl-CoA deficiency: outlines from newborn screening, in silico predictions, and molecular studies.

    PubMed

    Catarzi, Serena; Caciotti, Anna; Thusberg, Janita; Tonin, Rodolfo; Malvagia, Sabrina; la Marca, Giancarlo; Pasquini, Elisabetta; Cavicchi, Catia; Ferri, Lorenzo; Donati, Maria A; Baronio, Federico; Guerrini, Renzo; Mooney, Sean D; Morrone, Amelia

    2013-01-01

    Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a disorder of fatty acid oxidation characterized by hypoglycemic crisis under fasting or during stress conditions, leading to lethargy, seizures, brain damage, or even death. Biochemical acylcarnitines data obtained through newborn screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were confirmed by molecular analysis of the medium-chain acyl-CoA dehydrogenase (ACADM) gene. Out of 324.000 newborns screened, we identified 14 MCADD patients, in whom, by molecular analysis, we found a new nonsense c.823G>T (p.Gly275∗) and two new missense mutations: c.253G>C (p.Gly85Arg) and c.356T>A (p.Val119Asp). Bioinformatics predictions based on both phylogenetic conservation and functional/structural software were used to characterize the new identified variants. Our findings confirm the rising incidence of MCADD whose existence is increasingly recognized due to the efficacy of an expanded newborn screening panel by LC-MS/MS making possible early specific therapies that can prevent possible crises in at-risk infants. We noticed that the "common" p.Lys329Glu mutation only accounted for 32% of the defective alleles, while, in clinically diagnosed patients, this mutation accounted for 90% of defective alleles. Unclassified variants (UVs or VUSs) are especially critical when considering screening programs. The functional and pathogenic characterization of genetic variants presented here is required to predict their medical consequences in newborns. PMID:24294134

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2012-03-01

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

  6. Regioselective self-acylating cyclodextrins in organic solvent

    PubMed Central

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

    2016-01-01

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

  7. Regioselective self-acylating cyclodextrins in organic solvent

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  8. Regioselective self-acylating cyclodextrins in organic solvent.

    PubMed

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

    2016-01-01

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

  9. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

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

    2016-01-01

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

  10. Two fatty acyl reductases involved in moth pheromone biosynthesis.

    PubMed

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

    2016-01-01

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

  11. Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.

    PubMed

    Soupene, Eric; Fyrst, Henrik; Kuypers, Frans A

    2008-01-01

    The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.

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

    PubMed

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

    2016-09-15

    Snake venoms are rich and intriguing sources of biologically-active molecules that act on target cells, modulating a diversity of physiological functions and presenting promising pharmacological applications. Lys49 phospholipase A2 is one of the multifunctional proteins present in these complex secretions and, although catalytically inactive, has a variety of biological activities, including cytotoxic, antibacterial, inflammatory, antifungal activities. Herein, a Lys49 phospholipase A2, denominated CoaTx-II from Crotalus oreganus abyssus, was purified and structurally and pharmacologically characterized. CoaTx-II was isolated with a high degree of purity by a combination of two chromatographic steps; molecular exclusion and reversed-phase high performance liquid chromatography. This toxin is dimeric with a mass of 13868.2 Da (monomeric form), as determined by mass spectrometry. CoaTx-II is rich in Arg and Lys residues and displays high identity with other Lys49 PLA2 homologues, which have high isoelectric points. The structural model of dimeric CoaTx-II shows that the toxin is non-covalently stabilized. Despite its enzymatic inactivity, in vivo CoaTx-II caused local muscular damage, characterized by increased plasma creatine kinase and confirmed by histological alterations, in addition to an inflammatory activity, as demonstrated by mice paw edema induction and pro-inflammatory cytokine IL-6 elevation. CoaTx-II also presents antibacterial activity against gram negative (Pseudomonas aeruginosa 31NM, Escherichia coli ATCC 25922) and positive (Staphyloccocus aureus BEC9393 and Rib1) bacteria. Therefore, data show that this newly purified toxin plays a central role in mediating the degenerative events associated with envenomation, in addition to demonstrating antibacterial properties, with potential for use in the development of strategies for antivenom therapy and combating antibiotic-resistant bacteria. PMID:27530662

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

    PubMed

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

    2016-09-15

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

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

    NASA Technical Reports Server (NTRS)

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

    1972-01-01

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

  15. Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions.

    PubMed

    Machado, A; Nuñez de Castro, I; Mayor, F

    1975-02-28

    The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants. All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases. According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo". From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.

  16. [The PQQ-dehydrogenases. A novel example of bacterial quinoproteins].

    PubMed

    Flores-Encarnación, Marcos; Sánchez-Cuevas, Mariano; Ortiz-Gutiérrez, Felipe

    2004-01-01

    The word "quinoprotein" describes four groups of different enzymes which have cofactors containing o-quinones. Pyrrolo-quinoline quinone (PQQ) is not covalently attached. PQQ is the cofactor of several quinoprotein bacterial dehydrogenases including glucose dehydrogenase (G-DH), alcohol dehydrogenase (A-DH) and aldehyde dehydrogenase (AL-DH). These dehydrogenases are located in the periplasm of Gram-negative bacteria. This report summarises the structural properties of quinoprotein dehydrogenases, such as the biological functions and biotechnological aspects more important.

  17. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

    PubMed Central

    Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-01-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  18. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

    PubMed

    Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-10-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

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

    PubMed Central

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

    2014-01-01

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

  20. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  1. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  2. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  3. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  4. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  5. Quantum chemical study of penicillin: Reactions after acylation

    NASA Astrophysics Data System (ADS)

    Li, Rui; Feng, Dacheng; Zhu, Feng

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

  6. Diverse Activities of Histone Acylations Connect Metabolism to Chromatin Function.

    PubMed

    Dutta, Arnob; Abmayr, Susan M; Workman, Jerry L

    2016-08-18

    Modifications of histones play important roles in balancing transcriptional output. The discovery of acyl marks, besides histone acetylation, has added to the functional diversity of histone modifications. Since all modifications use metabolic intermediates as substrates for chromatin-modifying enzymes, the prevalent landscape of histone modifications in any cell type is a snapshot of its metabolic status. Here, we review some of the current findings of how differential use of histone acylations regulates gene expression as response to metabolic changes and differentiation programs. PMID:27540855

  7. Formate dehydrogenase from Pseudomonas oxalaticus.

    PubMed

    Müller, U; Willnow, P; Ruschig, U; Höpner, T

    1978-02-01

    Formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 315000) is a complex flavoprotein containing 2 FMN, 18--25 non-heme iron atoms and 15--20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD+, oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors. The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species. PMID:631130

  8. Opine dehydrogenases in marine invertebrates.

    PubMed

    Harcet, Matija; Perina, Drago; Pleše, Bruna

    2013-10-01

    It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD(+) ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

  9. Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei.

    PubMed

    Pasti, Claudia; Rinaldi, Eliana; Cervellati, Carlo; Dallocchio, Franco; Hardré, Renaud; Salmon, Laurent; Hanau, Stefania

    2003-04-01

    Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.

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

    PubMed Central

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

    2014-01-01

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

  11. Chemoenzymatic Synthesis of Acyl Coenzyme A Substrates Enables in Situ Labeling of Small Molecules and Proteins.

    PubMed

    Agarwal, Vinayak; Diethelm, Stefan; Ray, Lauren; Garg, Neha; Awakawa, Takayoshi; Dorrestein, Pieter C; Moore, Bradley S

    2015-09-18

    A chemoenzymatic approach to generate fully functional acyl coenzyme A molecules that are then used as substrates to drive in situ acyl transfer reactions is described. Mass spectrometry based assays to verify the identity of acyl coenzyme A enzymatic products are also illustrated. The approach is responsive to a diverse array of carboxylic acids that can be elaborated to their corresponding coenzyme A thioesters, with potential applications in wide-ranging chemical biology studies that utilize acyl coenzyme A substrates.

  12. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

    PubMed Central

    Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

    2015-01-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

  13. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase.

    PubMed

    Yao, Jiangwei; Dodson, V Joshua; Frank, Matthew W; Rock, Charles O

    2015-09-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

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

    PubMed

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

    2012-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  17. Thioesterification of 2-arylpropionic acids by recombinant acyl-coenzyme A synthetases (ACS1 and ACS2).

    PubMed

    Sevoz, C; Benoit, E; Buronfosse, T

    2000-04-01

    2-Arylpropionic acids are a class of frequently used nonsteroidal anti-inflammatory drugs exhibiting a potent inhibition of cyclooxygenase isoforms supported by the (+)S-enantiomer alone. Nevertheless, some of these compounds in the (-)R configuration may undergo extensive inversion of configuration to their antipode. The key molecular basis for this mechanism invokes the stereoselective formation of the coenzyme A (CoA) thioester of the 2-arylpropionic acid by long-chain acyl-CoA synthetases (ACSs). In this report, rat recombinant ACS1 and ACS2 enzymes, constitutively highly expressed in adult rat liver and brain, respectively, have been overproduced in Escherichia coli strains and purified to homogeneity to investigate the involvement of these enzymes in the thioesterification of fenoprofen and ibuprofen. Recombinant ACS1 efficiently catalyzed both nonsteroidal anti-inflammatory drugs with Michaelis-Menten parameters of K(M) = 1686 +/- 93 microM, V(max) = 353 +/- 45 nmol/min/mg protein for (-)R-ibuprofen and K(M) = 103 +/- 12 microM, V(max) = 267 +/- 10 nmol/min/mg protein for (-)R-fenoprofen, and exhibited a marked stereoselectivity in favor of the (-)R-enantiomer. Recombinant ACS2, a closely related sequence with ACS1, exhibited a lower enzymatic efficacy from 7- to 130-fold for (-)R-ibuprofen and (-)R-fenoprofen, respectively. On the basis of these findings and considering the level of tissue expression of the different long-chain ACSs, ACS1 appears to be the major enzyme involved in the first step of the chiral inversion of 2-arylpropionic acids. Nevertheless, the participation of other ACS isoforms of minor quantitative importance could not be excluded in the thioesterification of xenobiotics.

  18. QSAR and Molecular Docking Studies of Oxadiazole-Ligated Pyrrole Derivatives as Enoyl-ACP (CoA) Reductase Inhibitors

    PubMed Central

    Asgaonkar, Kalyani D.; Mote, Ganesh D.; Chitre, Trupti S.

    2014-01-01

    A quantitative structure-activity relationship model was developed on a series of compounds containing oxadiazole-ligated pyrrole pharmacophore to identify key structural fragments required for anti-tubercular activity. Two-dimensional (2D) and three-dimensional (3D) QSAR studies were performed using multiple linear regression (MLR) analysis and k-nearest neighbour molecular field analysis (kNN-MFA), respectively. The developed QSAR models were found to be statistically significant with respect to training, cross-validation, and external validation. New chemical entities (NCEs) were designed based on the results of the 2D- and 3D-QSAR. NCEs were subjected to Lipinski’s screen to ensure the drug-like pharmacokinetic profile of the designed compounds in order to improve their bioavailability. Also, the binding ability of the NCEs with enoyl-ACP (CoA) reductase was assessed by docking. PMID:24634843

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

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

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

    PubMed

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

    2015-09-01

    We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules. PMID:25846609

  2. Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

    Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid β-oxidation and phloem-mediated lipid transport is highlighted.

  3. Preservation of polyunsaturated fatty acyl glycerides via intramolecular antioxidant coupling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ferulic acid and its esters are known to be effective antioxidants. Feruloyl di-gamma-linolenoylglycerol was assessed for its ability to serve as an antioxidant for preventing the oxidation of its gamma-linolenoyl polyunsaturated fatty acyl groups in model membrane phospholipid vesicles. The molec...

  4. Lubricity characteristics of seed oils modified by acylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

    Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid β-oxidation and phloem-mediated lipid transport is highlighted. PMID:27023243

  6. Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS

    PubMed Central

    Okudaira, Michiyo; Inoue, Asuka; Shuto, Akira; Nakanaga, Keita; Kano, Kuniyuki; Makide, Kumiko; Saigusa, Daisuke; Tomioka, Yoshihisa; Aoki, Junken

    2014-01-01

    Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs. PMID:25114169

  7. Enhanced acyl-CoA dehydrogenase activity is associated with improved mitochondrial and contractile function in heart failure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heart failure is associated with decreased myocardial fatty acid oxidation capacity and has been likened to energy starvation. Increased fatty acid availability results in an induction of genes promoting fatty acid oxidation. The aim of the present study was to investigate possible mechanisms by whi...

  8. Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

    PubMed Central

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

    2013-01-01

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

  9. Succinate-dependent energy generation and pyruvate dehydrogenase complex activity in isolated Ascaris suum mitochondria

    SciTech Connect

    Campbell, T.A.

    1988-01-01

    Body wall muscle from the parasitic nematode, Ascaris suum, contain unique anaerobic mitochondria that preferentially utilize fumarate and branched-chain enoyl CoA's as terminal electron acceptors instead of oxygen. While electron transport in these organelles is well characterized, the role of oxygen in succinate-dependent phosphorylation is still not clearly defined. Therefore, the present study was designed to more fully characterize succinate metabolism in these organelles as well as the in vitro regulation of a key mitochondrial enzyme, the pyruvate dehydrogenase complex (PDC). In the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochrondrial ATP levels, but ATP/ADP ratios were considerably higher in incubations with malate. The stimulation of phosphorylation in aerobic incubations with succinate was rotenone sensitive and appears to be Site I dependent. Increase substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, {sup 14}CO{sub 2} evolution from 1,4-({sup 14}C)succinate was stimulated and NADH/NAD{sup +} ratios were elevated, but the formation of {sup 14}C propionate was unchanged.

  10. Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana.

    PubMed

    Nilsson, Anders K; Johansson, Oskar N; Fahlberg, Per; Kommuri, Murali; Töpel, Mats; Bodin, Lovisa J; Sikora, Per; Modarres, Masoomeh; Ekengren, Sophia; Nguyen, Chi T; Farmer, Edward E; Olsson, Olof; Ellerström, Mats; Andersson, Mats X

    2015-12-01

    The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response. PMID:26566971

  11. The Golgi S-acylation machinery comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity

    PubMed Central

    Lemonidis, Kimon; Gorleku, Oforiwa A.; Sanchez-Perez, Maria C.; Grefen, Christopher; Chamberlain, Luke H.

    2014-01-01

    S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins—synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes. PMID:25253725

  12. The effects of putative lipase and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase gene knockouts on triacylglycerol accumulation in Gordonia sp. KTR9.

    PubMed

    Indest, Karl J; Eberly, Jed O; Ringelberg, David B; Hancock, Dawn E

    2015-02-01

    Previously, we demonstrated triacylglycerol (TAG) accumulation and the in vivo ability to catalyze esters from exogenous short chain alcohol sources in Gordonia sp. strain KTR9. In this study, we investigated the effects that putative lipase (KTR9_0186) and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; KTR9_3844) gene knockouts had on TAG accumulation. Gene disruption of KTR9_0186 resulted in a twofold increase in TAG content in nitrogen starved cells. Lipase mutants subjected to carbon starvation, following nitrogen starvation, retained 75 % more TAGs and retained pigmentation. Transcriptome expression data confirmed the deletion of KTR9_0186 and identified the up-regulation of key genes involved in fatty acid degradation, a likely compensatory mechanism for reduced TAG mobilization. In vitro assays with purified KTR9_3844 demonstrated WS/DGAT activity with short chain alcohols and C16 and C18 fatty acid Co-As. Collectively, these results indicate that Gordonia sp. KTR9 has a suitable tractable genetic background for TAG production as well as the enzymatic capacity to catalyze fatty acid esters from short chain alcohols.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-01-01

    Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2. PMID:27274541

  16. Shikimate dehydrogenase from Pinu sylvestris L. needles

    SciTech Connect

    Osipov, V.I.; Shein, I.V.

    1986-07-10

    Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP/sup +/, but also with NAD/sup +/. The values of K/sub m/ for shikimate, when NADP/sup +/ and NAD/sup +/ are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed.

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    1995-03-01

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

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

    PubMed

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

    2016-01-01

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

  20. Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

    PubMed

    Perez-Siles, Gonzalo; Ly, Carolyn; Grant, Adrienne; Drew, Alexander P; Yiu, Eppie M; Ryan, Monique M; Chuang, David T; Tso, Shih-Chia; Nicholson, Garth A; Kennerson, Marina L

    2016-10-01

    Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

  1. Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

    PubMed

    Perez-Siles, Gonzalo; Ly, Carolyn; Grant, Adrienne; Drew, Alexander P; Yiu, Eppie M; Ryan, Monique M; Chuang, David T; Tso, Shih-Chia; Nicholson, Garth A; Kennerson, Marina L

    2016-10-01

    Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6. PMID:27388934

  2. Direct N-acylation of azoles via a metal-free catalyzed oxidative cross-coupling strategy.

    PubMed

    Zhao, Jingjing; Li, Pan; Xia, Chungu; Li, Fuwei

    2014-05-11

    The KI-catalyzed N-acylation of azoles via direct oxidative coupling of C-H and N-H bonds has been developed. It could be smoothly scaled up to gram synthesis of acyl azoles. The reaction occurred by the coupling of acyl radicals and azoles to form the acyl azole radical anion, followed by its further oxidation.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  5. Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

    PubMed

    Carpenter, K; Pollitt, R J; Middleton, B

    1992-03-16

    We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.

  6. An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates.

    PubMed

    Austin, J C; Wharton, C W; Hester, R E

    1989-02-21

    Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.

  7. Development of L-lactate dehydrogenase biosensor based on porous silicon resonant microcavities as fluorescence enhancers.

    PubMed

    Jenie, S N Aisyiyah; Prieto-Simon, Beatriz; Voelcker, Nicolas H

    2015-12-15

    The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution.

  8. Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

    PubMed

    Tsuji, Kohsei; Yoon, Ki-Seok; Ogo, Seiji

    2016-03-01

    Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE(S77)). Interestingly, the ADHE(S77) was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH(4))(2)SO(4) without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration.

  9. New acylated anthocyanins from purple yam and their antioxidant activity.

    PubMed

    Moriya, Chiemi; Hosoya, Takahiro; Agawa, Sayuri; Sugiyama, Yasumasa; Kozone, Ikuko; Shin-Ya, Kazuo; Terahara, Norihiko; Kumazawa, Shigenori

    2015-01-01

    Purple yam (Dioscorea alata L.), which is widely distributed in tropical and subtropical regions, is characterized by its color and viscosity. Previous studies have shown that purple yams contain a variety of acylated anthocyanins that exhibit higher levels of antioxidant activity than the corresponding nonacylated compounds. In this study, the pigments found in purple yams from the Philippines (D. alata) were isolated and evaluated in terms of antioxidant activity. Four new acylated anthocyanins, alanins (1-4) were isolated from the MeOH extracts of purple yam, which were subsequently determined to be cyanidin (1, 2, and 4) and peonidin (3) type compounds, along with four known anthocyanins (5-8). The structures of 1-4 were determined by spectroscopic methods, including NMR and MS analyses. The antioxidant activities of anthocyanins 1-8 were investigated using oxygen radical absorbing capacity and ferric reducing antioxidant power assays. PMID:25848974

  10. Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

    PubMed Central

    Borchardt, S. A.; Allain, E. J.; Michels, J. J.; Stearns, G. W.; Kelly, R. F.; McCoy, W. F.

    2001-01-01

    Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling. PMID:11425738

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

    PubMed

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

    2010-09-01

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

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

    PubMed

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

    2013-02-01

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

  13. A new acylated flavonol glycoside from Derris triofoliata.

    PubMed

    Xu, Lu-Rong; Wu, Jun; Zhang, Si

    2006-01-01

    A new acylated flavonol glycoside, kaempferol 3-O-[(6''''-feruloyl)-beta-D-glucopyranosyl-(1 --> 3)]-[alpha-L-rhamnopyranosyl-(1 --> 6)]-beta-D-glucopyranoside and two known cyclolignan glycosides, (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside and ( - )-lyoniresinol-3alpha-O-beta-D-glucopyranoside were isolated from n-BuOH extracts of the aerial parts of Derris triofoliata, their structures were determined from spectroscopic and chemical evidences.

  14. The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.

    PubMed

    Simic, Zeljko; Weiwad, Matthias; Schierhorn, Angelika; Steegborn, Clemens; Schutkowski, Mike

    2015-11-01

    Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl-CoA compounds. PMID:26382620

  15. The functional size of acyl-coenzyme A (CoA):cholesterol acyltransferase and acyl-CoA hydrolase as determined by radiation inactivation

    SciTech Connect

    Billheimer, J.T.; Cromley, D.A.; Kempner, E.S. )

    1990-05-25

    Frozen rat liver microsomes and rough endoplasmic reticulum were irradiated with high energy electrons. The surviving enzymatic activity of acyl-CoA:cholesterol acyltransferase and activity for esterification of 25-hydroxycholesterol decreased as a simple exponential function of radiation exposure, leading to a target size of 170-180 kDa. The loss of acyl-CoA hydrolase activity with a radiation dose was complex and resolved as a 45-kDa enzyme associated with a large inhibitor. It is interpreted that acyl-CoA hydrolase is the acyl-CoA-binding component and the inhibitor is the cholesterol-binding component of acyl-CoA:cholesterol acyltransferase.

  16. Metabolism of acyl-lipids in Chlamydomonas reinhardtii.

    PubMed

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

    2015-05-01

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

  17. Gastrointestinal uptake of nasunin, acylated anthocyanin in eggplant.

    PubMed

    Ichiyanagi, Takashi; Terahara, Norihiko; Rahman, M Mamunur; Konishi, Tetsuya

    2006-07-26

    We previously showed that nasunin, acylated anthocyanins in eggplant peel, comprises two isomers, cis-nasunin and trans-nasunin. In this study, gastrointestinal absorption of cis- and trans-nasunins was studied in rats. Orally administered nasunins were quickly absorbed in their original acylated forms and maximally appeared in blood plasma after 15 min. When the maximum plasma concentration and area under the plasma concentration curve were normalized by orally administered dose (micromoles per kilogram), there was no significant difference in the uptake efficiency between two isomers and both exhibited a plasma level almost identical to that of delphinidin 3-O-beta-D-glucopyranoside. However, metabolites such as 4'-O-methyl analogues and extended glucuronides which were observed for delphinidin 3-O-beta-D-glucopyranoside and cyanidin 3-O-beta-D-glucopyranoside metabolisms were not detected in urine or blood plasma. Moreover, deacylated and glycolytic products of nasunins such as delphinidin 3-O-beta-D-glucopyranoside or delphinidin (aglycone) were also not detected in blood plasma even after oral administration for 8 h. These results indicated that nasunins were absorbed in their original acylated forms and exhibit a bioavailability almost identical to that of nonacylated anthocyanins. PMID:16848510

  18. Naphthalene Derivatives Induce Acyl Chain Interdigitation in Dipalmitoylphosphatidylcholine Bilayers.

    PubMed

    Kamal, Md Arif; Raghunathan, V A

    2016-01-14

    The interdigitated phase of the lipid bilayer results when acyl chains from opposing monolayers fully interpenetrate such that the terminal methyl groups of the respective lipid chains are located at the interfacial region on the opposite sides of the bilayer. Usually, chain interdigitation is not encountered in a symmetric chain phosphatidylcholine (PC) membrane but can be induced under certain special conditions. In this article, we elucidate the contribution of small amphiphatic molecules in altering the physical properties of a symmetric chain PC bilayer membrane, which results in acyl chain interdigitation. Using small-angle X-ray scattering (SAXS), we have carried out a systematic investigation of the physical interactions of three naphthalene derivatives containing hydroxyl groups: β-naphthol, 2,3-dihydroxynaphthalene, and 2,7-dihydroxynaphthalene, with dipalmitoylphosphatidylcholine (DPPC) bilayers. On the basis of the diffraction patterns, we have determined the temperature-composition phase diagrams of these binary mixtures. The present study not only enables us to gain insight into the role played by small molecules in altering the packing arrangement of the acyl chains of the constituting PC lipids of the bilayer but also brings to light some important features that have not yet been reported hitherto. One such feature is the stabilization of the enigmatic asymmetric ripple phase over a wide temperature and concentration range. The results presented here strongly point toward a clear correlation between chain interdigitation and the stability of the ripple phase.

  19. Fatty acid acylation of salivary mucin in rat submandibular glands

    SciTech Connect

    Slomiany, B.L.; Murty, V.L.; Takagi, A.; Tsukada, H.; Kosmala, M.; Slomiany, A.

    1985-11-01

    The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with (/sup 3/H)palmitic acid and (/sup 3/H)proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of (/sup 3/H)palmitate and (/sup 3/H)proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.

  20. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

    Fernandes, Puja; Aldeborgh, Hannah; Carlucci, Lauren; Walsh, Lauren; Wasserman, Jordan; Zhou, Edward; Lefurgy, Scott T.; Mundorff, Emily C.

    2015-01-01

    The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates. PMID:25538307

  1. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  2. Nonthermal rotational distribution of CO/A 1Pi/ fragments produced by dissociative excitation of CO2 by electron impact. [in Mars atmosphere

    NASA Technical Reports Server (NTRS)

    Mumma, M. J.; Stone, E. J.; Zipf, E. C.

    1975-01-01

    Measurements were made of the rotational profiles of specific bands of the CO fourth-positive group (4PG). The CO 4PG bands were excited by electron impact dissociative excitation of CO2. The results are applicable to analysis of the Mariner observations of the CO 4PG in the dayglow of Mars. The results indicate that dissociative excitation of CO2 by electron impact leads to CO(A 1Pi) fragments with a rotational distribution that is highly nonthermal. The parent CO2 temperature was about 300 K in the experiment, while the fragment CO(A 1Pi) showed emission band profiles consistent with a rotational temperature greater than about 1500 K. Laboratory measurement of the reduced transmission of the hot bands by thermal CO appears to be the most direct way of determining the column density responsible for the CO(v',0) absorption of Mars.

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

    PubMed

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

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. PMID:23688416

  4. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  5. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  6. Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

    PubMed Central

    May, H D; Schauer, N L; Ferry, J G

    1986-01-01

    The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant. PMID:3700335

  7. NAD + -dependent Formate Dehydrogenase from Plants

    PubMed Central

    Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I.

    2011-01-01

    NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases. PMID:22649703

  8. Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts.

    PubMed

    Fukuoka, Tokuma; Morita, Tomotake; Konishi, Masaaki; Imura, Tomohiro; Kitamoto, Dai

    2007-07-01

    Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids. PMID:17417694

  9. Ciprofloxacin Therapy Results in Mitigation of ATP Loss after Irradiation Combined with Wound Trauma: Preservation of Pyruvate Dehydrogenase and Inhibition of Pyruvate Dehydrogenase Kinase 1.

    PubMed

    Swift, Joshua M; Smith, Joan T; Kiang, Juliann G

    2015-06-01

    Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited

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

    PubMed

    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

  11. Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

    PubMed

    Ding, Geng; Che, Ping; Ilarslan, Hilal; Wurtele, Eve S; Nikolau, Basil J

    2012-05-01

    3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

  12. Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.

    PubMed

    Soumya, Neelagiri; Tandan, Hitendra; Damre, Mangesh V; Gangwal, Rahul P; Sangamwar, Abhay T; Singh, Sushma

    2016-04-15

    AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340 nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme.

  13. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD.

    PubMed

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3',5'-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  14. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  15. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. PMID:27459246

  16. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the activity of the lactic dehydrogenase enzyme in serum. Increased levels of lactic dehydrogenase...

  17. Stimulation of bovine heart pyruvate dehydrogenase kinase by. cap alpha. -ketoisovalerate in the presence of thiamin pyrophosphate

    SciTech Connect

    Robertson, J.G.; Barron, L.L.; Olson, M.S.

    1987-05-01

    Purified bovine heart pyruvate dehydrogenase complex was used to study pyruvate dehydrogenase kinase (PDH kinase) regulation. Previously, they showed that KCl and NH4Cl stimulate PDH kinase, and that thiamin pyrophosphate over the range of 1-80 ..mu..M completely blocks PDH kinase stimulation by 20 mM KCl, whereas thiamin pyrophosphate has very little inhibitory effect in the absence of KCl. Under inhibitory conditions, 100 ..mu..M thiamin pyrophosphate and 20 mM KCl, addition of 1 mM ..cap alpha..-ketoisovalerate stimulated PDH kinase activity 1.5-fold. Addition of 1 mM ..cap alpha..-ketoisovalerate had a similar stimulatory effect in the presence of 1 mM NH4Cl and 100 ..mu..M thiamin pyrophosphate. Half-maximal stimulation occurred at about 200 ..mu..M ..cap alpha..-ketoisovalerate in both cases. Inhibition by thiamin pyrophosphate was uncompetitive, and the effect of ..cap alpha..-ketoisovalerate on thiamin pyrophosphate inhibition was to shift both Vm and Km toward control values without changing the slope of the reciprocal plot. After incubation of pyruvate dehydrogenase complex with ..cap alpha..-ketoisovalerate and gel filtration on Sephadex G-25 to remove ..cap alpha..-ketoisovalerate, PDH kinase activity remained stimulated relative to controls incubated with only buffer. In bovine heart mitochondria solubilized in 0.2% Triton X-100 and incubated with (..gamma..-/sup 32/P)ATP, ..cap alpha..-ketoisovalerate stimulated PDH phosphorylation in the presence of thiamin pyrophosphate, as detected by SDS-PAGE and autoradiography. The results indicate that acylation of pyruvate dehydrogenase complex results in PDH kinase stimulation, but only in the presence of monovalent cation and thiamin pyrophosphate. Furthermore, the results also suggest that this effect may be relevant under more in vivo conditions.

  18. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  19. Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice.

    PubMed

    Posch, K C; Napoli, J L

    1992-05-28

    Retinoic acid syntheses from retinol by cytosol from testes of alcohol dehydrogenase negative or positive deermice were similar in specific activity and in their insensitivity to 1 M ethanol or 100 mM 4-methylpyrazole. Anion-exchange followed by size-exclusion chromatography revealed multiple and similarly migrating peaks in each cytosol that had both retinol and retinal dehydrogenase activities. Thus, the effects of ethanol on testes cannot be caused by direct inhibition of cytosolic retinoic acid synthesis because retinoid dehydrogenases distinct from mouse class A2 alcohol dehydrogenases, which corresponds to human class I, occurred in testes and they were not inhibited by ethanol. These data also demonstrate the occurrence of multiple cytosolic retinoic acid synthesis activities and indicate that the two reactions of cytosolic retinoic acid synthesis, retinol and retinal dehydrogenation, may be catalyzed by enzymes that occur as complexes. PMID:1599517

  20. The physiological role of liver alcohol dehydrogenase.

    PubMed

    Krebs, H A; Perkins, J R

    1970-07-01

    1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores

  1. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a.

    PubMed

    Fang, M; Jin, A; Zhao, Y; Liu, X

    2016-02-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  2. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

    PubMed Central

    Fang, M.; Jin, A.; Zhao, Y.; Liu, X.

    2016-01-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  3. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic dehydrogenase... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactic dehydrogenase immunological test...

  4. Properties of formate dehydrogenase in Methanobacterium formicicum.

    PubMed Central

    Schauer, N L; Ferry, J G

    1982-01-01

    Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm. Images PMID:7061389

  5. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    SciTech Connect

    Girio, F.M.; Amaral-Collaco, M.T.; Pelica, F.

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  6. Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

    PubMed Central

    Tomatis, Vanesa M.; Trenchi, Alejandra; Gomez, Guillermo A.; Daniotti, Jose L.

    2010-01-01

    An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution. PMID:21152083

  7. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

    PubMed

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-07-19

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

  8. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes

    PubMed Central

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  9. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes.

    PubMed

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  10. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives' Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps.

    PubMed

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  11. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives’ Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps

    PubMed Central

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  12. Ortho C-H Acylation of Aryl Iodides by Palladium/Norbornene Catalysis.

    PubMed

    Dong, Zhe; Wang, Jianchun; Ren, Zhi; Dong, Guangbin

    2015-10-19

    Reported herein is a palladium/norbornene-catalyzed ortho-arene acylation of aryl iodides by a Catellani-type C-H functionalization. This transformation is enabled by isopropyl carbonate anhydrides, which serve as both an acyl cation equivalent and a hydride source.

  13. Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride.

    PubMed

    Li, Conghu; Tian, Zhenhua; Liu, Wentao; Li, Guoying

    2015-10-01

    The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen.

  14. Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers

    PubMed Central

    Bottomley, Peter J.

    2015-01-01

    Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS. PMID:26092466

  15. Synthesis of photoactivatable azido-acyl caged oxazine fluorophores for live-cell imaging.

    PubMed

    Anzalone, Andrew V; Chen, Zhixing; Cornish, Virginia W

    2016-07-19

    We report the design and synthesis of a photoactivatable azido-acyl oxazine fluorophore. Photoactivation is achieved cleanly and rapidly with UV light, producing a single fluorescent oxazine photoproduct. We demonstrate the utility of azido-acyl caged oxazines for protein specific labeling in living mammalian cells using the TMP-tag technology. PMID:27377037

  16. Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers.

    PubMed

    Mellbye, Brett L; Bottomley, Peter J; Sayavedra-Soto, Luis A

    2015-09-01

    Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

  17. Turnover of the 4'-phosphopantetheine prosthetic group of acyl carrier protein.

    PubMed

    Jackowski, S; Rock, C O

    1984-02-10

    Acyl carrier protein is an essential cofactor in fatty acid biosynthesis, and in contrast to the stability of the protein moiety during growth, its 4'-phosphopantetheine prosthetic group is metabolically active. The biosynthetic incorporation of deuterium into nonexchangeable positions of acyl carrier protein was found to enhance the sensitivity of the protein to pH-induced hydrodynamic expansion. This constitutional isotope effect was exploited to separate deuterated from normal acyl carrier protein by conformationally sensitive gel electrophoresis, thus providing the analytical framework for separating pre-existing (deuterated) from newly synthesized acyl carrier protein in pulse-chase experiments. The rate of acyl carrier protein prosthetic group turnover was found to depend on the intracellular concentration of coenzyme A. At low coenzyme A levels, prosthetic group turnover was four times faster than the rate of new acyl carrier protein biosynthesis but at the higher coenzyme A concentrations characteristic of logarithmic growth, turnover was an order of magnitude slower, amounting to approximately 25% of the acyl carrier protein pool per generation. These observations suggest that the acyl carrier protein prosthetic group turnover cycle may be related to coenzyme A metabolism rather than to lipid biosynthesis.

  18. Membrane Topology and Transient Acylation of Toxoplasma gondii Glycosylphosphatidylinositols

    PubMed Central

    Kimmel, Jürgen; Smith, Terry K.; Azzouz, Nahid; Gerold, Peter; Seeber, Frank; Lingelbach, Klaus; Dubremetz, Jean-François; Schwarz, Ralph T.

    2006-01-01

    Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-3H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-3H]mannose or UDP-[6-3H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER. PMID:16896225

  19. Fatty acid hydrolysis of acyl marinobactin siderophores by Marinobacter acylases.

    PubMed

    Kem, Michelle P; Naka, Hiroaki; Iinishi, Akira; Haygood, Margo G; Butler, Alison

    2015-01-27

    The marine bacteria Marinobacter sp. DS40M6 and Marinobacter nanhaiticus D15-8W produce a suite of acyl peptidic marinobactin siderophores to acquire iron under iron-limiting conditions. During late-log phase growth, the marinobactins are hydrolyzed to form the marinobactin headgroup with release of the corresponding fatty acid tail. The bntA gene, a homologue of the Pseudomonas aeruginosa pyoverdine acylase gene, pvdQ, was identified from Marinobacter sp. DS40M6. A bntA knockout mutant of Marinobacter sp. DS40M6 produced the suite of acyl marinobactins A-E, without the usual formation of the marinobactin headgroup. Another marinobactin-producing species, M. nanhaiticus D15-8W, is predicted to have two pvdQ homologues, mhtA and mhtB. MhtA and MhtB have 67% identical amino acid sequences. MhtA catalyzes hydrolysis of the apo-marinobactin siderophores as well as the quorum sensing signaling molecule, dodecanoyl-homoserine lactone. In contrast to hydrolysis of the suite of apo-marinobactins by MhtA, hydrolysis of the iron(III)-bound marinobactins was not observed. PMID:25588131

  20. Site‐Selective Acylations with Tailor‐Made Catalysts

    PubMed Central

    Huber, Florian

    2016-01-01

    Abstract The acylation of alcohols catalyzed by N,N‐dimethylamino pyridine (DMAP) is, despite its widespread use, sometimes confronted with substrate‐specific problems: For example, target compounds with multiple hydroxy groups may show insufficient selectivity for one hydroxyl, and the resulting product mixtures are hardly separable. Here we describe a concept that aims at tailor‐made catalysts for the site‐specific acylation. To this end, we introduce a catalyst library where each entry is constructed by connecting a variable and readily tuned peptide scaffold with a catalytically active unit based on DMAP. For selected examples, we demonstrate how library screening leads to the identification of optimized catalysts, and the substrates of interest can be converted with a markedly enhanced site‐selectivity compared with only DMAP. Furthermore, substrate‐optimized catalysts of this type can be used to selectively convert “their” substrate in the presence of structurally similar compounds, an important requisite for reactions with mixtures of substances. PMID:26970553

  1. The regulation of adipose tissue pyruvate dehydrogenase activity of dietary fiber.

    PubMed

    Ogunwole, J O; Knight, E M; Adkins, J S; Thomaskutty, K G; Pointer, R H

    1987-05-01

    In vitro studies have established that insulin enhances the oxidation of pyruvate to acetyl CoA by the stimulation of mitochondrial pyruvate dehydrogenase (PDH) activity through plasma membrane binding response (Jarett and Seals 1979; Kiechle, Jarett, Dennis and Kotagal 1980). In the present study adipose tissue PDH activity was utilized as a marker for insulin responsiveness. The metabolic response of this enzyme to exogenous insulin was employed to test the hypothesis that dietary fiber enhances tissue responsiveness to insulin using adipose tissue from male weanling Sprague Dawley rats. Eight groups of rats (n = 5 per group) were fed ad libitum various diets containing different levels of cellulose and protein as already reported elsewhere (Ogunwole, Knight, Adkins, Thomaskutty and Pointer 1985). Percent insulin stimulation of PDH from basal activity (PDS) was utilized as an index of insulin responsiveness. Compared to all fiber treated groups, both basal (PDB) and insulin stimulated (PDI) activities were significantly lower (P less than 0.05) in the fiber free groups at both low (10%) and high (20%) protein levels. At all fiber levels tested (0, 5, 15 and 30%) protein intake resulted in a significant increase in both PDB and PDI. Gradual increase in cellulose intake resulted in a biphasic increase in PDS in both protein groups at the 5% and 30% fiber levels. PDS was higher (P less than 0.05) in the 10% protein groups than the 20% protein group at all fiber levels tested. A significant interaction effect of protein and fiber was observed on PDB (P less than 0.001) and PDI (P less than 0.04) when caloric intake was held constant as a covariate.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum. Progress report, March 25, 1993--March 24, 1994

    SciTech Connect

    Ragsdale, S.W.

    1994-04-01

    Anaerobic acetogenic bacteria can convert carbon dioxide and carbon monoxide to cell carbon by a pathway which is called the acetyl-CoA pathway. With this pathway they convert monosaccharides and the methoxy group of lignin derived aromatics into acetic acid. The acetic acid is then used by a number of organisms, including methanogens, as a carbon and energy source. Therefore, the acetyl-CoA pathway links the biodegradation of complex macromolecules like cellulose and lignin to the utilization of simple two carbon units. The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. We have previously demonstrate that the conversion of CH{sub 3}-H{sub 4} folate, CO and CoA to acetyl-CoA involves enzyme-bound intermediates that the one- and two-carbon precursors of acetyl-CoA are organometallic complexes, and that the site for assembly of acetyl-CoA is a novel Ni-Fe-S cluster which binds CO as a terminal carbonyl, i.e., M-C {equivalent_to} O. In the past year we have shown that the activities for CO oxidation and acetyl-CoA synthesis sites occur at separate sites, that it is a Fe, not a Ni, site in the Ni-Fe-S cluster which binds CO for acetyl-CoA synthesis, and that carbon disulfide (CS2) reacts with CODH at the Ni-Fe-S site to generate an isolated Ni(I) species. We also determined that CS{sub 2} is competitive with CO at the acetyl-CoA synthesis site and does not bind to the CO oxidation/CO{sub 2} reduction site.

  3. Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement.

    PubMed

    Trier, Sofie; Linderoth, Lars; Bjerregaard, Simon; Strauss, Holger M; Rahbek, Ulrik L; Andresen, Thomas L

    2015-10-01

    Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may

  4. Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

    PubMed

    Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

    2013-01-17

    Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis.

  5. Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

    PubMed

    Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

    2013-01-01

    Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis. PMID:23344208

  6. In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: immunoblot identification of acylated PLP and DM-20

    SciTech Connect

    Garwood, M.M.; Gilbert, W.R.; Agrawal, H.C.

    1983-05-01

    The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of (/sup 3/H)palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.

  7. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting...

  8. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl...

  9. COA User's Guide

    SciTech Connect

    Fox, B.; Pautz, J.; Sellers, C.

    1999-01-28

    The Department of Energy (DOE) has one of the largest and most complete collections of information on crude oil composition that is available to the public. The computer program that manages this database of crude oil analyses has recently been rewritten to allow easier access to this information. This report describes how the new system can be accessed and how the information contained in the Crude Oil Analysis Data Bank can be obtained.

  10. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility.

  11. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility. PMID:5917779

  12. Steady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2014-05-01

    Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100% of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f(a) = 1.0) was significantly higher than cesium ions (f(a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f(a) = 0.54), whereas quenching with iodide ion was decreased (f(a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions. PMID:24322526

  13. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  14. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD

    PubMed Central

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3′,5′-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  15. Cardiac-specific deletion of acetyl CoA carboxylase 2 (ACC2) prevents metabolic remodeling during pressure-overload hypertrophy

    PubMed Central

    Kolwicz, Stephen C.; Olson, David P.; Marney, Luke C.; Garcia-Menendez, Lorena; Synovec, Robert E.; Tian, Rong

    2012-01-01

    Rationale Decreased fatty acid oxidation (FAO) with increased reliance on glucose are hallmarks of metabolic remodeling that occurs in pathological cardiac hypertrophy and is associated with decreased myocardial energetics and impaired cardiac function. To date, it has not been tested whether prevention of the metabolic switch that occurs during the development of cardiac hypertrophy has unequivocal benefits on cardiac function and energetics. Objectives Since malonyl CoA production via acetyl CoA carboxylase 2 (ACC2) inhibits mitochondrial fatty acid transport, we hypothesized that mice with a cardiac-specific deletion of ACC2 (ACC2H−/−) would maintain cardiac fatty acid oxidation (FAO) and improve function and energetics during the development of pressure-overload hypertrophy. Methods and Results ACC2 deletion led to a significant reduction in cardiac malonyl CoA levels. In isolated perfused heart experiments, left ventricular (LV) function and oxygen consumption were similiar in ACC2H−/− mice despite an ~60% increase in FAO compared to controls (CON). After 8 weeks of pressure-overload via transverse aortic constriction (TAC), ACC2H−/− mice exhibited a substrate utilization profile similar to sham animals while CON-TAC hearts had decreased FAO with increased glycolysis and anaplerosis. Myocardial energetics, assessed by 31P NMR spectroscopy, and cardiac function were maintained in ACC2H−/− after 8 weeks of TAC. Furthermore, ACC2H−/−-TAC demonstrated an attenuation of cardiac hypertrophy with a significant reduction in fibrosis relative to CON-TAC. Conclusions These data suggest that reversion to the fetal metabolic profile in chronic pathological hypertrophy is associated with impaired myocardial function and energetics and maintenance of the inherent cardiac metabolic profile and mitochondrial oxidative capacity is a viable therapeutic strategy. PMID:22730442

  16. Succinate dehydrogenase-deficient gastrointestinal stromal tumors

    PubMed Central

    Wang, Ya-Mei; Gu, Meng-Li; Ji, Feng

    2015-01-01

    Most gastrointestinal stromal tumors (GISTs) are characterized by KIT or platelet-derived growth factor alpha (PDGFRA) activating mutations. However, there are still 10%-15% of GISTs lacking KIT and PDGFRA mutations, called wild-type GISTs (WT GISTs). Among these so-called WT GISTs, a small subset is associated with succinate dehydrogenase (SDH) deficiency, known as SDH-deficient GISTs. In addition, GISTs that occur in Carney triad and Carney-Stratakis syndrome represent specific examples of SDH-deficient GISTs. SDH-deficient GISTs locate exclusively in the stomach, showing predilection for children and young adults with female preponderance. The tumor generally pursues an indolent course and exhibits primary resistance to imatinib therapy in most cases. Loss of succinate dehydrogenase subunit B expression and overexpression of insulin-like growth factor 1 receptor (IGF1R) are common features of SDH-deficient GISTs. In WT GISTs without succinate dehydrogenase activity, upregulation of hypoxia-inducible factor 1α may lead to increased growth signaling through IGF1R and vascular endothelial growth factor receptor (VEGFR). As a result, IGF1R and VEGFR are promising to be the novel therapeutic targets of GISTs. This review will update the current knowledge on characteristics of SDH-deficient GISTs and further discuss the possible mechanisms of tumorigenesis and clinical management of SDH-deficient GISTs. PMID:25741136

  17. Prenatal presentation of pyruvate dehydrogenase complex deficiency.

    PubMed

    Natarajan, Niranjana; Tully, Hannah M; Chapman, Teresa

    2016-08-01

    We present the case of a female infant referred for prenatal MR evaluation of ventriculomegaly, which had been attributed by the referring obstetrician to aqueductal stenosis. Fetal MR confirmed ventriculomegaly but also demonstrated cerebral volume loss and white matter abnormalities. After birth, the infant developed persistent lactic acidosis. A diagnosis of pyruvate dehydrogenase complex deficiency was made on the basis of metabolic and molecular genetic studies. Ventriculomegaly is a common referral reason for fetal MR, yet there are few published reports of the radiographic findings that accompany inborn errors of metabolism, one potentially under-recognized cause of enlarged ventricles. This case contributes to this small body of literature on the imaging features of pyruvate dehydrogenase complex deficiency by describing pre- and postnatal MR findings and key clinical details. Our report emphasizes the necessity of considering pyruvate dehydrogenase complex deficiency and other metabolic disorders as potential etiologies for fetal ventriculomegaly since prompt diagnosis may allow for early initiation of treatment and improve outcome. PMID:27026023

  18. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    SciTech Connect

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  19. Relationships within the aldehyde dehydrogenase extended family.

    PubMed

    Perozich, J; Nicholas, H; Wang, B C; Lindahl, R; Hempel, J

    1999-01-01

    One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.

  20. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed Central

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase. PMID:12228351

  1. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

  2. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

    PubMed Central

    Nguyen, Chi; Haushalter, Robert W.; Lee, D. John; Markwick, Phineus R. L.; Bruegger, Joel; Caldara-Festin, Grace; Finzel, Kara; Jackson, David R.; Ishikawa, Fumihiro; O’Dowd, Bing; McCammon, J. Andrew; Opella, Stanley J.; Tsai, Shiou-Chuan; Burkart, Michael D.

    2015-01-01

    Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious

  3. Acyl-Homoserine Lactone Quorum Sensing in the Roseobacter Clade

    PubMed Central

    Zan, Jindong; Liu, Yue; Fuqua, Clay; Hill, Russell T.

    2014-01-01

    Members of the Roseobacter clade are ecologically important and numerically abundant in coastal environments and can associate with marine invertebrates and nutrient-rich marine snow or organic particles, on which quorum sensing (QS) may play an important role. In this review, we summarize current research progress on roseobacterial acyl-homoserine lactone-based QS, particularly focusing on three relatively well-studied representatives, Phaeobacter inhibens DSM17395, the marine sponge symbiont Ruegeria sp. KLH11 and the dinoflagellate symbiont Dinoroseobacter shibae. Bioinformatic survey of luxI homologues revealed that over 80% of available roseobacterial genomes encode at least one luxI homologue, reflecting the significance of QS controlled regulatory pathways in adapting to the relevant marine environments. We also discuss several areas that warrant further investigation, including studies on the ecological role of these diverse QS pathways in natural environments. PMID:24402124

  4. Acylation in trypanosomatids: an essential process and potential drug target

    PubMed Central

    Goldston, Amanda M.; Sharma, Aabha I.; Paul, Kimberly S.; Engman, David M.

    2014-01-01

    Fatty acylation—the addition of fatty acid moieties such as myristate and palmitate to proteins—is essential for the survival, growth, and infectivity of the trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. Myristoylation and palmitoylation are critical for parasite growth, targeting and localization, and the intrinsic function of some proteins. The trypanosomatids possess a single N-myristoyltransferase (NMT) and multiple palmitoyl acyltransferases, and these enzymes and their cellular targets are only now being characterized. Global inhibition of either process leads to cell death in trypanosomatids, and genetic ablation of NMT compromises virulence. Moreover, NMT inhibitors effectively cure T. brucei infection in rodents. Thus, protein acylation represents an attractive target for the development of trypanocidal drugs. PMID:24954795

  5. Synthesis of acyl derivatives of salicin, salirepin, and arbutin.

    PubMed

    Stepanova, Elena V; Belyanin, Maxim L; Filimonov, Victor D

    2014-03-31

    The total synthesis of two natural phenolglycosides of the family Salicaceae, namely: populoside and 2-(β-d-glucopyranosyloxy)-5-hydroxy benzyl (3-methoxy-4-hydroxy) cinnamoate and nine not found yet in plants acyl derivatives of phenoglycosides: 2-(β-d-glucopyranosyloxy)-benzylcinnamoate, 2-(β-d-glucopyranosyloxy)-benzyl (4-hydroxy) benzoate, 2-(β-d-glucopyranosyloxy)-benzyl (3-methoxy-4-hydroxy) benzoate, 2-(β-d-glucopyranosyloxy)-5-hydroxy benzyl (3,4-dihydroxy) cinnamoate, 2-(β-d-glucopyranosyloxy)-5-hydroxy benzylcinnamoate, 2-(β-d-glucopyranosyloxy)-5-hydroxy benzyl (4-hydroxy) benzoate, 2-(β-d-glucopyranosyloxy)-5-hydroxy benzyl (3-methoxy-4-hydroxy) benzoate, 2-(β-d-glucopyranosyloxy)-5-benzoyloxy benzylbenzoate and 4-(β-d-glucopyranosyloxy)-phenylbenzoate, starting from readily available phenols and glucose was developed for the first time.

  6. Small Antimicrobial Agents Based on Acylated Reduced Amide Scaffold.

    PubMed

    Teng, Peng; Huo, Da; Nimmagadda, Alekhya; Wu, Jianfeng; She, Fengyu; Su, Ma; Lin, Xiaoyang; Yan, Jiyu; Cao, Annie; Xi, Chuanwu; Hu, Yong; Cai, Jianfeng

    2016-09-01

    Prevalence of drug-resistant bacteria has emerged to be one of the greatest threats in the 21st century. Herein, we report the development of a series of small molecular antibacterial agents that are based on the acylated reduced amide scaffold. These molecules display good potency against a panel of multidrug-resistant Gram-positive and Gram-negative bacterial strains. Meanwhile, they also effectively inhibit the biofilm formation. Mechanistic studies suggest that these compounds kill bacteria by compromising bacterial membranes, a mechanism analogous to that of host-defense peptides (HDPs). The mechanism is further supported by the fact that the lead compounds do not induce resistance in MRSA bacteria even after 14 passages. Lastly, we also demonstrate that these molecules have therapeutic potential by preventing inflammation caused by MRSA induced pneumonia in a rat model. This class of compounds could lead to an appealing class of antibiotic agents combating drug-resistant bacterial strains. PMID:27526720

  7. Detection of acyl-homoserine lactones by Escherichia and Salmonella

    PubMed Central

    Soares, Jitesh A.; Ahmer, Brian M. M.

    2011-01-01

    Escherichia and Salmonella do not synthesize quorum sensing signaling molecules of the N-acyl-L-homoserine lactone (AHL) type but they can detect AHLs produced by other species of bacteria. AHLs are present in the bovine rumen but not in the remainder of the gastrointestinal tract. Enterohemorrhagic E. coli (EHEC) responds to AHLs extracted from the bovine rumen. Salmonella fails to detect AHLs in the gastrointestinal tracts of pathogen-free mice or pigs, suggesting that AHLs are not present. However, Salmonella does detect the AHL production of Yersinia enterocolitica in mouse Peyer’s patches. In response to AHLs, EHEC represses flagellar genes and the LEE pathogenicity island while it activates the acid fitness island, whereas Salmonella activates the rck operon and a gene, srgE, encoding a putative Type III secreted effector. PMID:21353625

  8. Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

    PubMed

    Kite, Geoffrey C; Larsson, Sonny; Veitch, Nigel C; Porter, Elaine A; Ding, Ning; Simmonds, Monique S J

    2013-04-10

    LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

  9. Preparation and Characterization of O-Acylated Fucosylated Chondroitin Sulfate from Sea Cucumber

    PubMed Central

    Gao, Na; Wu, Mingyi; Liu, Shao; Lian, Wu; Li, Zi; Zhao, Jinhua

    2012-01-01

    Fucosylated chondroitin sulfate (FuCS), a kind of complex glycosaminoglycan from sea cucumber, has potent anticoagulant activity. In order to understand the relationship between structures and activity, the depolymerized FuCS (dFuCS) was chosen to prepare its derivates by selective substitution at OH groups. Its O-acylation was carried out in a homogeneous way using carboxylic acid anhydrides. The structures of O-acylated derivatives were characterized by NMR. The results indicated that the 4-O-sulfated fucose residues may be easier to be acylated than the other ones in the sulfated fucose branches. But the O-acylation was always accompanied by the β-elimination, and the degree of elimination was higher as that of acylation was higher. The results of clotting assay indicated that the effect of partial O-acylation of the dFuCS on their anticoagulant potency was not significant and the O-acylation of 2-OH groups of 4-O-sulfated fucose units did not affect the anticoagulant activity. PMID:23015767

  10. Preparation and characterization of O-acylated fucosylated chondroitin sulfate from sea cucumber.

    PubMed

    Gao, Na; Wu, Mingyi; Liu, Shao; Lian, Wu; Li, Zi; Zhao, Jinhua

    2012-08-01

    Fucosylated chondroitin sulfate (FuCS), a kind of complex glycosaminoglycan from sea cucumber, has potent anticoagulant activity. In order to understand the relationship between structures and activity, the depolymerized FuCS (dFuCS) was chosen to prepare its derivates by selective substitution at OH groups. Its O-acylation was carried out in a homogeneous way using carboxylic acid anhydrides. The structures of O-acylated derivatives were characterized by NMR. The results indicated that the 4-O-sulfated fucose residues may be easier to be acylated than the other ones in the sulfated fucose branches. But the O-acylation was always accompanied by the β-elimination, and the degree of elimination was higher as that of acylation was higher. The results of clotting assay indicated that the effect of partial O-acylation of the dFuCS on their anticoagulant potency was not significant and the O-acylation of 2-OH groups of 4-O-sulfated fucose units did not affect the anticoagulant activity.

  11. Synthesis and evaluation of novel acyl derivatives from jatropha oil as potential lubricant basestocks.

    PubMed

    Sammaiah, Arukali; Padmaja, Korlipara V; Prasad, Rachapudi B N

    2014-05-21

    A novel class of jatropha oil-based acylated derivatives from hydroxy alkyl esters of jatropha fatty acids (C1, C3, C4, and C8) and various anhydrides (C2, C3, C4, and C6) were synthesized and their physicochemical and lubricant properties reported. Jatropha fatty acid alkyl esters were dihydroxylated using the in situ performic acid method and further acylated with different anhydrides to produce acylated derivatives. Acylated derivatives of dihydroxy jatropha fatty acid alkyl esters were charaterized by NMR, FTIR, GC, and GC-MS analysis and were evaluated for their viscosity, viscosity index, pour and flash points, and oxidation stability. Most of the derivatives are either in ISO VG 22 or 32 viscosity grade with good viscosity index. It was observed that increase in acyl chain length and branching in the end-chain ester improved the pour point of the diacyl derivatives. All of the hexanoylated esters exhibited better oxidation stability compared to other acylated products, and their pour points are comparable to those of synthetic esters such as TMP trioleates. In general, isoalcohol esters with longer acyl chains showed promise as potential candidates for hydraulic fluids and metal-working fluids in ISO VG 22 and 32 viscosity range.

  12. N-Acylation During Glidobactin Biosynthesis by the Tridomain Nonribosomal Peptide Synthetase Module GlbF

    PubMed Central

    Imker, Heidi J.; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T.

    2011-01-01

    Summary Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on co-expression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr1 amino group and generate the fatty acyl-Thr1-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis. PMID:21035730

  13. N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF.

    PubMed

    Imker, Heidi J; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T

    2010-10-29

    Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on coexpression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr(1) amino group and generate the fatty acyl-Thr(1)-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis.

  14. Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions*

    PubMed Central

    Lager, Ida; Yilmaz, Jenny Lindberg; Zhou, Xue-Rong; Jasieniecka, Katarzyna; Kazachkov, Michael; Wang, Peng; Zou, Jitao; Weselake, Randall; Smith, Mark A.; Bayon, Shen; Dyer, John M.; Shockey, Jay M.; Heinz, Ernst; Green, Allan; Banas, Antoni; Stymne, Sten

    2013-01-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism. PMID:24189065

  15. A comparison of potato and vertebrate lactate dehydrogenases.

    PubMed Central

    Poerio, E; Davies, D D

    1980-01-01

    A 2000-fold purification of L(+)-lactate dehydrogenase from potatoes is reported. Five isoenzymes of lactate dehydrogenase can be detected in crude extracts of potato, and three of these are present in the purified preparation. The enzyme (mol.wt. 150 000), which is composed of four subunits (mol.wt. 37 500), is active with the same oxo acids and hydroxy acids that have been reported as substrates with the same oxo acids and hydroxy acids that have been reported as substrates for vertebrate lactate dehydrogenases. These similarities between potato and vertebrate lactate dehydrogenases contrast sharply with some other reports on potato lactate dehydrogenase. These discrepancies are discussed in relation to the proposition that vertebrate and potato lactate dehydrogenases share a common evolutionary origin. PMID:7236200

  16. Exploring Cooperative Effects in Oxidative NHC Catalysis: Regioselective Acylation of Carbohydrates.

    PubMed

    Cramer, David L; Bera, Srikrishna; Studer, Armido

    2016-05-23

    The utility of oxidative NHC catalysis for both the regioselective and chemoselective functionalization of carbohydrates is explored. Chiral NHCs allow for the highly regioselective oxidative esterification of various carbohydrates using aldehydes as acylation precursors. The transformation was also shown to be amenable to both cis/trans diol isomers, free amino groups, and selective for specific sugar epimers in competition experiments. Efficiency and regioselectivity of the acylation can be improved upon using two different NHC catalysts that act cooperatively. The potential of the method is documented by the regioselective acylation of an amino-linked neodisaccharide.

  17. Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

    PubMed Central

    Jörnvall, Hans

    1973-01-01

    The primary structure of about half of the protein chain of yeast alcohol dehydrogenase has been determined and compared with the amino-acid sequences of other dehydrogenases. The enzyme is found to be distantly related to horse-liver alcohol dehydrogenase, although these two proteins have different quaternary structures and subunit sizes. Some regions show no significant similarities, but long segments within the N-terminal parts of the molecules are homologous, suggesting a common and important function for these segments. Ancestral connections between some different dehydrogenases can be concluded and the degree of evolutionary changes may be estimated. PMID:4599620

  18. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  19. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  20. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  1. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  2. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  3. Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model.

    PubMed

    Tai, Akihiro; Goto, Satomi; Ishiguro, Yutaka; Suzuki, Kazuko; Nitoda, Teruhiko; Yamamoto, Itaru

    2004-02-01

    A series of novel lipophilic vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids possessing a branched-acyl chain of varying length from C(8) to C(16) (6-bAcyl-AA-2G), were evaluated as topical prodrugs of ascorbic acid (AA) with transdermal activity in a human living skin equivalent model. The permeability of 6-bAcyl-AA-2G was compared with those of the derivatives having a straight-acyl chain (6-sAcyl-AA-2G). Out of 10 derivatives of 6-sAcyl-AA-2G and 6-bAcyl-AA-2G, 6-sDode-AA-2G and 6-bDode-AA-2G exhibited most excellent permeability in this model. Measurement of the metabolites permeated from the skin model suggested that 6-bDode-AA-2G was mainly hydrolyzed via 6-O-acyl AA to AA by tissue enzymes, while 6-sDode-AA-2G was hydrolyzed via 2-O-alpha-D-glucopyranosyl-L-ascorbic acid to AA. The former metabolic pathway seems to be advantageous for a readily available source of AA, because 6-O-acyl AA, as well as AA, is able to show vitamin C activity.

  4. Enhanced Activity of Nanocrystalline Beta Zeolite for Acylation of Veratrole with Acetic Anhydride.

    PubMed

    Aisha Mahmood Abdulkareem, Al-Turkustani; Selvin, Rosilda

    2016-04-01

    Friedel-Craft acylation of veratrole using homogeneous acid catalysts such as AlCl3, FeCl3, ZnCl2, and HF etc. produces acetoveratrone, (3',4'-dimethoxyacetophenone), which is the intermediate for synthesis of papavarine alkaloids. The problems associated with these homogeneous catalysts can be overcome by using heterogeneous solid catalysts. Since acetoveratrone is a larger molecule, large pore Beta zeolites with smaller particle sizes are beneficial for the liquid-phase acylation of veratrole, for easy diffusion of reactants and products. The present study aims in the acylation of veratrole with acetic anhydride using nanocrystalline Beta Zeolite catalyst. A systematic investigation of the effects of various reaction parameters was done. The catalysts were characterized for their structural features by using XRD, TEM and DLS analyses. The catalytic activity of nanocrystalline Beta zeolite was compared with commercial Beta zeolite for the acylation and was found that nanocrystalline Beta zeolite possessed superior activity.

  5. The Acute Effects of Swimming on Appetite, Food Intake, and Plasma Acylated Ghrelin

    PubMed Central

    King, James A.; Wasse, Lucy K.; Stensel, David J.

    2011-01-01

    Swimming may stimulate appetite and food intake but empirical data are lacking. This study examined appetite, food intake, and plasma acylated ghrelin responses to swimming. Fourteen healthy males completed a swimming trial and a control trial in a random order. Sixty min after breakfast participants swam for 60 min and then rested for six hours. Participants rested throughout the control trial. During trials appetite was measured at 30 min intervals and acylated ghrelin was assessed periodically (0, 1, 2, 3, 4, 6, and 7.5 h. N = 10). Appetite was suppressed during exercise before increasing in the hours after. Acylated ghrelin was suppressed during exercise. Swimming did not alter energy or macronutrient intake assessed at buffet meals (total trial energy intake: control 9161 kJ, swimming 9749 kJ). These findings suggest that swimming stimulates appetite but indicate that acylated ghrelin and food intake are resistant to change in the hours afterwards. PMID:20953411

  6. Enhanced Activity of Nanocrystalline Beta Zeolite for Acylation of Veratrole with Acetic Anhydride.

    PubMed

    Aisha Mahmood Abdulkareem, Al-Turkustani; Selvin, Rosilda

    2016-04-01

    Friedel-Craft acylation of veratrole using homogeneous acid catalysts such as AlCl3, FeCl3, ZnCl2, and HF etc. produces acetoveratrone, (3',4'-dimethoxyacetophenone), which is the intermediate for synthesis of papavarine alkaloids. The problems associated with these homogeneous catalysts can be overcome by using heterogeneous solid catalysts. Since acetoveratrone is a larger molecule, large pore Beta zeolites with smaller particle sizes are beneficial for the liquid-phase acylation of veratrole, for easy diffusion of reactants and products. The present study aims in the acylation of veratrole with acetic anhydride using nanocrystalline Beta Zeolite catalyst. A systematic investigation of the effects of various reaction parameters was done. The catalysts were characterized for their structural features by using XRD, TEM and DLS analyses. The catalytic activity of nanocrystalline Beta zeolite was compared with commercial Beta zeolite for the acylation and was found that nanocrystalline Beta zeolite possessed superior activity. PMID:27451793

  7. Fat Metabolism in Higher Plants: LXII. Stearl-acyl Carrier Protein Desaturase from Spinach Chloroplasts.

    PubMed

    Jacobson, B S; Jaworski, J G; Stumpf, P K

    1974-10-01

    Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds.

  8. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    SciTech Connect

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H. )

    1991-04-15

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC.

  9. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

    Uhlinger, D.J.; Reed, L.J.

    1986-05-01

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg/sup 2 +/, and (..gamma..-/sup 32/P)ATP. The protein-bound radioactivity was localized in the PDH ..cap alpha.. subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg/sup 2 +/, and Ca/sup 2 +/. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the ..cap alpha.. subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

  10. Transcriptional regulation of pyruvate dehydrogenase kinase.

    PubMed

    Jeong, Ji Yun; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2012-10-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes. PMID:23130316

  11. New N-acyl taurine from the sea urchin Glyptocidaris crenularis.

    PubMed

    Zhou, Xuefeng; Xu, Tunhai; Wen, Kewei; Yang, Xian-Wen; Xu, Shi-Hai; Liu, Yonghong

    2010-01-01

    A new N-acyl taurine (1), together with a new natural product, l-(beta-D-ribofuranosyl)-1,2,4-triazole (4), and two known compounds (2 and 3), were isolated from the sea urchin, Glyptocidaris crenularis. The new N-acyl taurine was elucidated as 2-(5R,15S-dihydroxyeicosanoylamino) ethanesulfonic acid on the basis of spectroscopic (NMR, MS) analyses and the modified Mosher ester method. Compound 2 showed significant toxicity against brine shrimp larvae.

  12. LuxR homolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa.

    PubMed

    Chugani, Sudha; Greenberg, Everett Peter

    2010-06-01

    Pseudomonas aeruginosa quorum control of gene expression involves three LuxR-type signal receptors LasR, RhlR, and QscR that respond to the LasI- and RhlI-generated acyl-homoserine lactone (acyl-HSL) signals 3OC12-HSL and C4-HSL. We found that a LasR-RhlR-QscR triple mutant responds to acyl-HSLs by regulating at least 37 genes. LuxR homolog-independent activation of the representative genes antA and catB also occurs in the wild type. Expression of antA was influenced the most by C10-HSL and to a lesser extent by other acyl-HSLs, including the P. aeruginosa 3OC12-HSL and C4-HSL signals. The ant and cat operons encode enzymes for the degradation of anthranilate to tricarboxylic acid cycle intermediates. Our results indicate that LuxR homolog-independent acyl-HSL control of the ant and cat operons occurs via regulation of antR, which codes for the transcriptional activator of the ant operon. Although P. aeruginosa has multiple pathways for anthranilate synthesis, one pathway-the kynurenine pathway for tryptophan degradation-is required for acyl-HSL activation of the ant operon. The kynurenine pathway is also the critical source of anthranilate for energy metabolism via the antABC gene products, as well as the source of anthranilate for synthesis of the P. aeruginosa quinolone signal. Our discovery of LuxR homolog-independent responses to acyl-HSLs provides insight into acyl-HSL signaling. PMID:20498077

  13. Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

    PubMed Central

    Reinés, Mar; Llobet, Enrique; Dahlström, Käthe M.; Pérez-Gutiérrez, Camino; Llompart, Catalina M.; Torrecabota, Nuria; Salminen, Tiina A.; Bengoechea, José A.

    2012-01-01

    Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3′-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo2-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of

  14. Effect of water on exenatide acylation in poly(lactide-co-glycolide) microspheres.

    PubMed

    Liang, Rongcai; Li, Xiang; Shi, Yanan; Wang, Aiping; Sun, Kaoxiang; Liu, Wanhui; Li, Youxin

    2013-09-15

    Peptide or protein degradation often occurs when water flows into the dosage form. The aim of this study was to investigate the effect of water on exenatide acylation in poly(lactide-co-glycolide) (PLGA) microspheres. Exenatide-loaded PLGA microspheres were incubated at different relative humidities (RH) as well as in solutions of different pH for 20 days. The stability of exenatide was monitored using HPLC and HPLC-MS analysis. The alteration of exenatide conformation caused by water was investigated by FT-IR spectroscopy. Exenatide and glycolide were incubated in DMSO-water solutions to verify the effect of exenatide conformation state on the peptide acylation. Exenatide was relatively stable in microspheres at lower RH, and the absorbed water could act as a plasticizer and thus promote the peptide acylation by PLGA. However, when the microspheres were incubated at 100% RH, the excessively absorbed water could cause conformation recovery of exenatide and play an inhibitory effect on acylation. The formation of acylated exenatide incubated in acetate buffer saline of pH 6.0 was more than that of pH 4.5 and 3.0. Stability studies of exenatide in glycolide solutions showed that exenatide in nonnative monomer state was easier to be acylated by eletrophiles than that in aggregation state. PMID:23872225

  15. Jejunal administration of glucose enhances acyl ghrelin suppression in obese humans.

    PubMed

    Tamboli, Robyn A; Sidani, Reem M; Garcia, Anna E; Antoun, Joseph; Isbell, James M; Albaugh, Vance L; Abumrad, Naji N

    2016-07-01

    Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (iv) infusions of glucose. These "isoglycemic clamps" enabled us to determine the contribution of the GI tract and postabsorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared with gastric glucose administration (P < 0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P ≤ 0.01). However, gastric and isoglycemic iv glucose infusions resulted in similar degrees of acyl ghrelin suppression (P > 0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion. PMID:27279247

  16. Structural basis for acyl-group discrimination by human Gcn5L2

    PubMed Central

    Ringel, Alison E.; Wolberger, Cynthia

    2016-01-01

    Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications in vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group. PMID:27377381

  17. Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

    SciTech Connect

    Sato, Miho; Nakahara, Keiko; Goto, Shintaro; Kaiya, Hiroyuki; Miyazato, Mikiya . E-mail: a0d201u@cc.miyazaki-u.ac.jp; Date, Yukari; Nakazato, Masamitsu; Kangawa, Kenji; Murakami, Noboru

    2006-11-24

    Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [{sup 125}I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.

  18. ABHD4 regulates multiple classes of N-acyl phospholipids in the mammalian central nervous system

    PubMed Central

    Lee, Hyeon-Cheol; Simon, Gabriel M.; Cravatt, Benjamin F.

    2016-01-01

    N-acyl phospholipids are atypical components of cell membranes that bear three acyl chains and serve as potential biosynthetic precursors for lipid mediators such as endocannabinoids. Biochemical studies have implicated ABHD4 as a brain N-acyl phosphatidylethanolamine (NAPE) lipase, but in vivo evidence for this functional assignment is lacking. Here, we describe ABHD4−/− mice and their characterization using untargeted lipidomics to discover that ABHD4 regulates multiple classes of brain N-acyl phospholipids. In addition to showing reductions in brain glycerophospho-NAEs (GP-NAEs) and plasmalogen-based lyso-NAPEs (lyso-pNAPEs), ABHD4−/− mice exhibited decreases in a distinct set of brain lipids that were structurally characterized as N-acyl lysophosphatidylserines (lyso-NAPSs). Biochemical assays confirmed that NAPS lipids are direct substrates of ABHD4. These findings, taken together, designate ABHD4 as a principal regulator of N-acyl phospholipid metabolism in the mammalian nervous system. PMID:25853435

  19. Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids*

    PubMed Central

    Vats, Archana; Singh, Anil Kumar; Mukherjee, Raju; Chopra, Tarun; Ravindran, Madhu Sudhan; Mohanty, Debasisa; Chatterji, Dipankar; Reyrat, Jean-Marc; Gokhale, Rajesh S.

    2012-01-01

    Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs. PMID:22798073

  20. Effect of water on exenatide acylation in poly(lactide-co-glycolide) microspheres.

    PubMed

    Liang, Rongcai; Li, Xiang; Shi, Yanan; Wang, Aiping; Sun, Kaoxiang; Liu, Wanhui; Li, Youxin

    2013-09-15

    Peptide or protein degradation often occurs when water flows into the dosage form. The aim of this study was to investigate the effect of water on exenatide acylation in poly(lactide-co-glycolide) (PLGA) microspheres. Exenatide-loaded PLGA microspheres were incubated at different relative humidities (RH) as well as in solutions of different pH for 20 days. The stability of exenatide was monitored using HPLC and HPLC-MS analysis. The alteration of exenatide conformation caused by water was investigated by FT-IR spectroscopy. Exenatide and glycolide were incubated in DMSO-water solutions to verify the effect of exenatide conformation state on the peptide acylation. Exenatide was relatively stable in microspheres at lower RH, and the absorbed water could act as a plasticizer and thus promote the peptide acylation by PLGA. However, when the microspheres were incubated at 100% RH, the excessively absorbed water could cause conformation recovery of exenatide and play an inhibitory effect on acylation. The formation of acylated exenatide incubated in acetate buffer saline of pH 6.0 was more than that of pH 4.5 and 3.0. Stability studies of exenatide in glycolide solutions showed that exenatide in nonnative monomer state was easier to be acylated by eletrophiles than that in aggregation state.

  1. NADP-dehydrogenases from pepper fruits: effect of maturation.

    PubMed

    Mateos, Rosa M; Bonilla-Valverde, Daniel; del Río, Luis A; Palma, José M; Corpas, Francisco J

    2009-02-01

    NADPH is an important molecule in the redox balance of the cell. Pepper fruits are the second worldwide consumable vegetables and exhibit different phenotypes after maturation. In this paper, two pepper cultivars were studied: Vergasa whose fruits shift from green to red after maturation, and Biela that shifts to yellow. Using fresh fruits from the same plants of the two cultivars at distinct maturation stages, the activity and gene expression of the main NADPH-generating dehydrogenases was studied. The activity analysis of the main NADP-dehydrogenases, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-isocitrate dehydrogenase (NADP-ICDH) and NADP-malic enzyme (NADP-ME), showed that, except for the G6PDH, all the activities were enhanced (54-100%) in the mature pepper fruits from both cultivars (red or yellow) with respect to green pepper fruits. The content of NADPH and NADP in the mature fruits of both cultivars showed a noteworthy increase with respect to green fruits. For the transcript analysis, a partial cDNA of each NADP-dehydrogenase was obtained, and the NADP-ME was the only NADP-dehydrogenase that showed a significant induction. The increase in the content of NADPH in mature fruits because of the enhanced activity of NADP-dehydrogenases suggests that these NADPH-generating enzymes could be involved in the maturation of pepper fruits.

  2. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sorbitol dehydrogenase test system. 862.1670 Section 862.1670 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1670 Sorbitol dehydrogenase...

  3. Acylated iridoids from the roots of Valeriana officinalis var. latifolia.

    PubMed

    Han, Zhu-zhen; Yan, Zhao-hui; Liu, Qing-xin; Hu, Xian-qing; Ye, Ji; Li, Hui-liang; Zhang, Wei-dong

    2012-10-01

    Phytochemical investigation of the roots of Valeriana officinalis var. latifolia resulted in the isolation and characterization of six new acylated iridoids, (5S,7S,8S,9S)-7-hydroxy-8-isovaleroyloxy-Δ⁴,¹¹-dihyronepetalactone (1), (5S,7S,8S,9S)-7-hydroxy-10-isovaleroyloxy-Δ⁴,¹¹-dihyronepetalactone (2), (5S,8S,9S)-10-isovaleroyloxy-Δ⁴,¹¹-dihyronepetalactone (3), (5S,6S,8S,9R)-6-isovaleroyloxy-Δ⁴,¹¹-1,3-diol (4), (5S,6S,8S,9R)-1,3-isovaleroxy-Δ4,11-1,3-diol (5), and (5S,6S,8S,9R)-3-isovaleroxy-6-isovaleroyloxy-Δ⁴,¹¹-1,3-diol (6). Their structures were determined mainly by 1D and 2D NMR spectroscopic techniques. We also report herein for the first time the single crystal X-ray structure of compound 1. In addition, the cytotoxic activities of compounds 1-6 were evaluated against A549 (human lung adenocarcinoma), HCT116 (human colon carcinoma), SK-BR-3 (human breast carcinoma), and HepG2 (human hepatoma) cell lines. Compound 6 showed weak cell growth inhibition of A549, HCT116, SK-BR-3, and HepG2 cells.

  4. Marinopyrrole A target elucidation by acyl dye transfer.

    PubMed

    Hughes, Chambers C; Yang, Yu-Liang; Liu, Wei-Ting; Dorrestein, Pieter C; La Clair, James J; Fenical, William

    2009-09-01

    The targeting of marinopyrrole A to actin was identified using a fluorescent dye transfer strategy. The process began by appending a carboxylic acid terminal tag to a phenol in the natural product. The resulting probe was then studied in live cells to verify that it maintained activity comparable to marinopyrrole A. Two-color fluorescence microscopy confirmed that both unlabeled and labeled materials share comparable uptake and subcellular localization in HCT-116 cells. Subsequent immunoprecipitation studies identified actin as a putative target in HCT-116 cells, a result that was validated by mass spectral, affinity, and activity analyses on purified samples of actin. Further data analyses indicated that the dye in the marinopyrrole probe was selectively transferred to a single residue K(115), an event that did not occur with related acyl phenols and reactive labels. In this study, the combination of cell, protein, and amino acid analysis arose from a single sample of material, thereby, suggesting a means to streamline and reduce material requirements involved in mode of action studies.

  5. In vivo acylation of rat brain myelin proteolipid protein.

    PubMed

    Agrawal, H C; Randle, C L; Agrawal, D

    1982-04-25

    Examination of brain myelin proteins by sodium dodecyl sulfate-gel electrophoresis followed by fluorography clearly showed that both proteolipid protein (PLP) and DM-20 were acylated 24 h after the intracerebral injection of 30-day-old rats with [3H]palmitic acid. The radioactivity associated with PLP remained after purification, re-electrophoresis, and fluorography. Most of the radioactivity associated with PLP was removed when the gels were treated with hydroxylamine and then fluorographed, indicating that fatty acids were bound to PLP by ester linkage. Cleavage of purified PLP with methanolic sodium hydroxide readily released almost all protein-bound radioactivity. Thin layer chromatography of this material on both silver nitrate and reverse-phase plates provided evidence that most of the radioactivity co-migrated with methyl palmitate (77%) and methyl stearate (19%); however, some radioactivity was associated with methyl oleate (4%). Gas-liquid chromatography of the fatty acids associated with PLP distinctly revealed the presence of methyl palmitate and a detectable peak of methyl stearate. PMID:7068653

  6. Endogenous N-acyl taurines regulate skin wound healing.

    PubMed

    Sasso, Oscar; Pontis, Silvia; Armirotti, Andrea; Cardinali, Giorgia; Kovacs, Daniela; Migliore, Marco; Summa, Maria; Moreno-Sanz, Guillermo; Picardo, Mauro; Piomelli, Daniele

    2016-07-26

    The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs-N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]-as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy. PMID:27412859

  7. Role of intraamygdaloid acylated-ghrelin in spatial learning.

    PubMed

    Tóth, Krisztián; László, Kristóf; Lénárd, László

    2010-01-15

    According to recently published papers acylated-ghrelin (A-Ghr) modifies memory and learning. The basolateral nucleus of amygdala (ABL) participates in the regulation of memory and learning mechanisms. Previously we verified A-Ghr responsive neurons in the ABL by electrophysiological methods. In male Wistar rats effects of bilateral intraamygdaloid microinfusion of 50 ng, 100 ng A-Ghr, 15 ng Ghr receptor antagonist d-Lys3-GHRP-6 (ANT) or ANT+50 ng A-Ghr [dissolved in 0.15M sterile saline], or vehicle in 0.4 microl volume were investigated in Morris water maze paradigm. 50 ng A-Ghr significantly reduced latency to find the platform located in one of the quadrants of the maze. Effect of 50 ng A-Ghr was blocked by ANT pretreatment. ANT alone had no effect. Our results show that place learning linked memory processes are facilitated by A-Ghr in the rat ABL. It is a specific effect, because it could be eliminated by ANT pretreatment.

  8. Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice.

    PubMed

    Porporato, Paolo E; Filigheddu, Nicoletta; Reano, Simone; Ferrara, Michele; Angelino, Elia; Gnocchi, Viola F; Prodam, Flavia; Ronchi, Giulia; Fagoonee, Sharmila; Fornaro, Michele; Chianale, Federica; Baldanzi, Gianluca; Surico, Nicola; Sinigaglia, Fabiola; Perroteau, Isabelle; Smith, Roy G; Sun, Yuxiang; Geuna, Stefano; Graziani, Andrea

    2013-02-01

    Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

  9. Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

    PubMed Central

    Lee, Chi-Yu; Eichner, Ronald D.; Kaplan, Nathan O.

    1973-01-01

    The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN+ and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN+ were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alchol dehydrogenase (EC 1.1.1.1.). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form. PMID:4351183

  10. Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases.

    PubMed

    Pojer, Florence; Ferrer, Jean-Luc; Richard, Stéphane B; Nagegowda, Dinesh A; Chye, Mee-Len; Bach, Thomas J; Noel, Joseph P

    2006-08-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  11. Structural Basis for the Design of Potent and Species-specific Inhibitors of 3-hydroxy-3-methylglutaryl CoA Synthases

    SciTech Connect

    Pojer,F.; Ferrer, J.; Richard, S.; Nagegowda, D.; Chye, M.; Bach, T.; Noel, J.

    2006-01-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  12. Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae).

    PubMed

    Kite, Geoffrey C; Rowe, Emily R; Lewis, Gwilym P; Veitch, Nigel C

    2011-04-01

    The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar

  13. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  14. Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

    PubMed

    Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

    2016-02-26

    We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition. PMID:26773867

  15. Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

    PubMed

    Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

    2016-02-26

    We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition.

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

    PubMed

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. PMID:27372278

  17. Interactions between heparinoids and alcohol dehydrogenase.

    PubMed

    Paulíková, H; Valusová, E; Antalík, M

    1997-07-01

    The interaction between polysulfated polysaecharides (low-molecular-weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 muM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD-binding region of YADH.

  18. The Aldehyde Dehydrogenase Gene Superfamily Resource Center

    PubMed Central

    2009-01-01

    The website http://www.aldh.org is a publicly available database for nomenclature and functional and molecular sequence information for members of the aldehyde dehydrogenase (ALDH) gene superfamily for animals, plants, fungi and bacteria. The site has organised gene-specific records. It provides synopses of ALDH gene records, marries trivial terms to correct nomenclature and links global accession identifiers with source data. Server-side alignment software characterises the integrity of each sequence relative to the latest genomic assembly and provides identifier-specific detail reports, including a graphical presentation of the transcript's exon - intron structure, its size, coding sequence, genomic strand and locus. Also included are a summary of substrates, inhibitors and enzyme kinetics. The site provides reference lists and is designed to facilitate data mining by interested investigators. PMID:20038501

  19. Mitochondrial aldehyde dehydrogenase and cardiac diseases

    PubMed Central

    Chen, Che-Hong; Sun, Lihan; Mochly-Rosen, Daria

    2010-01-01

    Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular ‘aldehydic load’. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases. PMID:20558439

  20. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  1. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

    Monkenbusch, M; Stadler, A; Biehl, R; Ollivier, J; Zamponi, M; Richter, D

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  2. NADH electrochemical sensor coupled with dehydrogenase enzymes

    SciTech Connect

    Yamanaka, Hideko; Mascini, Marco )

    1992-06-01

    A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

  3. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  4. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  5. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  6. Structure-Function Relationships in Lactate Dehydrogenase

    PubMed Central

    Adams, Margaret J.; Buehner, Manfred; Chandrasekhar, K.; Ford, Geoffrey C.; Hackert, Marvin L.; Liljas, Anders; Rossmann, Michael G.; Smiley, Ira E.; Allison, William S.; Everse, Johannes; Kaplan, Nathan O.; Taylor, Susan S.

    1973-01-01

    The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate. PMID:4146647

  7. Molybdenum and tungsten-dependent formate dehydrogenases.

    PubMed

    Maia, Luisa B; Moura, José J G; Moura, Isabel

    2015-03-01

    The prokaryotic formate metabolism is considerably diversified. Prokaryotes use formate in the C1 metabolism, but also evolved to exploit the low reduction potential of formate to derive energy, by coupling its oxidation to the reduction of numerous electron acceptors. To fulfil these varied physiological roles, different types of formate dehydrogenase (FDH) enzymes have evolved to catalyse the reversible 2-electron oxidation of formate to carbon dioxide. This review will highlight our present knowledge about the diverse physiological roles of FDH in prokaryotes, their modular structural organisation and active site structures and the mechanistic strategies followed to accomplish the formate oxidation. In addition, the ability of FDH to catalyse the reverse reaction of carbon dioxide reduction, a potentially relevant reaction for carbon dioxide sequestration, will also be addressed.

  8. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  9. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti.

    PubMed

    Inoue, T; Sunagawa, M; Mori, A; Imai, C; Fukuda, M; Takagi, M; Yano, K

    1989-06-01

    A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.

  10. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    PubMed

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  11. Chromatographic investigation on acyl migration in betacyanins and their decarboxylated derivatives.

    PubMed

    Wybraniec, Sławomir

    2008-01-01

    Chemopreventive and antioxidant action of betalain pigments can differ in dependence on their stereoselective properties, therefore, it is necessary to use relevant methods for monitoring of their possible stereoisomers. Chromatographic characterisation of a group of new isomers of various 6'-O-acylated betacyanins and decarboxylated betacyanins which were generated at low concentration by intramolecular pH-dependent acyl migration was studied in aqueous solutions by HPLC separation with diode-array and mass spectrometric detection. Under alkaline conditions (pH 10.5) the rate of migration was dramatically accelerated, however, always favouring the 6'-O-position and it was much less prominent at lower pH (under 7.0). The possible products of the partial rearrangement were tentatively identified as the 3'-O- and 4'-O-acylated forms and their relative retention times were provided. In malonylated betacyanins and 17-decarboxy-betacyanins the 4'-O-forms were characterised in RP-HPLC by higher retention than the 6'-O forms, whereas the 3'-O-forms were always the most polar. In contrast, the isomerisation of hylocerenin and 17-decarboxy-hylocerenin resulted in different chromatographic profiles of the migration products. In 2-decarboxy- and 2,17-bidecarboxy-betacyanins the 3'-O- and 4'-O-acylated forms eluted always before the 6'-O-acylated betacyanins. The investigations on acyl migration in isolated 4'-O-malonyl-betanin confirmed the strong tendency of reverse acyl migration (4'-->6') and also partial 4'-->3' rearrangement which were leading to the final monoester regioisomeric distribution (%) close to 87:7:6 (6'-O-, 4'-O-, 3'-O-).

  12. A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

    PubMed Central

    Bijtenhoorn, Patrick; Mayerhofer, Hubert; Müller-Dieckmann, Jochen; Utpatel, Christian; Schipper, Christina; Hornung, Claudia; Szesny, Matthias; Grond, Stephanie; Thürmer, Andrea; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Dierking, Katja; Schulenburg, Hinrich; Streit, Wolfgang R.

    2011-01-01

    In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies. PMID:22046268

  13. A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants.

    PubMed

    Vialart, Guilhem; Hehn, Alain; Olry, Alexandre; Ito, Kyoko; Krieger, Celia; Larbat, Romain; Paris, Cedric; Shimizu, Bun-Ichi; Sugimoto, Yukihiro; Mizutani, Masaharu; Bourgaud, Frederic

    2012-05-01

    Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.

  14. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

  15. Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L 1

    PubMed Central

    Williams, Michael; Randall, Douglas D.

    1979-01-01

    Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg2+ and thiamine pyrophosphate. Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants. Images PMID:16661100

  16. Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids.

    PubMed

    Reid, E E; Thompson, P; Lyttle, C R; Dennis, D T

    1977-05-01

    The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s(20,w) of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD(+) and pyruvate; NADP(+) and other keto acids give no reaction. Mg(2+), thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria. Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg(2+), cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations.

  17. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. PMID:25542170

  18. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB’) from fatty acid degradation operon of Ralstonia eutropha H16

    PubMed Central

    2014-01-01

    In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB’, gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB’ is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB’ comprises an NAD+ binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB’ was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD+. The Km value for acetoacetyl-CoA was 48 μM and Vmax 149 μmol mg−1 min−1. NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB’ exhibited optimal activity at pH 6–7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB’. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB’ and fatty acid metabolism, FadB’ might be also interesting for biotechnological applications. PMID:25401070

  19. Vertebrate fatty acyl desaturase with Δ4 activity

    PubMed Central

    Li, Yuanyou; Monroig, Oscar; Zhang, Liang; Wang, Shuqi; Zheng, Xiaozhong; Dick, James R.; You, Cuihong; Tocher, Douglas R.

    2010-01-01

    Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 18∶2n - 6 to produce 18∶3n - 6 that is elongated to 20∶3n - 6 followed by Δ5 desaturation. Synthesis of EPA from 18∶3n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22∶5n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22∶5n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above. PMID:20826444

  20. Arabidopsis PIZZA Has the Capacity to Acylate Brassinosteroids

    PubMed Central

    Schneider, Katja; Breuer, Christian; Kawamura, Ayako; Jikumaru, Yusuke; Hanada, Atsushi; Fujioka, Shozo; Ichikawa, Takanari; Kondou, Youichi; Matsui, Minami; Kamiya, Yuji; Yamaguchi, Shinjiro; Sugimoto, Keiko

    2012-01-01

    Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis. PMID:23071642

  1. Arabidopsis PIZZA has the capacity to acylate brassinosteroids.

    PubMed

    Schneider, Katja; Breuer, Christian; Kawamura, Ayako; Jikumaru, Yusuke; Hanada, Atsushi; Fujioka, Shozo; Ichikawa, Takanari; Kondou, Youichi; Matsui, Minami; Kamiya, Yuji; Yamaguchi, Shinjiro; Sugimoto, Keiko

    2012-01-01

    Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

  2. Biochemical Competition Makes Fatty-Acid β-Oxidation Vulnerable to Substrate Overload

    PubMed Central

    van Eunen, Karen; Simons, Sereh M. J.; Gerding, Albert; Bleeker, Aycha; den Besten, Gijs; Touw, Catharina M. L.; Houten, Sander M.; Groen, Bert K.; Krab, Klaas; Reijngoud, Dirk-Jan; Bakker, Barbara M.

    2013-01-01

    Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD+]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration. PMID:23966849

  3. Biochemical competition makes fatty-acid β-oxidation vulnerable to substrate overload.

    PubMed

    van Eunen, Karen; Simons, Sereh M J; Gerding, Albert; Bleeker, Aycha; den Besten, Gijs; Touw, Catharina M L; Houten, Sander M; Groen, Bert K; Krab, Klaas; Reijngoud, Dirk-Jan; Bakker, Barbara M

    2013-01-01

    Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD⁺]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration.

  4. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    PubMed

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

  5. Role of quinate dehydrogenase in quinic acid metabolism in conifers

    SciTech Connect

    Osipov, V.I.; Shein, I.V.

    1986-08-10

    Quinate dehydrogenase was isolated from young needles of the Siberian larch and partially purified by ammonium sulfate fractionation. It was found that in conifers, in contrast to other plants, quinate dehydrogenase is active both with NAD and with NADP. The values of K/sub m/ for quinate and NADP were 1.8 and 0.18 mM. The enzyme exhibits maximum activity at pH 9.0. It was assumed that NADP-dependent quinate dehydrogenase is responsible for quinic acid synthesis. The special features of the organization and regulation of the initial stages of the shikimate pathway in conifers are discussed.

  6. Topo-optical reactions for the identification of O-acyl sugars in amyloid deposits.

    PubMed

    Richter, Susann; Makovitzky, Josef

    2009-01-01

    The aldehyde bisulfite toluidine blue (ABT) reaction with former saponification (KOH-ABT) and periodic acid-borohydride reduction-saponification (PB-KOH-ABT) were applied to sections of human amyloid deposits in the respiratory tract. The saponification-induced increase in ABT-reactivity was confined to the presence of O-acyl sugars associated with the amyloid fibrils. The anisotropic and metachromatic effect in the ABT and KOH-ABT reaction was reduced in the corresponding PB-KOH-ABT reaction, a difference attributed to the removal of staining due to neutral carbohydrate residues. Since the periodic acid-borohydride reduction abolishes all pre-existing ABT-reactivity of neutral sugar vicinal diols, the isolated KOH-effect could be shown using the PB-KOH-ABT reaction. By application of this sequence, the problem identifying small quantities of O-acyl sugars was solved. It is suggested that the KOH-effect depends upon the removal of O-acyl substituents located on the polyhydroxy side chain (C7, C8, C9) of sialic acid residues. An advantage of such topo-optical reactions over biochemical techniques is the exact localization of O-acyl sugars in tissue sites. By means of the KOH-ABT and PB-KOH-ABT reactions we have demonstrated, for the first time, that O-acyl sugars occur within amyloid deposits.

  7. Computational Prediction of acyl-coA Binding Proteins Structure in Brassica napus.

    PubMed

    Raboanatahiry, Nadia Haingotiana; Lu, Guangyuan; Li, Maoteng

    2015-01-01

    Acyl-coA binding proteins could transport acyl-coA esters from plastid to endoplasmic reticulum, prior to fatty acid biosynthesis, leading to the formation of triacylglycerol. The structure and the subcellular localization of acyl-coA binding proteins (ACBP) in Brassica napus were computationally predicted in this study. Earlier, the structure analysis of ACBPs was limited to the small ACBPs, the current study focused on all four classes of ACBPs. Physicochemical parameters including the size and the length, the intron-exon structure, the isoelectric point, the hydrophobicity, and the amino acid composition were studied. Furthermore, identification of conserved residues and conserved domains were carried out. Secondary structure and tertiary structure of ACBPs were also studied. Finally, subcellular localization of ACBPs was predicted. The findings indicated that the physicochemical parameters and subcellular localizations of ACBPs in Brassica napus were identical to Arabidopsis thaliana. Conserved domain analysis indicated that ACBPs contain two or three kelch domains that belong to different families. Identical residues in acyl-coA binding domains corresponded to eight amino acid residues in all ACBPs of B. napus. However, conserved residues of common ACBPs in all species of animal, plant, bacteria and fungi were only inclusive in small ACBPs. Alpha-helixes were displayed and conserved in all the acyl-coA binding domains, representing almost the half of the protein structure. The findings confirm high similarities in ACBPs between A. thaliana and B. napus, they might share the same functions but loss or gain might be possible.

  8. Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

    SciTech Connect

    de Vetten, M.P.; Agre, P.

    1988-12-05

    The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with (3H)palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.

  9. The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure[S

    PubMed Central

    Son, Mijin; London, Erwin

    2013-01-01

    Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains. PMID:23093551

  10. Altered hepatic retinyl ester concentration and acyl composition in response to alcohol consumption.

    PubMed

    Clugston, Robin D; Jiang, Hongfeng; Lee, Man Xia; Berk, Paul D; Goldberg, Ira J; Huang, Li-Shin; Blaner, William S

    2013-07-01

    Retinoids (vitamin A and its metabolites) are essential micronutrients that regulate many cellular processes. Greater than 70% of the body's retinoid reserves are stored in the liver as retinyl ester (RE). Chronic alcohol consumption induces depletion of hepatic retinoid stores, and the extent of this has been correlated with advancing stages of alcoholic liver disease. The goal of this study was to analyze the mechanisms responsible for depletion of hepatic RE stores by alcohol consumption A change in the fatty-acyl composition of RE in alcohol-fed mice was observed within two weeks after the start of alcohol consumption. Specifically, alcohol-feeding was associated with a significant decline in hepatic retinyl palmitate levels; however, total RE levels were maintained by a compensatory increase in levels of usually minor RE species, particularly retinyl oleate. Our data suggests that alcohol feeding initially stimulates a futile cycle of RE hydrolysis and synthesis, and that the change in RE acyl composition is associated with a change in the acyl composition of hepatic phosphatidylcholine. The alcohol-induced change in RE acyl composition was specific to the liver, and was not seen in lung or white adipose tissue. This shift in hepatic RE fatty acyl composition is a sensitive indicator of alcohol consumption and may be an early biomarker for events associated with the development of alcoholic liver disease. PMID:24046868

  11. Altered hepatic retinyl ester concentration and acyl composition in response to alcohol consumption.

    PubMed

    Clugston, Robin D; Jiang, Hongfeng; Lee, Man Xia; Berk, Paul D; Goldberg, Ira J; Huang, Li-Shin; Blaner, William S

    2012-07-01

    Retinoids (vitamin A and its metabolites) are essential micronutrients that regulate many cellular processes. Greater than 70% of the body's retinoid reserves are stored in the liver as retinyl ester (RE). Chronic alcohol consumption induces depletion of hepatic retinoid stores, and the extent of this has been correlated with advancing stages of alcoholic liver disease. The goal of this study was to analyze the mechanisms responsible for depletion of hepatic RE stores by alcohol consumption. A change in the fatty-acyl composition of RE in alcohol-fed mice was observed within two weeks after the start of alcohol consumption. Specifically, alcohol-feeding was associated with a significant decline in hepatic retinyl palmitate levels; however, total RE levels were maintained by a compensatory increase in levels of usually minor RE species, particularly retinyl oleate. Our data suggests that alcohol feeding initially stimulates a futile cycle of RE hydrolysis and synthesis, and that the change in RE acyl composition is associated with a change in the acyl composition of hepatic phosphatidylcholine. The alcohol-induced change in RE acyl composition was specific to the liver, and was not seen in lung or white adipose tissue. This shift in hepatic RE fatty acyl composition is a sensitive indicator of alcohol consumption and may be an early biomarker for events associated with the development of alcoholic liver disease. PMID:23583843

  12. Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia.

    PubMed

    Yamada, Takahiro; Takemura, Yoshizumi; Niisato, Naomi; Mitsuyama, Etsuko; Iwasaki, Yoshinobu; Marunaka, Yoshinori

    2009-03-13

    We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

  13. Novel deletion in a patient with an isolated peroxisoml acyl-CoA oxidase deficiency

    SciTech Connect

    Poll-The, B.T.; Fournier, B.; Clevers, H.; Wanders, R.J.A.

    1994-09-01

    Disorders with defective peroxisome assembly are associated with multiple peroxisomal enzymatic abnormalities. Besides these diseases patients have been described suspected of having a single enzyme defect in the peroxisomal {beta}-oxidation pathway. Laboratory findings for these patients include elevated plasma very long chain fatty acids (VLCFA) and impaired VLCFA oxidation in fibroblasts. Complementation analysis between these patients and those with a proven single enzyme deficiency, using peroxisomal {beta}-oxidation of VLCFA as the criterion for complementation, has been used to show whether the patients are deficient in acyl-CoA oxidase, peroxisomal trifunctional protein or thiolase activity. Fibroblasts from a patient showing the clinical and biochemical abnormalities of isolated acyl-CoA oxidase deficiency (using cell complementation) were analyzed at the molecular level. Isolation of RNA from patient`s fibroblasts was followed by random reverse transcription of RNA and PCR amplification. PCR products were blotted and hybridized with the human acyl-CoA oxidase cDNA. A fragment 150 bp shorter than normal was found. Upon sequencing, exon 7 was found to be deleted leading to a frameshift in the acyl-CoA oxidase mRNA. Southern blot analysis of the patient`s DNA did not reveal any deletion in contrast to two siblings previously reported as having a deletion of at least 17 kb in the acyl-CoA oxidase gene.

  14. Carbohydrate conformation and lipid condensation in monolayers containing glycosphingolipid Gb3: influence of acyl chain structure.

    PubMed

    Watkins, Erik B; Gao, Haifei; Dennison, Andrew J C; Chopin, Nathalie; Struth, Bernd; Arnold, Thomas; Florent, Jean-Claude; Johannes, Ludger

    2014-09-01

    Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3's influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3's capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment's impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.

  15. Identification and distribution of simple and acylated betacyanins in the Amaranthaceae.

    PubMed

    Cai, Y; Sun, M; Corke, H

    2001-04-01

    Red-colored plants in the family Amaranthaceae are recognized as a rich source of diverse and unique betacyanins. The distribution of betacyanins in 37 species of 8 genera in the Amaranthaceae was investigated. A total of 16 kinds of betacyanins were isolated and characterized by HPLC, spectral analyses, and MS. They consisted of 6 simple (nonacylated) betacyanins and 10 acylated betacyanins, including 8 amaranthine-type pigments, 6 gomphrenin-type pigments, and 2 betanin-type pigments. Acylated betacyanins were identified as betanidin 5-O-beta-glucuronosylglucoside or betanidin 6-O-beta-glucoside acylated with ferulic, p-coumaric, or 3-hydroxy-3-methylglutaric acids. Total betacyanin content in the 37 species ranged from 0.08 to 1.36 mg/g of fresh weight. Simple betacyanins (such as amaranthine, which averaged 91.5% of total peak area) were widespread among all species of 8 genera. Acylated betacyanins were distributed among 11 species of 6 genera, with the highest proportion occurring in Iresine herbstii (79.6%) and Gomphrena globosa (68.4%). Some cultivated species contained many more acylated betacyanins than wild species, representing a potential new source of these pigments as natural colorants.

  16. Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

    PubMed Central

    Watkins, Erik B.; Gao, Haifei; Dennison, Andrew J.C.; Chopin, Nathalie; Struth, Bernd; Arnold, Thomas; Florent, Jean-Claude; Johannes, Ludger

    2014-01-01

    Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding. PMID:25185550

  17. Glucose-6-Phosphate Dehydrogenase and NADPH Redox Regulates Cardiac Myocyte L-Type Calcium Channel Activity and Myocardial Contractile Function

    PubMed Central

    Rawat, Dhwajbahadur K.; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J.; Okada, Takao; Edwards, John G.; Gupte, Sachin A.

    2012-01-01

    We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca2+ currents (ICa-L) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit ICa-L and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca2+ channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and ICa-L were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of −80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO2 and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak ICa-L amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of ICa-L by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca2+ channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure). PMID:23071515

  18. Simultaneous involvement of a tungsten-containing aldehyde:ferredoxin oxidoreductase and a phenylacetaldehyde dehydrogenase in anaerobic phenylalanine metabolism.

    PubMed

    Debnar-Daumler, Carlotta; Seubert, Andreas; Schmitt, Georg; Heider, Johann

    2014-01-01

    Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.

  19. Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde-alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica.

    PubMed

    Pineda, Erika; Encalada, Rusely; Rodríguez-Zavala, José S; Olivos-García, Alfonso; Moreno-Sánchez, Rafael; Saavedra, Emma

    2010-08-01

    The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O(2) exposure (IC(50) 0.034 mm), whereas short-term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe(2+). CoA and acetyl-CoA prevented, whereas pyruvate exacerbated, inactivation induced by short-term saturating O(2) exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe(2+)-reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe-S clusters. After O(2) exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde-alcohol dehydrogenase (EhADH2), whereas acetyl-CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress. PMID:20629749

  20. Pyruvate dehydrogenase complex deficiency and its relationship with epilepsy frequency--An overview.

    PubMed

    Bhandary, Suman; Aguan, Kripamoy

    2015-10-01

    The pyruvate dehydrogenase complex (PDHc) is a member of a family of multienzyme complexes that provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the physiologically irreversible decarboxylation of various 2-oxoacid substrates to their corresponding acyl-CoA derivatives, NADH and CO2. PDHc deficiency is a metabolic disorder commonly associated with lactic acidosis, progressive neurological and neuromuscular degeneration that vary with age and gender. In this review, we aim to discuss the relationship between occurrence of epilepsy and PDHc deficiency associated with the pyruvate dehydrogenase complex (E1α subunit (PDHA1) and E1β subunit (PDHB)) and PDH phosphatase (PDP) deficiency. PDHc plays a crucial role in the aerobic carbohydrate metabolism and regulates the use of carbohydrate as the source of oxidative energy. In severe PDHc deficiency, the energy deficit impairs brain development in utero resulting in physiological and structural changes in the brain that contributes to the subsequent onset of epileptogenesis. Epileptogenesis in PDHc deficiency is linked to energy failure and abnormal neurotransmitter metabolism that progressively alters neuronal excitability. This metabolic blockage might be restricted via inclusion of ketogenic diet that is broken up by β-oxidation and directly converting it to acetyl-CoA, and thereby improving the patient's health condition. Genetic counseling is essential as PDHA1 deficiency is X-linked. The demonstration of the X-chromosome localization of PDHA1 resolved a number of questions concerning the variable phenotype displayed by patients with E1 deficiency. Most patients show a broad range of neurological abnormalities, with the severity showing some dependence on the nature of the mutation in the Elα gene, while PDHB and PDH phosphatase (PDP) deficiencies are of autosomal recessive inheritance. However, in females, the disorder is further complicated by the pattern of X