Science.gov

Sample records for acyl chain substrates

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

    SciTech Connect

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

    2008-08-26

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

  2. Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate.

    PubMed

    Cheriyan, Manoj; Walters, Matthew J; Kang, Brian D; Anzaldi, Laura L; Toone, Eric J; Fierke, Carol A

    2011-11-01

    The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.

  3. Fatty Acyl Chains of Mycobacterium marinum Lipooligosaccharides

    PubMed Central

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

    2011-01-01

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

  4. The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.

    PubMed

    Koetsier, Martijn J; Gombert, Andreas K; Fekken, Susan; Bovenberg, Roel A L; van den Berg, Marco A; Kiel, Jan A K W; Jekel, Peter A; Janssen, Dick B; Pronk, Jack T; van der Klei, Ida J; Daran, Jean-Marc

    2010-01-01

    Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

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

    PubMed Central

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

    2000-01-01

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

  6. Medium-chain acyl-CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low carnitine levels.

    PubMed

    Stanley, C A; Hale, D E; Coates, P M; Hall, C L; Corkey, B E; Yang, W; Kelley, R I; Gonzales, E L; Williamson, J R; Baker, L

    1983-11-01

    Three children in two families presented in early childhood with episodes of illness associated with fasting which resembled Reye's syndrome: coma, hypoglycemia, hyperammonemia, and fatty liver. One child died with cerebral edema during an episode. Clinical studies revealed an absence of ketosis on fasting (plasma beta-hydroxybutyrate less than 0.4 mmole/liter) despite elevated levels of free fatty acids (2.6-4.2 mmole/liter) which suggested that hepatic fatty acid oxidation was impaired. Urinary dicarboxylic acids were elevated during illness or fasting. Total carnitine levels were low in plasma (18-25 mumole/liter), liver (200-500 nmole/g), and muscle (500-800 nmole/g); however, treatment with L-carnitine failed to correct the defect in ketogenesis. Studies on ketone production from fatty acid substrates by liver tissue in vitro showed normal rates from short-chain fatty acids, but very low rates from all medium and long-chain fatty acid substrates. These results suggested that the defect was in the mid-portion of the intramitochondrial beta-oxidation pathway at the medium-chain acyl-CoA dehydrogenase step. A new assay for the electron transfer flavoprotein-linked acyl-CoA dehydrogenases was used to test this hypothesis. This assay follows the decrease in electron transfer flavoprotein fluorescence as it is reduced by acyl-CoA-acyl-CoA dehydrogenase complex. Results with octanoyl-CoA as substrate indicated that patients had less than 2.5% normal activity of medium-chain acyl-CoA dehydrogenase. The activities of short-chain and isovaleryl acyl-CoA dehydrogenases were normal; the activity of long-chain acyl-CoA dehydrogenase was one-third normal. These results define a previously unrecognized inherited metabolic disorder of fatty acid oxidation due to deficiency of medium-chain acyl-CoA dehydrogenase.

  7. Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

    PubMed

    Voelker, T A; Davies, H M

    1994-12-01

    The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.

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

    PubMed

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

    2016-08-08

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

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

    PubMed Central

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

    1989-01-01

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

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

    PubMed

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

    2017-02-21

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

  12. Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.

    PubMed

    Shahi, Puja; Kumar, Ish; Sharma, Ritu; Sanger, Shefali; Jolly, Ravinder S

    2006-06-01

    A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.

  13. Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases.

    PubMed

    Jost, Marco; Born, David A; Cracan, Valentin; Banerjee, Ruma; Drennan, Catherine L

    2015-11-06

    Acyl-CoA mutases are a growing class of adenosylcobalamin-dependent radical enzymes that perform challenging carbon skeleton rearrangements in primary and secondary metabolism. Members of this class of enzymes must precisely control substrate positioning to prevent oxidative interception of radical intermediates during catalysis. Our understanding of substrate specificity and catalysis in acyl-CoA mutases, however, is incomplete. Here, we present crystal structures of IcmF, a natural fusion protein variant of isobutyryl-CoA mutase, in complex with the adenosylcobalamin cofactor and four different acyl-CoA substrates. These structures demonstrate how the active site is designed to accommodate the aliphatic acyl chains of each substrate. The structures suggest that a conformational change of the 5'-deoxyadenosyl group from C2'-endo to C3'-endo could contribute to initiation of catalysis. Furthermore, detailed bioinformatic analyses guided by our structural findings identify critical determinants of acyl-CoA mutase substrate specificity and predict new acyl-CoA mutase-catalyzed reactions. These results expand our understanding of the substrate specificity and the catalytic scope of acyl-CoA mutases and could benefit engineering efforts for biotechnological applications ranging from production of biofuels and commercial products to hydrocarbon remediation.

  14. Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

    PubMed Central

    Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

    2012-01-01

    Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

  15. 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

  16. Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin.

    PubMed

    Demant, E J; Nystrøm, B T

    2001-08-01

    The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.

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

    PubMed

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

    1995-11-07

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

  18. Acyl-CoA dehydrogenase 9 (ACAD 9) is the long-chain acyl-CoA dehydrogenase in human embryonic and fetal brain.

    PubMed

    Oey, N A; Ruiter, J P N; Ijlst, L; Attie-Bitach, T; Vekemans, M; Wanders, R J A; Wijburg, F A

    2006-07-21

    We recently reported the expression and activity of several fatty acid oxidation enzymes in human embryonic and fetal tissues including brain and spinal cord. Liver and heart showed expression of both very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) mRNA. However, while mRNA expression of LCHAD could be clearly detected in the retina and spinal cord, expression of VLCAD mRNA was low to undetectable in these tissues. Nevertheless, abundant acyl-CoA dehydrogenase (ACAD) activity was detected with palmitoyl-CoA as substrate in fetal central nervous tissue. These conflicting data suggested the presence of a different long-chain ACAD in human embryonic and fetal brain. In this study, using in situ hybridization as well as enzymatic studies, we identified acyl-CoA dehydrogenase 9 (ACAD 9) as the long-chain ACAD in human embryonic and fetal central nervous tissue. Until now, no clinical signs and symptoms of central nervous system involvement have been reported in VLCAD deficiency. A novel long-chain FAO defect, i.e., ACAD 9 deficiency with only central nervous system involvement, could, if not lethal during intra uterine development, easily escape proper diagnosis, since probably no classical signs and symptoms of FAO deficiency will be observed. Screening for ACAD 9 deficiency in patients with undefined neurological symptoms and/or impairment in neurological development of unknown origin is necessary to establish if ACAD 9 deficiency exists as a separate disease entity.

  19. 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.

  20. Defluoridation potential of jute fibers grafted with fatty acyl chain

    NASA Astrophysics Data System (ADS)

    Manna, Suvendu; Saha, Prosenjit; Roy, Debasis; Sen, Ramkrishna; Adhikari, Basudam

    2015-11-01

    Waterborne fluoride is usually removed from water by coagulation, adsorption, ion exchange, electro dialysis or reverse osmosis. These processes are often effective over narrow pH ranges, release ions considered hazardous to human health or produce large volumes of toxic sludge that are difficult to handle and dispose. Although plant matters have been shown to remove waterborne fluoride, they suffer from poor removal efficiency. Following from the insight that interaction between microbial carbohydrate biopolymers and anionic surfaces is often facilitated by lipids, an attempt has been made to enhance fluoride adsorption efficiency of jute by grafting the lignocellulosic fiber with fatty acyl chains found in vegetable oils. Fluoride removal efficiency of grafted jute was found to be comparable or higher than those of alternative defluoridation processes. Infrared and X-ray photoelectron spectroscopic evidence indicated that hydrogen bonding, protonation and C-F bonding were responsible for fluoride accumulation on grafted jute. Adsorption based on grafted jute fibers appears to be an economical, sustainable and eco-friendly alternative technique for removing waterborne fluoride.

  1. The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure[S

    PubMed Central

    Son, Mijin; London, Erwin

    2013-01-01

    Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains. PMID:23093551

  2. Acyl chain preference and inhibitor identification of Moraxella catarrhalis LpxA: Insight through crystal structure and computational studies.

    PubMed

    Pratap, Shivendra; Kesari, Pooja; Yadav, Ravi; Dev, Aditya; Narwal, Manju; Kumar, Pravindra

    2017-03-01

    Lipopolysaccharide (LPS) is an important surface component and a potential virulence factor in the pathogenesis of Gram-negative bacteria. UDP-N-acetylglucosamine acyltransferase (LpxA) enzyme catalyzes the first reaction of LPS biosynthesis, reversible transfer of R-3-hydroxy-acyl moiety from donor R-3-hydroxy-acyl-acyl carrier protein to the 3' hydroxyl position of UDP-N-acetyl-glucosamine. LpxA enzyme's essentiality in bacterial survival and absence of any homologous protein in humans makes it a promising target for anti-bacterial drug development. Herein, we present the crystal structure of Moraxella catarrhalis LpxA (McLpxA). We propose that L171 is responsible for limiting the acyl chain length in McLpxA to 10C or 12C. The study reveals the plausible interactions between the highly conserved clusters of basic residues at the C-terminal end of McLpxA and acidic residues of acyl carrier protein (ACP). Furthermore, the crystal structure of McLpxA was used to screen potential inhibitors from NCI open database using various computational approaches viz. pharmacophore mapping, virtual screening and molecular docking. Molecules Mol212032, Mol609399 and Mol152546 showed best binding affinity with McLpxA among all screened molecules. These molecules mimic the substrate-LpxA binding interactions.

  3. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.

    PubMed

    Mashek, Douglas G; Bornfeldt, Karin E; Coleman, Rosalind A; Berger, Johannes; Bernlohr, David A; Black, Paul; DiRusso, Concetta C; Farber, Steven A; Guo, Wen; Hashimoto, Naohiro; Khodiyar, Varsha; Kuypers, Frans A; Maltais, Lois J; Nebert, Daniel W; Renieri, Alessandra; Schaffer, Jean E; Stahl, Andreas; Watkins, Paul A; Vasiliou, Vasilis; Yamamoto, Tokuo T

    2004-10-01

    By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

  4. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

    PubMed Central

    Kawelke, Steffen; Feussner, Ivo

    2015-01-01

    Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity. PMID:26714272

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

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

    2000-01-01

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

  7. Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson’s disease.

    PubMed

    Abbott, Sarah K; Li, Hongyun; Muñoz, Sonia Sanz; Knoch, Bianca; Batterham, Marijka; Murphy, Karen E; Halliday, Glenda M; Garner, Brett

    2014-04-01

    Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson’s disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.

  8. 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

  9. Accumulation of acyl-enzyme in DD-peptidase-catalysed reactions with analogues of peptide substrates.

    PubMed Central

    Jamin, M; Adam, M; Damblon, C; Christiaens, L; Frère, J M

    1991-01-01

    Thioester substrates can be used to study the hydrolysis and transfer reactions catalysed by beta-lactamases and DD-peptidases. With the latter enzymes, accumulation of the acyl-enzyme can be detected directly. The efficiency of various amines as acceptor substrates was in excellent agreement with previous results obtained with peptide substrates of the DD-peptidases. The results indicated the presence of a specific binding site for the acceptor substrates. Although most of the results agreed well with a simple partition model, more elaborate hypotheses will be needed to account for all the data presented. PMID:1747125

  10. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    PubMed Central

    Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

    2012-01-01

    We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

  11. Substrate Effects for Atomic Chain Electronics

    NASA Technical Reports Server (NTRS)

    Yamada, Toshishige; Saini, Subhash (Technical Monitor)

    1998-01-01

    A substrate for future atomic chain electronics, where adatoms are placed at designated positions and form atomically precise device components, is studied theoretically. The substrate has to serve as a two-dimensional template for adatom mounting with a reasonable confinement barrier and also provide electronic isolation, preventing unwanted coupling between independent adatom structures. For excellent structural stability, we demand chemical bonding between the adatoms and substrate atoms, but then good electronic isolation may not be guaranteed. Conditions are clarified for good isolation. Because of the chemical bonding, fundamental adatom properties are strongly influenced: a chain with group IV adatoms having two chemical bonds, or a chain with group III adatoms having one chemical bond is semiconducting. Charge transfer from or to the substrate atoms brings about unintentional doping, and the electronic properties have to be considered for the entire combination of the adatom and substrate systems even if the adatom modes are well localized at the surface.

  12. 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 ...

  13. Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis.

    PubMed

    Goblirsch, Brandon R; Jensen, Matthew R; Mohamed, Fatuma A; Wackett, Lawrence P; Wilmot, Carrie M

    2016-12-23

    Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolase-are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys(143)) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ(117)) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.

  14. Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

    SciTech Connect

    Goblirsch, Brandon R.; Jensen, Matthew R.; Mohamed, Fatuma A.; Wackett, Lawrence P.; Wilmot, Carrie M.

    2016-11-04

    Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety—unusual for a thiolase—are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.

  15. Acyl chain conformations in phospholipid bilayers: a comparative study of docosahexaenoic acid and saturated fatty acids.

    PubMed

    Feller, Scott E

    2008-05-01

    A variety of experimental methods indicate unique biophysical properties of membranes containing the highly polyunsaturated omega-3 fatty acid, docosahexaenoic acid (DHA). In the following we review the atomically detailed picture of DHA acyl chains structure and dynamics that has emerged from computational studies of this system in our lab. A comprehensive approach, beginning with ab-initio quantum chemical studies of model compounds representing segments of DHA and ending with large scale classical molecular dynamics simulations of DHA-containing bilayers, is described with particular attention paid to contrasting the properties of DHA with those of saturated fatty acids. Connection with experiment is made primarily through comparison with Nuclear Magnetic Resonance (NMR) studies, particularly those that probe details of the chain structure and dynamics. Our computational results suggest that low torsional energy barriers, comparable to kT at physiological conditions, for the rotatable bonds in the DHA chain are the key to the differences observed between polyunsaturated and saturated acyl chains.

  16. Cationic amphiphiles with fatty acyl chain asymmetry of coconut oil deliver genes selectively to mouse lung.

    PubMed

    Chandrashekhar, Voshavar; Srujan, Marepally; Prabhakar, Rairala; Reddy, Rakesh C; Sreedhar, Bojja; Rentam, Kiran K R; Kanjilal, Sanjit; Chaudhuri, Arabinda

    2011-03-16

    Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhancing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. Herein, we demonstrate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent.

  17. Insights into Medium-chain Acyl-CoA Dehydrogenase Structure by Molecular Dynamics Simulations.

    PubMed

    Bonito, Cátia A; Leandro, Paula; Ventura, Fátima V; Guedes, Rita C

    2016-08-01

    The medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that catalyzes the first step of mitochondrial fatty acid β-oxidation (mFAO) pathway. Its deficiency is the most common genetic disorder of mFAO. Many of the MCAD disease-causing variants, including the most common p.K304E variant, show loss of function due to protein misfolding. Herein, we used molecular dynamics simulations to provide insights into the structural stability and dynamic behavior of MCAD wild-type (MCADwt) and validate a structure that would allow reliable new studies on its variants. Our results revealed that in both proteins the flavin adenine dinucleotide (FAD) has an important structural role on the tetramer stability and also in maintaining the volume of the enzyme catalytic pockets. We confirmed that the presence of substrate changes the dynamics of the catalytic pockets and increases FAD affinity. A comparison between the porcine MCADwt (pMCADwt) and human MCADwt (hMCADwt) structures revealed that both proteins are essentially similar and that the reversion of the double mutant E376G/T255E of hMCAD enzyme does not affect the structure of the protein neither its behavior in simulation. Our validated hMCADwt structure is crucial for complementing and accelerating the experimental studies aiming for the discovery and development of potential stabilizers of MCAD variants as candidates for the treatment of MCAD deficiency (MCADD).

  18. Enzymatic Transesterification of Kraft Lignin with Long Acyl Chains in Ionic Liquids.

    PubMed

    Hulin, Lise; Husson, Eric; Bonnet, Jean-Pierre; Stevanovic, Tatjana; Sarazin, Catherine

    2015-09-09

    Valorization of lignin is essential for the economic viability of the biorefinery concept. For example, the enhancement of lignin hydrophobicity by chemical esterification is known to improve its miscibility in apolar polyolefin matrices, thereby helping the production of bio-based composites. To this end and due to its many reactive hydroxyl groups, lignin is a challenging macromolecular substrate for biocatalyzed esterification in non-conventional media. The present work describes for the first time the lipase-catalyzed transesterification of Kraft lignin in ionic liquids (ILs). Three lipases, three 1-butyl-3-methylimidazolium based ILs and ethyl oleate as long chain acyl donor were selected. Best results were obtained with a hydrophilic/hydrophobic binary IL system (1-butyl-3-methylimidazolium trifluoromethanesulfonate/1-butyl-3-methylimidazolium hexafluoro- phosphate, 1/1 v/v) and the immobilized lipase B from Candida antarctica (CALB) that afforded a promising transesterification yield (ca. 30%). Similar performances were achieved by using 1-butyl-3-methylimidazolium hexafluorophosphate as a coating agent for CALB rather than as a co-solvent in 1-butyl-3-methylimidazolium trifluoromethane-sulfonate thus limiting the use of hydrophobic IL. Structural characterization of lignin oleate was performed by spectroscopic studies (FTIR and ¹H-NMR). The synthesized lignin oleate exhibited interesting thermal and textural properties, different from those of the original Kraft lignin.

  19. On Substrate for Atomic Chain Electronics

    NASA Technical Reports Server (NTRS)

    Yamada, Toshishige; Bauschlicher, Charles W., Jr.; Partridge, Harry; Saini, Subhash (Technical Monitor)

    1998-01-01

    A substrate for future atomic chain electronics, where adatoms are placed at designated positions and form atomically precise device components, is studied theoretically. The substrate has to serve as a two-dimensional template for adatom mounting with a reasonable confinement barrier and also provide electronic isolation, preventing unwanted coupling between independent adatom structures. However, the two requirements conflict. For excellent electronic isolation, we may seek adatom confinement via van der Waals interaction without chemical bonding to the substrate atoms, but the confinement turns out to be very weak and hence unsatisfactory. An alternative chemical bonding scheme with excellent structural strength is examined, but even fundamental adatom chain properties such as whether chains are semiconducting or metallic are strongly influenced by the nature of the chemical bonding, and electronic isolation is not always achieved. Conditions for obtaining semiconducting chains with well-localized surface-modes, leading to good isolation, are clarified and discussed.

  20. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.

    PubMed Central

    Faergeman, N J; Knudsen, J

    1997-01-01

    The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase. PMID:9173866

  1. Comparison of the lipid acyl chain dynamics between small and large unilamellar vesicles.

    PubMed Central

    Lepore, L S; Ellena, J F; Cafiso, D S

    1992-01-01

    13C NMR spin-lattice relaxation (T1) rates and 13C-1H nuclear Overhauser effects (NOEs) were measured in an identical fashion in two lipid preparations having dramatically different curvatures. The T1 times that were obtained at four magnetic field strengths were fit along with the NOEs to simple models for lipid molecular dynamics. The results indicate that phospholipid chain ordering and dynamics are virtually identical in small and large unilamellar vesicles at the time scales sampled by these 13C-NMR studies. The order parameters and reorientational correlation times that characterize the amplitudes and rates of internal acyl chain motions were equal within experimental error for the methylene segments in the middle of the chains. The only significant differences in order parameters and correlation times between the two vesicle types were small and appeared at the ends of the acyl chains. At the carbonyl end the order was slightly higher in small vesicles than large vesicles, and at the methyl end the order was slightly lower for small vesicles. This indicates that in the more planar systems the acyl chains exhibit a slightly flatter order profile than in more highly curved membranes. The use of the same experimental approach in both small and large vesicle systems provided a more reliable and accurate assessment of the effect of curvature on molecular order than has been previously obtained. PMID:1504247

  2. A severe genotype with favourable outcome in very long chain acyl-CoA dehydrogenase deficiency

    PubMed Central

    Touma, E; Rashed, M; Vianey-Saban, C; Sakr, A; Divry, P; Gregersen, N; Andresen, B

    2001-01-01

    A patient with very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is reported. He had a severe neonatal presentation and cardiomyopathy. He was found to be homozygous for a severe mutation with no residual enzyme activity. Tandem mass spectrometry on dried blood spots revealed increased long chain acylcarnitines. VLCAD enzyme activity was severely decreased to 2% of control levels. Dietary management consisted of skimmed milk supplemented with medium chain triglycerides and L-carnitine. Outcome was good and there was no acute recurrence.

 PMID:11124787

  3. Probing the Mechanism of the Mycobacterium tuberculosis [beta]-Ketoacyl-Acyl Carrier Protein Synthase III mtFabH: Factors Influencing Catalysis and Substrate Specificity

    SciTech Connect

    Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent; Lindenberg, Sandra; Dover, Lynn G.; Sacchettini, James C.; Besra, Gurdyal S.

    2010-11-30

    Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.

  4. Effects of Nanoparticle Morphology and Acyl Chain Length on Spontaneous Lipid Transfer Rates

    SciTech Connect

    Xia, Yan; Li, Ming; Charubin, Kamil; Liu, Ying; Heberle, Frederick A.; Katsaras, John; Jing, Benxin; Zhu, Yingxi; Nieh, Mu-Ping

    2015-11-05

    In this paper, we report on studies of lipid transfer rates between different morphology nanoparticles and lipids with different length acyl chains. The lipid transfer rate of dimyristoylphosphatidylcholine (di-C14, DMPC) in discoidal “bicelles” (0.156 h–1) is 2 orders of magnitude greater than that of DMPC vesicles (ULVs) (1.1 × 10–3 h–1). For both bicellar and ULV morphologies, increasing the acyl chain length by two carbons [going from di-C14 DMPC to di-C16, dipalmitoylphosphatidylcholine (DPPC)] causes lipid transfer rates to decrease by more than 2 orders of magnitude. Results from small angle neutron scattering (SANS), differential scanning calorimetry (DSC), and fluorescence correlation spectroscopy (FCS) are in good agreement. Finally, the present studies highlight the importance of lipid dynamic processes taking place in different morphology biomimetic membranes.

  5. Effects of Nanoparticle Morphology and Acyl Chain Length on Spontaneous Lipid Transfer Rates

    DOE PAGES

    Xia, Yan; Li, Ming; Charubin, Kamil; ...

    2015-11-05

    In this paper, we report on studies of lipid transfer rates between different morphology nanoparticles and lipids with different length acyl chains. The lipid transfer rate of dimyristoylphosphatidylcholine (di-C14, DMPC) in discoidal “bicelles” (0.156 h–1) is 2 orders of magnitude greater than that of DMPC vesicles (ULVs) (1.1 × 10–3 h–1). For both bicellar and ULV morphologies, increasing the acyl chain length by two carbons [going from di-C14 DMPC to di-C16, dipalmitoylphosphatidylcholine (DPPC)] causes lipid transfer rates to decrease by more than 2 orders of magnitude. Results from small angle neutron scattering (SANS), differential scanning calorimetry (DSC), and fluorescence correlationmore » spectroscopy (FCS) are in good agreement. Finally, the present studies highlight the importance of lipid dynamic processes taking place in different morphology biomimetic membranes.« less

  6. A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica.

    PubMed

    Pollard, M R; Anderson, L; Fan, C; Hawkins, D J; Davies, H M

    1991-02-01

    Umbellularia californica (California Bay) seeds accumulate 10:0 and 12:0 as principal reserve fatty acyl groups. An in vitro fatty acid synthesis system from the developing cotyledons produces chiefly 10:0 and 12:0, in approximately the same proportions as the intact tissue. The kinetics of acyl thioester and free fatty acid formation in this system suggest that a medium-chain specific acyl-acyl-carrier protein (ACP) hydrolysis mechanism is responsible for the preponderance of medium-chain products. A crude extract of the developing cotyledons exhibits hydrolytic activity toward acyl-ACPs, with marked preference for 12:0-ACP and 18:1-ACP in the test series 6:0, 8:0, 10:0, 11:0, 12:0, 14:0, 16:0, and 18:1-ACPs. Partial purification of the 12:0-ACP hydrolytic activity has resulted in its separation from the 18:1-ACP hydrolase(s) and the 12:0-coenzyme A hydrolase(s) that are also present, thereby demonstrating its specificity for the 12-carbon acyl chain length and the ACP derivative. During cotyledon development, as the proportion of medium-chain to other fatty acyl groups increases, the extractable yield of this activity also increases substantially. Collectively these results suggest a role for this 12-ACP thioesterase in medium-chain production in vivo.

  7. Syntheses of Papyracillic Acids: Application of the Tandem Chain Extension-Acylation Reaction

    PubMed Central

    Mazzone, Jennifer R.; Zercher, Charles K.

    2012-01-01

    A synthetic approach to the papyracillic acid family of natural products has been developed. The spiroacetal core is rapidly assembled through an unprecedented zinc carbenoid-mediated tandem chain extension-acylation reaction. Subsequent functional group manipulation provided access to papyracillic acid B and 4-epi-papyracillic acid C. The successful preparation of these molecules resulted in the clarification of structural assignments of members of this family of natural products. PMID:23013246

  8. Effect of salicylic acid and diclofenac on the medium-chain and long-chain acyl-CoA formation in the liver and brain of mouse.

    PubMed

    Kasuya, Fumiyo; Kazumi, Maya; Tatsuki, Takao; Suzuki, Risa

    2009-07-01

    Medium-chain and long-chain acyl-CoA esters are key metabolites in fatty acid metabolism. Effects of salicylic acid on the in vivo formation of acyl-CoAs in mouse liver and brain were investigated. Further, inhibition of the medium-chain and long-chain acyl-CoA synthetases by salicylic acid and diclofenac was determined in mouse liver and brain mitochondria. Acyl-CoA esters were analyzed by liquid chromatography-tandem mass spectrometry. The amounts of medium-chain acyl-CoAs (C(6), C(8) and C(10)) were less than long-chain acyl-CoAs (C(16:0), C(18:0), C(18:1) and C(20:4)) in both liver and brain. The administration of salicylic acid decreased the levels of both the medium-chain (C(6), C(8) and C(10)) and long-chain acyl-CoAs (C(16:0), C(18:0), C(18:1) and C(20:4)) in liver. In brain, however, only long-chain acyl-CoAs were decreased. The level of salicylyl-CoA detected in brain was about 12% of that in liver. Salicylic acid had a strong inhibitory activity (IC(50) = 0.1 mm) for the liver mitochondrial formation of hexanoyl-CoA from hexanoic acid, whereas diclofenac was weak (IC(50) = 4.4 mm). In contrast, diclofenac (IC(50) = 1.4 mm) inhibited the liver mitochondrial long-chain acyl-CoA synthetases more potently than salicylic acid (IC(50) = 25.5 mm). Similar inhibitory activities for the acyl-CoA synthetases were obtained in the case of the brain and liver mitochondria, except for the weak inhibition of brain medium-chain acyl-CoA synthetases by salicylic acid (IC(50) = 1.8 mm). These findings suggest that salicylic acid and diclofenac exhibit different mechanisms of inhibition of fatty acid metabolism depending on the length of the acyl chain and tissues, and they may contribute to the further understanding of the toxic effects associated with these drugs.

  9. Hepatic fatty acid uptake is regulated by the sphingolipid acyl chain length

    PubMed Central

    Park, Woo-Jae; Park, Joo-Won; Merrill, Alfred H.; Storch, Judith; Pewzner-Jung, Yael; Futerman, Anthony H.

    2015-01-01

    Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in liver but not in adipose tissue or skeletal muscle in the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [3H]-palmitate were also abrogated in hepatocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids. PMID:25241943

  10. Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry[S

    PubMed Central

    Maccarone, Alan T.; Duldig, Jackson; Mitchell, Todd W.; Blanksby, Stephen J.; Duchoslav, Eva; Campbell, J. Larry

    2014-01-01

    Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation. PMID:24939921

  11. 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

  12. Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa.

    PubMed

    Hu, Zhaohui; Wu, Qian; Dalal, Jyoti; Vasani, Naresh; Lopez, Harry O; Sederoff, Heike W; Qu, Rongda

    2017-01-01

    With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

  13. Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa

    PubMed Central

    Dalal, Jyoti; Vasani, Naresh; Lopez, Harry O.; Sederoff, Heike W.

    2017-01-01

    With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production. PMID:28212406

  14. Structural identification of skin ceramides containing ω-hydroxy acyl chains using mass spectrometry.

    PubMed

    Wu, Zhexue; Shon, Jong Cheol; Kim, Jong Yei; Cho, Yunhi; Liu, Kwang-Hyeon

    2016-10-01

    The stratum corneum (SC) acts as a barrier that protects organisms against the environment and from transepidermal water loss. It consists of corneocytes embedded in a matrix of lipid metabolites (ceramides, cholesterol, and free fatty acids). Of these lipids, ceramides are sphingolipids consisting of sphingoid bases, linked to fatty acyl chains. Typical fatty acid acyl chains are composed of α-hydroxy fatty acids (A), esterified ω-hydroxy fatty acids (EO), non-hydroxy fatty acids (N), and ω-hydroxy fatty acids (O). Of these, O-type ceramides are ester-linked via their ω-hydroxyl group to proteins in the cornified envelope and can be released and extracted following mild alkaline hydrolysis. Tandem mass spectrometry (MS/MS) analysis of O-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, suggesting that this method could be applied to the structural identification of O-type ceramides. Based on the MS/MS fragmentation patterns of O-type ceramides, comprehensive fragmentation schemes are proposed. In addition, we have also developed a method for identifying and profiling O-type ceramides in the mouse and guinea pig SC. This information may be used to identify O-type ceramides in the SC of animal skin.

  15. Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA

    PubMed Central

    Albesa-Jové, David; Svetlíková, Zuzana; Tersa, Montse; Sancho-Vaello, Enea; Carreras-González, Ana; Bonnet, Pascal; Arrasate, Pedro; Eguskiza, Ander; Angala, Shiva K.; Cifuente, Javier O.; Korduláková, Jana; Jackson, Mary; Mikušová, Katarína; Guerin, Marcelo E.

    2016-01-01

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. PMID:26965057

  16. Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA.

    PubMed

    Albesa-Jové, David; Svetlíková, Zuzana; Tersa, Montse; Sancho-Vaello, Enea; Carreras-González, Ana; Bonnet, Pascal; Arrasate, Pedro; Eguskiza, Ander; Angala, Shiva K; Cifuente, Javier O; Korduláková, Jana; Jackson, Mary; Mikušová, Katarína; Guerin, Marcelo E

    2016-03-11

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.

  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

    2016-08-09

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

  18. Acyl chain length and charge effect on Tamoxifen-lipid model membrane interactions

    NASA Astrophysics Data System (ADS)

    Bilge, Duygu; Kazanci, Nadide; Severcan, Feride

    2013-05-01

    Tamoxifen (TAM), which is an antiestrogenic agent, is widely used during chemotherapy of breast, pancreas, brain and liver cancers. In this study, TAM and model membrane interactions in the form of multilamellar vesicles (MLVs) were studied for lipids containing different acyl chain length and different charge status as a function of different TAM (1, 6, 9 and 15 mol%) concentrations. Zwitterionic lipids namely dipalmitoyl phosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC) lipids were used to see the acyl chain length effect and anionic dipalmitoyl phosphtidylglycerol (DPPG) lipid was used to see the charge effect. For this purpose Fourier transform-infrared (FTIR) spectroscopic and differential scanning calorimetric (DSC) techniques have been conducted. For zwitterionic lipid, concentration dependent different action of TAM was observed both in the gel and liquid crystalline phases by significantly increasing the lipid order and decreasing the dynamics for 1 mol% TAM, while decreasing the lipid order and increasing the dynamics of the lipids for higher concentrations (6, 9 and 15 mol%). However, different than neutral lipids, the dynamics and disorder of DPPG liposome increased for all TAM concentrations. The interactions between TAM and head group of multilamellar liposomes was monitored by analyzing the Cdbnd O stretching and PO2- antisymmetric double bond stretching bands. Increasing Tamoxifen concentrations led to a dehydration around these functional groups in the polar part of the lipids. DSC studies showed that for all types of lipids, TAM eliminates the pre-transition, shifts the main phase transition to lower temperatures and broadened the phase transition curve. The results indicate that not the acyl chain length but the charge status of the polar head group induces different effects on lipid membranes order and dynamics.

  19. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    PubMed

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  20. 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

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

    PubMed Central

    2011-01-01

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

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

    PubMed

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

    2016-04-18

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

  3. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

    SciTech Connect

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2014-03-07

    Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

  4. Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates.

    PubMed

    Gustafson, W G; Feinberg, B A; McFarland, J T

    1986-06-15

    We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.

  5. Long-chain bases of sphingolipids are transported into cells via the acyl-CoA synthetases

    PubMed Central

    Narita, Tomomi; Naganuma, Tatsuro; Sase, Yurie; Kihara, Akio

    2016-01-01

    Transport of dietary lipids into small-intestinal epithelial cells is pathologically and nutritionally important. However, lipid uptake remains an almost unexplored research area. Although we know that long-chain bases (LCBs), constituents of sphingolipids, can enter into cells efficiently, the molecular mechanism of LCB uptake is completely unclear. Here, we found that the yeast acyl-CoA synthetases (ACSs) Faa1 and Faa4 are redundantly involved in LCB uptake. In addition to fatty acid-activating activity, transporter activity toward long-chain fatty acids (LCFAs) has been suggested for ACSs. Both LCB and LCFA transports were largely impaired in faa1Δ faa4Δ cells. Furthermore, LCB and LCFA uptakes were mutually competitive. However, the energy dependency was different for their transports. Sodium azide/2-deoxy-D-glucose treatment inhibited import of LCFA but not that of LCB. Furthermore, the ATP-AMP motif mutation FAA1 S271A largely impaired the metabolic activity and LCFA uptake, while leaving LCB import unaffected. These results indicate that only LCFA transport requires ATP. Since ACSs do not metabolize LCBs as substrates, Faa1 and Faa4 are likely directly involved in LCB transport. Furthermore, we revealed that ACSs are also involved in LCB transport in mammalian cells. Thus, our findings provide strong support for the hypothesis that ACSs directly transport LCFAs. PMID:27136724

  6. Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

    PubMed

    Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

    2014-03-01

    Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.

  7. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.

    PubMed Central

    Van Hove, J L; Zhang, W; Kahler, S G; Roe, C R; Chen, Y T; Terada, N; Chace, D H; Iafolla, A K; Ding, J H; Millington, D S

    1993-01-01

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species. PMID:8488845

  8. Suppression of long chain acyl-CoA synthetase 3 decreases hepatic de novo fatty acid synthesis through decreased transcriptional activity.

    PubMed

    Bu, So Young; Mashek, Mara T; Mashek, Douglas G

    2009-10-30

    Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-gamma, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-alpha and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-(14)C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis.

  9. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    PubMed Central

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-01-01

    The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-­hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination. PMID:16511283

  10. Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes.

    PubMed

    Westerlund, Bodil; Grandell, Pia-Maria; Isaksson, Y Jenny E; Slotte, J Peter

    2010-07-01

    Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T (m) and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X (Cer) = 0.05. These broad components had higher T (m) values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X (Cer) = 0.05-0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.

  11. Lipid transfer between phosphatidylcholine vesicles and human erythrocytes: exponential decrease in rate with increasing acyl chain length.

    PubMed

    Ferrell, J E; Lee, K J; Huestis, W H

    1985-06-04

    The rate of phospholipid transfer from sonicated phospholipid vesicles to human erythrocytes has been studied as a function of membrane concentration and lipid acyl chain composition. Phospholipid transfer exhibits saturable first-order kinetics with respect to both cell and vesicle membrane concentrations. This kinetic behavior is consistent either with transfer during transient contact between cell and vesicle surfaces (but only if the fraction of the cell surface susceptible to such interaction is small) or with transfer of monomers through the aqueous phase. The acyl chain composition of the transferred phospholipid affects the transfer kinetics profoundly; for homologous saturated phosphatidylcholines, the rate of transfer decreases exponentially with increasing acyl chain length. This behavior is consistent with passage of phospholipid monomers through a polar phase, which might be the bulk aqueous phase( as in the monomer transfer model) or the hydrated head-group regions of a cell-vesicle complex (transient collision model). Collisional transfer also predicts that intercell transfer of phospholipids should be slow compared to cell-vesicle transfer, as surface charge and steric effects should prevent close apposition of donor and acceptor membranes. This is not found; dilauroylphosphatidylcholine transfers rapidly between red cells. Thus, the observed relationship between acyl chain length and intermembrane phospholipid transfer rates likely reflects the energetics of monomer transfer through the aqueous phase.

  12. Genomic analysis of branched chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acyl sugars are extracellular epidermal lipids that are exuded from glandular trichomes and coat the aerial organs of many species in the Solanaceae. These highly viscous surfactants, which often contain branched-chain fatty acids (BCFA), play an important defensive role against pest and insects. ...

  13. Unsaturated acyl chains dramatically enhanced cellular uptake by direct translocation of a minimalist oligo-arginine lipopeptide.

    PubMed

    Swiecicki, J-M; Di Pisa, M; Lippi, F; Chwetzoff, S; Mansuy, C; Trugnan, G; Chassaing, G; Lavielle, S; Burlina, F

    2015-10-07

    The recurring issue with cell penetrating peptides is how to increase direct translocation vs. endocytosis, to avoid premature degradation. Acylation by a cis unsaturated chain (C22:6) of a short cationic peptide provides a new rational design to favour diffuse cytosolic and dense Golgi localisations.

  14. Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases.

    PubMed

    Tong, Fumin; Black, Paul N; Coleman, Rosalind A; DiRusso, Concetta C

    2006-03-01

    Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.

  15. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    SciTech Connect

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-02-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

  16. 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.

  17. Identification of Middle Chain Fatty Acyl-CoA Ligase Responsible for the Biosynthesis of 2-Alkylmalonyl-CoAs for Polyketide Extender Unit*

    PubMed Central

    Miyazawa, Takeshi; Takahashi, Shunji; Kawata, Akihiro; Panthee, Suresh; Hayashi, Teruo; Shimizu, Takeshi; Nogawa, Toshihiko; Osada, Hiroyuki

    2015-01-01

    Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8–C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via β-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the β-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-13C]octanoic acid. 13C NMR analysis clearly demonstrated incorporation of the [3,4-13C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit. PMID:26378232

  18. Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes.

    PubMed

    Janůšová, Barbora; Zbytovská, Jarmila; Lorenc, Petr; Vavrysová, Helena; Palát, Karel; Hrabálek, Alexandr; Vávrová, Kateřina

    2011-03-01

    Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.

  19. Retrospective study of the medium-chain acyl-CoA dehydrogenase deficiency in Portugal.

    PubMed

    Ventura, F V; Leandro, P; Luz, A; Rivera, I A; Silva, M F B; Ramos, R; Rocha, H; Lopes, A; Fonseca, H; Gaspar, A; Diogo, L; Martins, E; Leão-Teles, E; Vilarinho, L; Tavares de Almeida, I

    2014-06-01

    Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the commonest genetic defect of mitochondrial fatty acid β-oxidation. About 60% of MCADD patients are homozygous for the c.985A>G (p.Lys329Glu) mutation in the ACADM gene (G985 allele). Herein, we present the first report on the molecular and biochemical spectrum of Portuguese MCADD population. From the 109 patients studied, 83 were diagnosed after inclusion of MCADD in the national newborn screening, 8 following the onset of symptoms and 18 through segregation studies. Gypsy ancestry was identified in 85/109 patients. The G985 allele was found in homozygosity in 102/109 patients, in compound heterozygosity in 6/109 and was absent in one patient. Segregation studies in the Gypsy families showed that 93/123 relatives were carriers of the G985 allele, suggesting its high prevalence in this ethnic group. Additionally, three new substitutions-c.218A>G (p.Tyr73Cys), c.503A>T (p.Asp168Val) and c.1205G>T (p.Gly402Val)-were identified. Despite the particularity of the MCADD population investigated, the G985 allele was found in linkage disequilibrium with H1(112) haplotype. Furthermore, two novel haplotypes, H5(212) and H6(122) were revealed.

  20. Medium chain acyl-CoA dehydrogenase deficiency detected among Hispanics by New Jersey newborn screening.

    PubMed

    Anderson, Sharon; Botti, Christina; Li, Bo; Millonig, James H; Lyon, Elaine; Millson, Alison; Karabin, Suzanne S M; Brooks, Susan Sklower

    2012-09-01

    In the follow-up of New Jersey newborn screens suggestive of medium chain acyl-CoA dehydrogenase deficiency (MCADD) during a 30-month period, we identified five patients of Hispanic American ethnicity. With information provided by the New Jersey Department of Health and Human Services Newborn Screening program we calculated an overall cumulative incidence of approximately 7.20/100,000 for MCADD; 7.58/100,000 among Hispanic Americans and 7.08/100,000 among non-Hispanic Americans. Among the five Hispanic American infants who screened positive, a common variant (c.443G>A [p.R148K]) was identified which accounted for 30% of the alleles; c.799G>A (p.G267R) and c.985A>G (p.K329E) each accounted for an additional 20%; and a novel variant c.302G>A (p.G101E) was identified in one patient. Although treated prospectively during interim illnesses to prevent unwanted sequelae; till date, none of the patients carrying the c.443G>A variant have been symptomatic.

  1. Concentrations of long-chain acyl-acyl carrier proteins during fatty acid synthesis by chloroplasts isolated from pea (Pisum sativum), safflower (Carthamus tinctoris), and amaranthus (Amaranthus lividus) leaves

    SciTech Connect

    Roughan, G.; Nishida, I. )

    1990-01-01

    Fatty acid synthesis from (1-14C)acetate by chloroplasts isolated from peas and amaranthus was linear for at least 15 min, whereas incorporation of the tracer into long-chain acyl-acyl carrier protein (ACP) did not increase after 2-3 min. When reactions were transferred to the dark after 3-5 min, long-chain acyl-ACPs lost about 90% of their radioactivity and total fatty acids retained all of theirs. Half-lives of the long-chain acyl-ACPs were estimated to be 10-15 s. Concentrations of palmitoyl-, stearoyl-, and oleoyl-ACP as indicated by equilibrium labeling during steady-state fatty acid synthesis, ranged from 0.6-1.1, 0.2-0.7, and 0.4-1.6 microM, respectively, for peas and from 1.6-1.9, 1.3-2.6, and 0.6-1.4 microM, respectively, for amaranthus. These values are based on a chloroplast volume of 47 microliters/mg chlorophyll and varied according to the mode of the incubation. A slow increase in activity of the fatty acid synthetase in safflower chloroplasts resulted in long-chain acyl-ACPs continuing to incorporate labeled acetate for 10 min. Upon re-illumination following a dark break, however, both fatty acid synthetase activity and acyl-ACP concentrations increased very rapidly. Palmitoyl-ACP was present at concentrations up to 2.5 microM in safflower chloroplasts, whereas those of stearoyl- and oleoyl-ACPs were in the lower ranges measured for peas. Acyl-ACPs were routinely separated from extracts of chloroplasts that had been synthesising long-chain fatty acids from labeled acetate by a minor modification of the method of Mancha et al. The results compared favorably with those obtained using alternative analytical methods such as adsorption to filter paper and partition chromatography on silicic acid columns.

  2. Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH).

    PubMed

    Jiang, Wen; Jiang, Yanfang; Bentley, Gayle J; Liu, Di; Xiao, Yi; Zhang, Fuzhong

    2015-08-01

    Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids.

  3. Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry

    PubMed Central

    Clayton, P.; Doig, M.; Ghafari, S.; Meaney, C.; Taylor, C.; Leonard, J.; Morris, M.; Johnson, A.

    1998-01-01

    OBJECTIVE—To establish criteria for the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in the UK population using a method in which carnitine species eluted from blood spots are butylated and analysed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS).
DESIGN—Four groups were studied: (1) 35 children, aged 4 days to 16.2 years, with proven MCAD deficiency (mostly homozygous for the A985G mutation, none receiving carnitine supplements); (2) 2168control children; (3) 482 neonates; and (4) 15 MCAD heterozygotes.
RESULTS—All patients with MCAD deficiency had an octanoylcarnitine concentration ([C8-Cn]) > 0.38 µM and no accumulation of carnitine species > C10 or < C6. Among the patients with MCAD deficiency, the [C8-Cn] was significantly lower in children > 10 weeks old and in children with carnitine depletion (free carnitine < 20 µM). Neonatal blood spots from patients with MCAD deficiency had a [C8-Cn] > 1.5 µM, whereas in heterozygotes and other normal neonates the [C8-Cn] was < 1.0 µM. In contrast, the blood spot [C8-Cn] in eight of 27 patients with MCAD deficiency > 10 weeks old fell within the same range as five of 15 MCAD heterozygotes (0.38-1.0 µM). However, the free carnitine concentrations were reduced (< 20 µM) in the patients with MCAD deficiency but normal in the heterozygotes.
CONCLUSIONS—Criteria for the diagnosis of MCAD deficiency using ESI-MS/MS must take account of age and carnitine depletion. If screening is undertaken at 7-10 days, the number of false positive and negative results should be negligible. Because there have been no instances of death or neurological damage following diagnosis of MCAD deficiency in our patient group, a strong case can be made for neonatal screening for MCAD deficiency in the UK.

 PMID:9797589

  4. Unusual Heme Iron-Lipid Acyl Chain Coordination in Escherichia coli Flavohemoglobin

    PubMed Central

    D'Angelo, Paola; Lucarelli, Debora; della Longa, Stefano; Benfatto, Maurizio; Hazemann, Jean Louis; Feis, Alessandro; Smulevich, Giulietta; Ilari, Andrea; Bonamore, Alessandra; Boffi, Alberto

    2004-01-01

    Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 Å in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 Å (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 Å resolution obtained in the present work. Thus, the contributions at 2.7 Å distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid. PMID:15189885

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

    PubMed Central

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

    2016-01-01

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

  6. Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenases in isoleucine and valine metabolism.

    PubMed

    Andresen, B S; Christensen, E; Corydon, T J; Bross, P; Pilgaard, B; Wanders, R J; Ruiter, J P; Simonsen, H; Winter, V; Knudsen, I; Schroeder, L D; Gregersen, N; Skovby, F

    2000-11-01

    Acyl-CoA dehydrogenase (ACAD) defects in isoleucine and valine catabolism have been proposed in clinically diverse patients with an abnormal pattern of metabolites in their urine, but they have not been proved enzymatically or genetically, and it is unknown whether one or two ACADs are involved. We investigated a patient with isolated 2-methylbutyrylglycinuria, suggestive of a defect in isoleucine catabolism. Enzyme assay of the patient's fibroblasts, using 2-methylbutyryl-CoA as substrate, confirmed the defect. Sequence analysis of candidate ACADs revealed heterozygosity for the common short-chain ACAD A625 variant allele and no mutations in ACAD-8 but a 100-bp deletion in short/branched-chain ACAD (SBCAD) cDNA from the patient. Our identification of the SBCAD gene structure (11 exons; >20 kb) enabled analysis of genomic DNA. This showed that the deletion was caused by skipping of exon 10, because of homozygosity for a 1228G-->A mutation in the patient. This mutation was not present in 118 control chromosomes. In vitro transcription/translation experiments and overexpression in COS cells confirmed the disease-causing nature of the mutant SBCAD protein and showed that ACAD-8 is an isobutyryl-CoA dehydrogenase and that both wild-type proteins are imported into mitochondria and form tetramers. In conclusion, we report the first mutation in the SBCAD gene, show that it results in an isolated defect in isoleucine catabolism, and indicate that ACAD-8 is a mitochondrial enzyme that functions in valine catabolism.

  7. Ceramides with a pentadecasphingosine chain and short acyls have strong permeabilization effects on skin and model lipid membranes.

    PubMed

    Školová, Barbora; Janůšová, Barbora; Vávrová, Kateřina

    2016-02-01

    The composition and organization of stratum corneum lipids play an essential role in skin barrier function. Ceramides represent essential components of this lipid matrix; however, the importance of the individual structural features in ceramides is not fully understood. To probe the structure-permeability relationships in ceramides, we prepared analogs of N-lignoceroylsphingosine with shortened sphingosine (15 and 12 carbons) and acyl chains (2, 4 and 6 carbons) and studied their behavior in skin and in model lipid membranes. Ceramide analogs with pentadecasphingosine (15C) chains were more barrier-perturbing than 12C- and 18C-sphingosine ceramides; the greatest effects were found with 4 to 6C acyls (up to 15 times higher skin permeability compared to an untreated control and up to 79 times higher permeability of model stratum corneum lipid membranes compared to native very long-chain ceramides). Infrared spectroscopy using deuterated lipids and X-ray powder diffraction showed surprisingly similar behavior of the short ceramide membranes in terms of lipid chain order and packing, phase transitions and domain formation. The high- and low-permeability membranes differed in their amide I band shape and lamellar organization. These skin and membrane permeabilization properties of some short ceramides may be explored, for example, for the rational design of permeation enhancers for transdermal drug delivery.

  8. Substrate Effects on Electronic Properties of Atomic Chains

    NASA Technical Reports Server (NTRS)

    Yamada, Toshishige; Saini, Subhash (Technical Monitor)

    1998-01-01

    Atomic chains, precise structures of adatoms created on an atomically regulated surface, are candidates for constituent elements in future electronics. It was predicted that Si chains were metallic and Mg chains were semiconducting, and a doping method was also discussed. The substrate was assumed to work as a non-interacting template holding the adatoms. However, this scheme requires a low-temperature environment so that the adatoms will not displace from their ideal positions due to unwanted thermal agitation. For better structural stability, we may seek a scheme to allow the adatoms to form chemical bonding with the substrate atoms and secure their positions. The chemical bonding has two major effects on the chain electronic properties. First, only the remaining s and p orbitals in an adatom not used for the chemical bonding can decide chain band structures, rather than the full set of orbitals previously assumed. Second, because of a possible HOMO energy difference between adatom and substrate atom, semiconducting chains are unintentionally doped. These effects are studied with a self-consistent tight-binding method with universal parameters. With one adatom per unit cell, adatom chains are semiconducting: (1) if adatoms are of group III and form one chemical bond per adatom, or (2) if adatoms are of group IV and form two chemical bonds. 'Me previous result [I I of realizing semiconducting chains by group II adatoms without chemical bonds is consistent with this picture. When the HOMO energy in an adatom is shallower than that in a substrate atom, the entire chain is positively polarized, unintentionally achieving p-type doping. When deeper, the chain is negatively charged, achieving n-type doping.

  9. Acyl chain length and saturation modulate interleaflet coupling in asymmetric bilayers: effects on dynamics and structural order.

    PubMed

    Chiantia, Salvatore; London, Erwin

    2012-12-05

    A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.

  10. α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase: Exploration of Conformational Constraints in the Acyl Side Chain

    PubMed Central

    Duncan, Katharine K.; Otrubova, Katerina; Boger, Dale L.

    2014-01-01

    A series of α-ketooxazoles containing heteroatoms embedded within conformational constraints in the C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydrolase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candidate inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively inhibit FAAH. PMID:24690529

  11. Modulation of cellulase activity by charged lipid bilayers with different acyl chain properties for efficient hydrolysis of ionic liquid-pretreated cellulose.

    PubMed

    Mihono, Kai; Ohtsu, Takeshi; Ohtani, Mai; Yoshimoto, Makoto; Kamimura, Akio

    2016-10-01

    The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains.

  12. Phospholipid profiling identifies acyl chain elongation as a ubiquitous trait and potential target for the treatment of lung squamous cell carcinoma

    PubMed Central

    Marien, Eyra; Meister, Michael; Muley, Thomas; del Pulgar, Teresa Gomez; Derua, Rita; Spraggins, Jeffrey M.; Van de Plas, Raf; Vanderhoydonc, Frank; Machiels, Jelle; Binda, Maria Mercedes; Dehairs, Jonas; Willette-Brown, Jami; Hu, Yinling; Dienemann, Hendrik; Thomas, Michael; Schnabel, Philipp A.; Caprioli, Richard M.; Lacal, Juan Carlos; Waelkens, Etienne; Swinnen, Johannes V.

    2016-01-01

    Lung cancer is the leading cause of cancer death. Beyond first line treatment, few therapeutic options are available, particularly for squamous cell carcinoma (SCC). Here, we have explored the phospholipidomes of 30 human SCCs and found that they almost invariably (in 96.7% of cases) contain phospholipids with longer acyl chains compared to matched normal tissues. This trait was confirmed using in situ 2D-imaging MS on tissue sections and by phospholipidomics of tumor and normal lung tissue of the L-IkkαKA/KA mouse model of lung SCC. In both human and mouse, the increase in acyl chain length in cancer tissue was accompanied by significant changes in the expression of acyl chain elongases (ELOVLs). Functional screening of differentially expressed ELOVLs by selective gene knockdown in SCC cell lines followed by phospholipidomics revealed ELOVL6 as the main elongation enzyme responsible for acyl chain elongation in cancer cells. Interestingly, inhibition of ELOVL6 drastically reduced colony formation of multiple SCC cell lines in vitro and significantly attenuated their growth as xenografts in vivo in mouse models. These findings identify acyl chain elongation as one of the most common traits of lung SCC discovered so far and pinpoint ELOVL6 as a novel potential target for cancer intervention. PMID:26862848

  13. 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

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

    PubMed

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

    2010-06-01

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

  15. Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

    PubMed Central

    Recuero-Checa, Maria A.; Sharma, Manu; Lau, Constance; Watkins, Paul A.; Gaydos, Charlotte A.; Dean, Deborah

    2016-01-01

    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs. PMID:26988341

  16. Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

    PubMed Central

    Andresen, B S; Olpin, S; Poorthuis, B J; Scholte, H R; Vianey-Saban, C; Wanders, R; Ijlst, L; Morris, A; Pourfarzam, M; Bartlett, K; Baumgartner, E R; deKlerk, J B; Schroeder, L D; Corydon, T J; Lund, H; Winter, V; Bross, P; Bolund, L; Gregersen, N

    1999-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established. PMID:9973285

  17. Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs).

    PubMed

    Recuero-Checa, Maria A; Sharma, Manu; Lau, Constance; Watkins, Paul A; Gaydos, Charlotte A; Dean, Deborah

    2016-03-18

    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

  18. Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

    PubMed Central

    Martín, J; Pérez-Gil, J; Acebal, C; Arche, R

    1990-01-01

    A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a 'hysteretic' pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied. PMID:2310381

  19. Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains

    PubMed Central

    Wattelet-Boyer, Valérie; Brocard, Lysiane; Jonsson, Kristoffer; Esnay, Nicolas; Joubès, Jérôme; Domergue, Frédéric; Mongrand, Sébastien; Raikhel, Natasha; Bhalerao, Rishikesh P.; Moreau, Patrick; Boutté, Yohann

    2016-01-01

    The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells. PMID:27681606

  20. Differential effects of cholesterol on acyl chain order in erythrocyte membranes as a function of depth from the surface. An electron paramagnetic resonance (EPR) spin label study.

    PubMed

    Cassera, M B; Silber, A M; Gennaro, A M

    2002-10-16

    The purpose of this work is to analyze the effects of cholesterol modulation on acyl chain ordering in the membrane of human erythrocytes as a function of depth from the surface. Partial cholesterol depletion was achieved by incubation of erythrocytes with liposomes containing saturated phospholipids, or with methyl-beta-cyclodextrin (MbetaCD). Cholesterol enrichment was achieved by incubation with liposomes formed by phospholipids/cholesterol, or with the complex MbetaCD/cholesterol. Acyl chain order was studied with electron paramagnetic resonance spectroscopy (EPR) using spin labels that sense the lipid bilayer at different depths. It is shown that the increase in cholesterol stiffens acyl chains but decreases the interaction among lipid headgroups, while cholesterol depletion causes the opposite behavior. It is likely that the observed cholesterol effects are related to those stabilizing the cholesterol-rich detergent-insoluble membrane domains (rafts), recently shown to exist in erythrocytes.

  1. AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

    PubMed

    González, Alex; Bellenberg, Sören; Mamani, Sigde; Ruiz, Lina; Echeverría, Alex; Soulère, Laurent; Doutheau, Alain; Demergasso, Cecilia; Sand, Wolfgang; Queneau, Yves; Vera, Mario; Guiliani, Nicolas

    2013-04-01

    Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

  2. Ontogeny of mRNA expression and activity of long-chain acyl-CoA synthetase (ACSL) isoforms in Mus musculus heart.

    PubMed

    de Jong, Hendrik; Neal, Andrea C; Coleman, Rosalind A; Lewin, Tal M

    2007-01-01

    Long-chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for virtually every metabolic pathway that catabolizes FA or synthesizes complex lipids. We have hypothesized that each of the five cloned ACSL isoforms partitions FA towards specific downstream pathways. Adult heart expresses all five cloned ACSL isoforms, but their independent functional roles have not been elucidated. Studies implicate ACSL1 in both oxidative and lipid synthetic pathways. To clarify the functional role of ACSL1 and the other ACSL isoforms (3-6), we examined ACS specific activity and Acsl mRNA expression in the developing mouse heart which increases FA oxidative pathways for energy production after birth. Compared to the embryonic heart, ACS specific activity was 14-fold higher on post-natal day 1 (P1). On P1, as compared to the fetus, only Acsl1 mRNA increased, whereas transcripts for the other Acsl isoforms remained the same, suggesting that ACSL1 is the major isoform responsible for activating long-chain FA for myocardial oxidation after birth. In contrast, the mRNA abundance of Acsl3 was highest on E16, and decreased dramatically by P7, suggesting that ACSL3 may play a critical role during the development of the fetal heart. Our data support the hypothesis that each ACSL has a specific role in the channeling of FA towards distinct metabolic fates.

  3. Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169.

    PubMed

    Allen, James W; DiRusso, Concetta C; Black, Paul N

    2017-01-06

    Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-(13)C]glucose, (13)CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions.

  4. Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

    PubMed Central

    Carter, Michael S.

    2015-01-01

    ABSTRACT Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR). IMPORTANCE Short-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling

  5. Ghrelin acylation and metabolic control.

    PubMed

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

    2011-11-01

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

  6. Modified acyl-ACP desaturase

    DOEpatents

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

    1998-01-06

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

  7. Substrate Effects on Electronic Properties of Atomic Chains

    NASA Technical Reports Server (NTRS)

    Yamada, Toshishige; Saini, Subhash (Technical Monitor)

    1998-01-01

    When the device size is reduced down to 0.07 micrometers, the number of dopant atoms in the channel will no longer be macroscopic, typically less than a hundred. A spatial distribution of these dopant atoms will fluctuate statistically from device to device even in identically designed devices, and this places a serious limitation for integration. It is, however, impractical to control dopant positions within atomic dimension. One fundamental solution to this problem is to create electronics with atomically precise, but preferably simple structures. Atomic chains, precise structures of adatoms created on an atomically regulated surface, are candidates for constituent components in future electronics. All the adatoms will be placed at designated positions on the substrate, and all the device structures will be precise, free from any deviations. It was predicted using the tight-binding calculation with universal parameters that silicon chains were metallic and magnesium chains were semiconducting regardless of the lattice spacing, and a possible doping method was also proposed. In these treatments, the substrate was assumed to serve as a non-interacting template holding the adatoms without a formation of chemical bonding with substrate atoms. However, this scheme may not be easy to implement experimentally. Adatoms will have to be fixed with a van der Waals force on the substrate, but the force is generally weak and an extremely low temperature environment has to be prepared to suppress their unwanted thermal displacement. It may be logical to seek a scheme to allow the adatoms to form chemical bonding with the substrate atoms and secure their positions. The substrate effects are studied in detail.

  8. A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome.

    PubMed

    Santiago-Tirado, Felipe H; Peng, Tao; Yang, Meng; Hang, Howard C; Doering, Tamara L

    2015-05-01

    Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter's opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.

  9. 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

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

    PubMed

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

    2012-06-05

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

  11. Medium-chain triglycerides impair lipid metabolism and induce hepatic steatosis in very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient mice.

    PubMed

    Tucci, Sara; Primassin, Sonja; Ter Veld, Frank; Spiekerkoetter, Ute

    2010-09-01

    A medium-chain-triglyceride (MCT)-based diet is mainstay of treatment in very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), a long-chain fatty acid beta-oxidation defect. Beneficial effects have been reported with an MCT-bolus prior to exercise. Little is known about the impact of a long-term MCT diet on hepatic lipid metabolism. Here we investigate the effects of MCT-supplementation on liver and blood lipids in the murine model of VLCADD. Wild-type (WT) and VLCAD-knock-out (KO) mice were fed (1) a long-chain triglyceride (LCT)-diet over 5weeks, (2) an MCT diet over 5 weeks and (3) an LCT diet plus MCT-bolus. Blood and liver lipid content were determined. Expression of genes regulating lipogenesis was analyzed by RT-PCR. Under the LCT diet, VLCAD-KO mice accumulated significantly higher blood cholesterol concentrations compared to WT mice. The MCT-diet induced severe hepatic steatosis, significantly higher serum free fatty acids and impaired hepatic lipid mobilization in VLCAD-KO mice. Expression at mRNA level of hepatic lipogenic genes was up-regulated. The long-term MCT diet stimulates lipogenesis and impairs hepatic lipid metabolism in VLCAD-KO mice. These results suggest a critical reconsideration of a long-term MCT-modified diet in human VLCADD. In contrast, MCT in situations of increased energy demand appears to be a safer treatment alternative.

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

    PubMed

    Li, Yaxiao; Qi, Baoxiu

    2017-01-01

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

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

    PubMed Central

    Li, Yaxiao; Qi, Baoxiu

    2017-01-01

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

  15. 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 ...

  16. 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 ...

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

    PubMed Central

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

    2015-01-01

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

  18. Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.

    PubMed

    Petković, Hrvoje; Sandmann, Axel; Challis, Iain R; Hecht, Hans-Jürgen; Silakowski, Barbara; Low, Lindsey; Beeston, Nicola; Kuscer, Enej; Garcia-Bernardo, Jose; Leadlay, Peter F; Kendrew, Steven G; Wilkinson, Barrie; Müller, Rolf

    2008-02-07

    The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.

  19. Protoxenicins A and B, Cytotoxic Long-Chain Acylated Xenicanes from the Soft Coral Protodendron repens.

    PubMed

    Urda, Carlos; Fernández, Rogelio; Pérez, Marta; Rodríguez, Jaime; Jiménez, Carlos; Cuevas, Carmen

    2017-03-24

    Two new xenicanes, named protoxenicins A (1) and B (2), were isolated from an organic extract of the soft coral Protodendron repens, collected off the coast of Okuza (Tanzania), being the first chemical study of an organism belonging to this genus. Their planar structures were determined by 1D and 2D NMR and HRESIMS techniques, while the relative configurations were elucidated by comparison of their chemical shifts and coupling constants with the literature values of their congeners, as well as by ROESY experiments, chemical derivatization, and molecular mechanics calculations. This is the first report of a xenicin acylated with a long saturated fatty acid. Furthermore, the absolute configuration of the stereogenic centers of the cyclononane ring and at C-1 in 1 was determined by Mosher's method. Protoxenicin B (2) is present in solution as a mixture of two conformers in a 2:1 ratio deduced by (1)H NMR. Both xenicanes display significant cytotoxic activity against a panel of different tumor cell lines.

  20. Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse

    PubMed Central

    van Vlies, Naomi; Tian, Liqun; Overmars, Henk; Bootsma, Albert H.; Kulik, Willem; Wanders, Ronald J. A.; Wood, Philip A.; Vaz, Frédéric M.

    2004-01-01

    In the present paper, we describe a novel method which enables the analysis of tissue acylcarnitines and carnitine biosynthesis intermediates in the same sample. This method was used to investigate the carnitine and fatty acid metabolism in wild-type and LCAD−/− (long-chain acyl-CoA dehydrogenase-deficient) mice. In agreement with previous results in plasma and bile, we found accumulation of the characteristic C14:1-acylcarnitine in all investigated tissues from LCAD−/− mice. Surprisingly, quantitatively relevant levels of 3-hydroxyacylcarnitines were found to be present in heart, muscle and brain in wild-type mice, suggesting that, in these tissues, long-chain 3-hydroxyacyl-CoA dehydrogenase is rate-limiting for mitochondrial β-oxidation. The 3-hydroxyacylcarnitines were absent in LCAD−/− tissues, indicating that, in this situation, the β-oxidation flux is limited by the LCAD deficiency. A profound deficiency of acetylcarnitine was observed in LCAD−/− hearts, which most likely corresponds with low cardiac levels of acetyl-CoA. Since there was no carnitine deficiency and only a marginal elevation of potentially cardiotoxic acylcarnitines, we conclude from these data that the cardiomyopathy in the LCAD−/− mouse is caused primarily by a severe energy deficiency in the heart, stressing the important role of LCAD in cardiac fatty acid metabolism in the mouse. PMID:15535801

  1. Hepatic expression of long-chain acyl-CoA synthetase 3 is upregulated in hyperlipidemic hamsters.

    PubMed

    Wu, Minhao; Liu, Haiyan; Chen, Wei; Fujimoto, Yasuyuki; Liu, Jingwen

    2009-11-01

    Members of the mammalian long-chain acyl-CoA synthetase (ACSL) family are key enzymes for cellular fatty acid metabolism that catalyze the initial step in activation of long-chain fatty acids. However, the specificity of individual isoforms of ACSL to the lipid metabolic process is not well studied. In addition, the regulation of expression of individual ACSL isoforms under hyperlipidemic conditions is largely unknown. We cloned the hamster ACSL3 cDNA coding region and generated specific antibodies recognizing the ACSL3 protein. We next observed the changes in ACSL3 mRNA and protein expression in hamsters fed a standard chow diet or a high fat and high cholesterol (HFHC) diet. HFHC feeding significantly increased ACSL3 mRNA and protein expression in liver and to a lesser extent in muscle but not in adipose, brain, heart, or testis. Additionally, ACSL3 mRNA abundance was differentially regulated by the nutritional status in different tissues with liver, muscle, and adipose being the most sensitive tissues. Importantly, the hepatic ACSL3 mRNA expression pattern in response to fasting and refeeding in hyperlipidemic hamsters differed from that observed in normal chow-fed hamsters. Together, these results provide the first in vivo evidence of altered regulation of hepatic ACSL3 expression under hyperlipidemic conditions and suggest important regulatory roles for this enzyme in lipid metabolism.

  2. Surface active molecules: preparation and properties of long chain n-acyl-l-alpha-amino-omega-guanidine alkyl acid derivatives.

    PubMed

    Infante, R; Dominguez, J G; Erra, P; Julia, R; Prats, M

    1984-12-01

    Synopsis A new route for the synthesis of long chain N(alpha)-acyl-l-alpha-amino-omega-guamdine alkyl acid derivatives, with cationic or amphoteric character has been established. The general formula of these compounds is shown below. A physico-chemical and antimicrobial study of these products as a function of the alkyl ester or sodium salt (R), the straight chain length of the fatty acid residue (x) and the number of carbons between the omega-guanidine and omega-carboxyl group (n) has been investigated. The water solubility, surface tension, critical micelle concentration (c.m.c.) and minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative bacteria (including Pseudomonas) has been determined. Dicyclohexylcarbodiimide has been used to condense fatty acids and alpha-amino-omega-guanidine alkyl acids. In these conditions protection of the omega-guanidine group is not necessary. The main characteristic of this synthetic procedure is the use of very mild experimental conditions (temperature, pH) to form the amide linkage which leads to pure optical compounds in high yield in the absence of electrolytes. The results show that some structural modifications, particularly the protection of the carboxyl group, promote variations of the surfactant and antimicrobial properties. Only those molecules with the blocked carboxyl group (cationic molecules, where R = Me, Et or Pr) showed a good surfactant and antimicrobial activity. When the carboxyl group was unprotected (amphoteric molecules, where R = Na(+)) the resulting compounds were inactive.

  3. 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.

  4. The adjuvant activity of fatty acid esters. The role of acyl chain length and degree of saturation.

    PubMed Central

    Bomford, R

    1981-01-01

    Water-in-oil emulsions of metabolizable fatty acid esters, with the non-toxic surfactant Pluronic L122 as emulsifying agent, potentiated the humoral response to bovine serum albumin and staphylococcal toxoid in the mouse. Adjuvant activity was increased by changing the chemical nature of the esters as follows: (i) using a series of ethyl esters, adjuvant activity appeared when the acyl chain length of the fatty acid component was 16 or greater; (ii) isobutyl and isopropyl esters of palmitic acid (C16:0) were superior to ethyl; (iii) the ethyl esters of oleic (C18:1) and linoleic (C18:2) acids were better than stearic (C18:0). Since emulsions prepared with longer chain saturated esters are very viscous or solid at room temperature, and unsaturated esters are chemically reactive, emulsions were prepared with differing proportions of ethyl caprate (C10:0) and butyl stearate. At a ratio of 9:1 the emulsions possessed the low viscosity of ethyl caprate, but gained the adjuvant activity of butyl stearate. 125I-labelled BSA was retained in the footpad to a significantly greater extent than with a caprate emulsion, but reasons are given for believing that slow release of antigen is not the only mechanism of adjuvant activity. The ester emulsions caused more acute but less chronic local inflammation (footpad swelling) than Freund's incomplete adjuvant. PMID:7275184

  5. Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

    PubMed Central

    Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

    2012-01-01

    Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

  6. Only one of the five Ralstonia solanacearum long-chain 3-ketoacyl-acyl carrier protein synthase homologues functions in fatty acid synthesis.

    PubMed

    Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E; Wang, Haihong

    2012-03-01

    Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C(16:0)), palmitoleic (C(16:1)) and cis-vaccenic (C(18:1)) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I.

  7. Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase.

    PubMed

    Vandevoorde, Séverine; Saha, Bijali; Mahadevan, Anu; Razdan, Raj K; Pertwee, Roger G; Martin, Billy R; Fowler, Christopher J

    2005-11-11

    Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.2 microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and 21 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 12 and 32 microM, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.

  8. Clarification of the Mechanism of Acylation Reaction and Origin of Substrate Specificity of the Serine-Carboxyl Peptidase Sedolisin through QM/MM Free Energy Simulations

    SciTech Connect

    Xu, Qin; Yao, Jianzhuang; Wiodawer, Alexander; Guo, Hong

    2011-01-01

    Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzyme-catalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers as well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N H of P1 -Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P1-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P1-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P1-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton transfers

  9. Interfacial interactions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acyl chain.

    PubMed

    Holopainen, J M; Brockman, H L; Brown, R E; Kinnunen, P K

    2001-02-01

    The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.

  10. Interfacial interactions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acyl chain.

    PubMed Central

    Holopainen, J M; Brockman, H L; Brown, R E; Kinnunen, P K

    2001-01-01

    The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties. PMID:11159444

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

    PubMed

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

    2009-08-01

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

  12. Elevated Temperature Enhances Short to Medium Chain Acyl Homoserine Lactone Production by Black Band Disease Associated Vibrios.

    PubMed

    Bhedi, Chinmayee D; Prevatte, Carson W; Lookadoo, Maggie S; Waikel, Patricia A; Gillevet, Patrick M; Sikaroodi, Masoumeh; Campagna, Shawn R; Richardson, Laurie L

    2017-01-29

    Black band disease (BBD) of corals is a horizontally migrating, pathogenic, polymicrobial mat community which is active above a temperature threshold of 27.5°C on the reef. Bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of healthy corals, and SML of healthy areas of BBD infected corals were tested for production of short to medium chain acyl homoserine lactones (AHLs) using the Chromobacterium violaceum CV026 reporter strain. Of 110 bacterial isolates tested, 19 produced AHLs and 15 of these were from BBD. Eight AHLs were identified using LC-MS/MS, with 3OHC4 the most commonly produced, followed by C6. AHL-producing isolates exposed to three temperatures (24°, 27°, 30°C) revealed that production of three AHLs (3OHC4, 3OHC5, and 3OHC6) significantly increased at 30°C when compared to 24°C. 16S rRNA gene sequencing revealed that all of the AHL producing BBD isolates were vibrios. Metagenomic data of BBD communities showed the presence of AHL (and autoinducer-2) genes, many of which are known to be associated with vibrios. These findings suggest that quorum sensing may be involved in BBD pathobiology and community structure due to enhanced production of quorum sensing signal molecules (AHLs) above the temperature threshold of this globally distributed coral disease.

  13. 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

  14. 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.

  15. Functional effects of different medium-chain acyl-CoA dehydrogenase genotypes and identification of asymptomatic variants.

    PubMed

    Sturm, Marga; Herebian, Diran; Mueller, Martina; Laryea, Maurice D; Spiekerkoetter, Ute

    2012-01-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the ACADM gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%-60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new ACADM genotypes associated with asymptomatic disease variants.

  16. Separation of isomeric short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Purves, Randy W; Ambrose, Stephen J; Clark, Shawn M; Stout, Jake M; Page, Jonathan E

    2015-02-01

    Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants.

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

    PubMed Central

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

    1995-01-01

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

  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.

  19. 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.

  20. Genetic association of long-chain acyl-CoA synthetase 1 variants with fasting glucose, diabetes, and subclinical atherosclerosis[S

    PubMed Central

    Manichaikul, Ani; Wang, Xin-Qun; Zhao, Wei; Wojczynski, Mary K.; Siebenthall, Kyle; Stamatoyannopoulos, John A.; Saleheen, Danish; Borecki, Ingrid B.; Reilly, Muredach P.; Rich, Stephen S.; Bornfeldt, Karin E.

    2016-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) converts free fatty acids into acyl-CoAs. Mouse studies have revealed that ACSL1 channels acyl-CoAs to β-oxidation, thereby reducing glucose utilization, and is required for diabetes-accelerated atherosclerosis. The role of ACSL1 in humans is unknown. We therefore examined common variants in the human ACSL1 locus by genetic association studies for fasting glucose, diabetes status, and preclinical atherosclerosis by using the MAGIC and DIAGRAM consortia; followed by analyses in participants from the Multi-Ethnic Study of Atherosclerosis, the Penn-T2D consortium, and a meta-analysis of subclinical atherosclerosis in African Americans; and finally, expression quantitative trait locus analysis and identification of DNase I hypersensitive sites (DHS). The results show that three SNPs in ACSL1 (rs7681334, rs735949, and rs4862423) are associated with fasting glucose or diabetes status in these large (>200,000 subjects) data sets. Furthermore, rs4862423 is associated with subclinical atherosclerosis and coincides with a DHS highly accessible in human heart. SNP rs735949 is in strong linkage disequilibrium with rs745805, significantly associated with ACSL1 levels in skin, suggesting tissue-specific regulatory mechanisms. This study provides evidence in humans of ACSL1 SNPs associated with fasting glucose, diabetes, and subclinical atherosclerosis and suggests links among these traits and acyl-CoA synthesis. PMID:26711138

  1. FAT/CD36 is located on the outer mitochondrial membrane, upstream of long-chain acyl-CoA synthetase, and regulates palmitate oxidation.

    PubMed

    Smith, Brennan K; Jain, Swati S; Rimbaud, Stéphanie; Dam, Aaron; Quadrilatero, Joe; Ventura-Clapier, Renée; Bonen, Arend; Holloway, Graham P

    2011-07-01

    FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36), a plasma membrane fatty-acid transport protein, has been found on mitochondrial membranes; however, it remains unclear where FAT/CD36 resides on this organelle or its functional role within mitochondria. In the present study, we demonstrate, using several different approaches, that in skeletal muscle FAT/CD36 resides on the OMM (outer mitochondrial membrane). To determine the functional role of mitochondrial FAT/CD36 in this tissue, we determined oxygen consumption rates in permeabilized muscle fibres in WT (wild-type) and FAT/CD36-KO (knockout) mice using a variety of substrates. Despite comparable muscle mitochondrial content, as assessed by unaltered mtDNA (mitochondrial DNA), citrate synthase, β-hydroxyacyl-CoA dehydrogenase, cytochrome c oxidase complex IV and respiratory capacities [maximal OXPHOS (oxidative phosphorylation) respiration] in WT and KO mice, palmitate-supported respiration was 34% lower in KO animals. In contrast, palmitoyl-CoA-supported respiration was unchanged. These results indicate that FAT/CD36 is key for palmitate-supported respiration. Therefore we propose a working model of mitochondrial fatty-acid transport, in which FAT/CD36 is positioned on the OMM, upstream of long-chain acyl-CoA synthetase, thereby contributing to the regulation of mitochondrial fatty-acid transport. We further support this model by providing evidence that FAT/CD36 is not located in mitochondrial contact sites, and therefore does not directly interact with carnitine palmitoyltransferase-I as original proposed.

  2. Prolonged QT interval and lipid alterations beyond β-oxidation in very long-chain acyl-CoA dehydrogenase null mouse hearts

    PubMed Central

    Gélinas, Roselle; Thompson-Legault, Julie; Bouchard, Bertrand; Daneault, Caroline; Mansour, Asmaa; Gillis, Marc-Antoine; Charron, Guy; Gavino, Victor; Labarthe, François

    2011-01-01

    Patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency frequently present cardiomyopathy and heartbeat disorders. However, the underlying factors, which may be of cardiac or extra cardiac origins, remain to be elucidated. In this study, we tested for metabolic and functional alterations in the heart from 3- and 7-mo-old VLCAD null mice and their littermate counterparts, using validated experimental paradigms, namely, 1) ex vivo perfusion in working mode, with concomitant evaluation of myocardial contractility and metabolic fluxes using 13C-labeled substrates under various conditions; as well as 2) in vivo targeted lipidomics, gene expression analysis as well as electrocardiogram monitoring by telemetry in mice fed various diets. Unexpectedly, when perfused ex vivo, working VLCAD null mouse hearts maintained values similar to those of the controls for functional parameters and for the contribution of exogenous palmitate to β-oxidation (energy production), even at high palmitate concentration (1 mM) and increased energy demand (with 1 μM epinephrine) or after fasting. However, in vivo, these hearts displayed a prolonged rate-corrected QT (QTc) interval under all conditions examined, as well as the following lipid alterations: 1) age- and condition-dependent accumulation of triglycerides, and 2) 20% lower docosahexaenoic acid (an omega-3 polyunsaturated fatty acid) in membrane phospholipids. The latter was independent of liver but affected by feeding a diet enriched in saturated fat (exacerbated) or fish oil (attenuated). Our finding of a longer QTc interval in VLCAD null mice appears to be most relevant given that such condition increases the risk of sudden cardiac death. PMID:21685264

  3. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains

    PubMed Central

    Soupene, Eric; Kuypers, Frans A

    2006-01-01

    Background The formation of acyl-CoA by the action of acyl-CoA synthetases plays a crucial role in membrane lipid turnover, including the plasma membrane of erythrocytes. In human, five Acyl-CoA Synthetase Long-chain (ACSL) genes have been identified with as many as 3 different transcript variants for each. Results Acyl-CoA Synthetase Long-chain member 6 (ACSL6) is responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants were also isolated from brain and testis. We report the expression in reticulocytes of two new variants and of the one isolated from brain. All three represented different spliced variants of a mutually exclusive exon pair. They encode a slightly different short motif which contains a conserved structural domain, the fatty acid Gate domain. The motifs differ in the presence of either the aromatic residue phenylalanine (Phe) or tyrosine (Tyr). Based on homology, two new isoforms for the closely related ACSL1 were predicted and characterized. One represented a switch of the Phe- to the Tyr-Gate domain motif, the other resulted from the exclusion of both. Swapping of this motif also appears to be common in all mammalian ACSL member 1 and 6 homologs. Conclusion We propose that a Phe to Tyr substitution or deletion of the Gate domain, is the structural reason for the conserved alternative splicing that affects these motifs. Our findings support our hypothesis that this region is structurally important to define the activity of these enzymes. PMID:16834775

  4. Successful Treatment of Cardiomyopathy due to Very Long-Chain Acyl-CoA Dehydrogenase Deficiency: First Case Report from Oman with Literature Review

    PubMed Central

    Sharef, Sharef Waadallah; Al-Senaidi, Khalfan; Joshi, Surendra Nath

    2013-01-01

    Very long-chain acyl-CoA dehydrogenase deficiency (MIM 201475) is a severe defect of mitochondrial energy production from oxidation of very long-chain fatty acids. This inherited metabolic disorder often presents in early neonatal period with episodes of symptomatic hypoglycemia usually responding well to intravenous glucose infusion. These babies are often discharged without establishment of diagnosis but return by 2-5 months of age with severe and progressive cardiac failure due to hypertrophic cardiomyopathy with or without hepatic failure and steatosis. An early diagnosis and treatment with high concentration medium chain triglycerides based feeding formula can be life saving in such patients. Here, we report the first diagnosed and treated case of Very long-chain acyl-CoA dehydrogenase deficiency in Oman. This infant developed heart failure with left ventricular dilation, hypertrophy and pericardial effusion at the age of 7 weeks. Prompt diagnosis and subsequent intervention with medium chain triglycerides-based formula resulted in a reversal of severe clinical symptoms with significant improvement of cardiac status. This treatment also ensured normal growth and neurodevelopment. It is stressed that the disease must be recognized by the pediatricians and cardiologists since the disease can be identified by Tandem Mass Spectrometry; therefore, it should be considered to be included in expanded newborn screening program, allowing early diagnosis and intervention in order to ensure better outcome and prevent complications. PMID:24044064

  5. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

    PubMed Central

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2017-01-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells. PMID:25645621

  6. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro.

    PubMed

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2015-05-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

  7. 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/.

  8. Insertion of apoLp-III into a lipid monolayer is more favorable for saturated, more ordered, acyl-chains

    SciTech Connect

    Rathnayake, Sewwandi S.; Mirheydari, Mona; Schulte, Adam; Gillahan, James E.; Gentit, Taylor; Phillips, Ashley N.; Okonkwo, Rose K.; Burger, Koert N.J.; Mann, Elizabeth K.; Vaknin, David; Bu, Wei; Agra-Kooijman, Dena Mae; Kooijman, Edgar E.

    2013-10-04

    Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic a-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic a:helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an a-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic a-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic cc-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein-lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts. (C) 2013 Elsevier B.V. All rights reserved.

  9. Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N- myristoylation

    PubMed Central

    1992-01-01

    Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N- myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp. PMID:1572893

  10. Structural Milestones in the Reaction Pathway of an Amide Hydrolase: Substrate, Acyl, and Product Complexes of Cephalothin with AmpC [beta]-Lactamase

    SciTech Connect

    Beadle, Beth M.; Trehan, Indi; Focia, Pamela J.; Shoichet, Brian K.

    2010-03-05

    {beta}-lactamases hydrolyze {beta}-lactam antibiotics and are the leading cause of bacterial resistance to these drugs. Although {beta}-lactamases have been extensively studied, structures of the substrate-enzyme and product-enzyme complexes have proven elusive. Here, the structure of a mutant AmpC in complex with the {beta}-lactam cephalothin in its substrate and product forms was determined by X-ray crystallography to 1.53 {angstrom} resolution. The acyl-enzyme intermediate between AmpC and cephalothin was determined to 2.06 {angstrom} resolution. The ligand undergoes a dramatic conformational change as the reaction progresses, with the characteristic six-membered dihydrothiazine ring of cephalothin rotating by 109{sup o}. These structures correspond to all three intermediates along the reaction path and provide insight into substrate recognition, catalysis, and product expulsion.

  11. Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Delta6-desaturase with omega3-preference from the marine microalga Micromonas pusilla.

    PubMed

    Petrie, James R; Shrestha, Pushkar; Mansour, Maged P; Nichols, Peter D; Liu, Qing; Singh, Surinder P

    2010-05-01

    Long-chain (> or = C20) polyunsaturated fatty acids (LC-PUFA) EPA and DHA (20:5(Delta5,8,11,14,17) and 22:6(Delta4,7,10,13,16,19)) have well-documented health benefits against coronary heart disease, rheumatoid arthritis and other disorders. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but research is being conducted to ensure that a sustainable, land-based production system can be developed. We here describe the metabolic engineering of an artificial pathway that produces 26% EPA in leaf triacylglycerol using a newly-identified Delta6-desaturase from the marine microalga Micromonas pusilla. We also demonstrate that this enzyme appears to function as an acyl-CoA desaturase that has preference for omega3 substrates both in planta and in yeast. Phylogenetic analysis indicates that this desaturase shares highly conserved motifs with previously described acyl-CoA Delta6-desaturases.

  12. Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus.

    PubMed

    Alves-Bezerra, Michele; Klett, Eric L; De Paula, Iron F; Ramos, Isabela B; Coleman, Rosalind A; Gondim, Katia C

    2016-07-01

    Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 μM), but different Km for arachidonic acid (0.5 and 6 μM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid β-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for β-oxidation in the fat body, and is also required for normal reproduction.

  13. Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

    PubMed Central

    Keeler, Allison M; Conlon, Thomas; Walter, Glenn; Zeng, Huadong; Shaffer, Scott A; Dungtao, Fu; Erger, Kirsten; Cossette, Travis; Tang, Qiushi; Mueller, Christian; Flotte, Terence R

    2012-01-01

    Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans. PMID:22395529

  14. [A two-year-old infant with a myopathic form of very-long-chain Acyl-CoA dehydrogenase deficiency].

    PubMed

    Ito, Yasushi; Nakano, Kazutoshi; Shishikura, Keiko; Suzuki, Haruko; Iida, Norihisa; Sasaki, Nobutaka; Kimura, Masahiko; Hasegawa, Yuki; Yamaguchi, Seiji; Osawa, Makiko

    2003-11-01

    A two-year-three-month old girl was hospitalized for detailed examination following repeated hyper-creatine kinasemia and cervical muscle cramps induced by pyrexia and persistent hypertonicity of the cervical muscles. Physical examination showed mild hypotonia but no muscle weakness. Induction of symptoms by continuous cervical muscular exercise and the appearance of dicarboxylic aciduria during the fasting test indicated a disorder of fatty acid oxidation. Free fatty acid and acyl carnitine analyses using dried blood spots, and acyl-CoA dehydrogenase activity assays using cultured skin fibroblasts established a diagnosis of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. Currently VLCAD deficiency has been divided into three phenotypes; a severe childhood form, a milder childhood form, and an adult form. However, we suggest that the severe and milder childhood forms would be better described as a systemic form, and the adult form and our infant case as a myopathic form. An early onset of the myopathic form within the first year of life, as well as its diagnosis in early infancy, has never been described in the literature.

  15. 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

  16. Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation

    PubMed Central

    Grevengoed, Trisha J.; Cooper, Daniel E.; Young, Pamela A.; Ellis, Jessica M.; Coleman, Rosalind A.

    2015-01-01

    Because hearts with a temporally induced knockout of acyl-CoA synthetase 1 (Acsl1T−/−) are virtually unable to oxidize fatty acids, glucose use increases 8-fold to compensate. This metabolic switch activates mechanistic target of rapamycin complex 1 (mTORC1), which initiates growth by increasing protein and RNA synthesis and fatty acid metabolism, while decreasing autophagy. Compared with controls, Acsl1T−/− hearts contained 3 times more mitochondria with abnormal structure and displayed a 35–43% lower respiratory function. To study the effects of mTORC1 activation on mitochondrial structure and function, mTORC1 was inhibited by treating Acsl1T−/− and littermate control mice with rapamycin or vehicle alone for 2 wk. Rapamycin treatment normalized mitochondrial structure, number, and the maximal respiration rate in Acsl1T−/− hearts, but did not improve ADP-stimulated oxygen consumption, which was likely caused by the 33–51% lower ATP synthase activity present in both vehicle- and rapamycin-treated Acsl1T−/− hearts. The turnover of microtubule associated protein light chain 3b in Acsl1T−/− hearts was 88% lower than controls, indicating a diminished rate of autophagy. Rapamycin treatment increased autophagy to a rate that was 3.1-fold higher than in controls, allowing the formation of autophagolysosomes and the clearance of damaged mitochondria. Thus, long-chain acyl-CoA synthetase isoform 1 (ACSL1) deficiency in the heart activated mTORC1, thereby inhibiting autophagy and increasing the number of damaged mitochondria.—Grevengoed, T. J., Cooper, D. E., Young, P. A., Ellis, J. M., Coleman, R. A. Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation. PMID:26220174

  17. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner.

    PubMed

    Erikson, Elina; Wratil, Paul R; Frank, Martin; Ambiel, Ina; Pahnke, Katharina; Pino, Maria; Azadi, Parastoo; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Meier, Chris; Schnaar, Ronald L; Crocker, Paul R; Reutter, Werner; Keppler, Oliver T

    2015-11-06

    Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.

  18. The membrane interaction of amphiphilic model peptides affects phosphatidylserine headgroup and acyl chain order and dynamics. Application of the phospholipid headgroup electrometer concept to phosphatidylserine

    SciTech Connect

    de Kroon, A.I.P.M.; Killian, J.A.; de Gier, J.; de Kruijff, B. )

    1991-01-29

    Deuterium nuclear magnetic resonance ({sup 2}H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the {beta}-position of the serine moiety ((2-{sup 2}H)DOPS) or at the 11-position of the acyl chains ((11,11-{sup 2}H{sub 2})DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH and H-Arg-Met-Leu-Trp-Ala-OH and contain a positive charge of +1 or +2 at the amino terminus or one positive charge at each end of the molecule. Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilyers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting of both (2-{sup 2}H)DOPS and (11,11-{sup 2}H{sub 2})DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. Titrations of DOPS with poly(L-lysine){sub 100}, which were included for reasons of comparison, reveal increased {Delta}v{sub q} values. When the peptide-lipid titrations are carried out without applying a freeze-thaw procedure to achieve full equilibration, two-component {sup 2}H NMR spectra occur. The apparently limited accessibility of the lipid to the peptides under these circumstances is discussed in relation to the ability of the peptides to exhibit transbilayer movement. {sup 2}H spin-lattice relaxation time T1 measurements demonstrate a decrease of the rates of motion of both headgroup and acyl chains of DOPS in the presence of the peptides.

  19. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner*

    PubMed Central

    Erikson, Elina; Wratil, Paul R.; Frank, Martin; Ambiel, Ina; Pahnke, Katharina; Pino, Maria; Azadi, Parastoo; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Meier, Chris; Schnaar, Ronald L.; Crocker, Paul R.; Reutter, Werner; Keppler, Oliver T.

    2015-01-01

    Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction. PMID:26370074

  20. Cardiac failure in very long chain acyl-CoA dehydrogenase deficiency requiring extracorporeal membrane oxygenation (ECMO) treatment: A case report and review of the literature.

    PubMed

    Katz, Sharon; Landau, Yuval; Pode-Shakked, Ben; Pessach, Itai M; Rubinshtein, Marina; Anikster, Yair; Salem, Yishay; Paret, Gideon

    2017-03-01

    Fatty acid oxidation (FAO) defects often present with multi-system involvement, including several life-threatening cardiac manifestations, such as cardiomyopathy, pericardial effusion and arrhythmias. We report herein a fatal case of cardiac dysfunction and rapid-onset tamponade following an acute illness in a neonate with molecularly proven very long chain acyl-CoA dehydrogenase (VLCAD) deficiency (harboring the known del799_802 mutation), requiring 15 days of extracorporeal membrane oxygenation (ECMO) treatment. As data regarding the use of ECMO in FAO defects in general, and VLCAD in particular, are scarce, we review the literature and discuss insights from in vitro models and several successful reported cases.

  1. Human plasma lecithin:cholesterol acyltransferase. On the substrate efficiency of cholest-5-ene-3 beta-thiol as a fatty acyl acceptor.

    PubMed

    Zhou, G; Dolphin, P J

    1995-09-14

    Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyses cholesteryl ester formation from lecithin and cholesterol present in the surface of plasma lipoproteins. Sterol fatty acid acceptors have previously been shown to require the presence of a trans conformation of the A/B ring and a 3 beta-OH group. Our laboratory has, however, demonstrated that two thiol sites within LCAT can become fatty acylated following lecithin cleavage although this does not appear to be essential for catalysis. In order to assess the ability of LCAT to donate a fatty acid derived from the sn-2 position of lecithin and present as an acyl enzyme intermediate (linked via an oxyester bond to Ser-181) to a sulfhydryl residue, we evaluated the ability of cholest-5-ene-3 beta-thiol to act as a substrate for cholesterol ester formation by LCAT. Thiocholesterol was a good terminal fatty acyl acceptor when incorporated into synthetic proteoliposomes containing lecithin/thiocholesterol/apo A-I in the molar ratios of 250:15:0.8. The Km for thiocholesterol was 203.6 microM with a Vmax of 5.3 nmol thiocholesteryl ester formed/h per microgram. The Km for cholesterol when substituted for thiocholesterol in the proteoliposomes was 29.5 microM with a Vmax of 8.8 nmol cholesteryl ester formed/h per microgram. Thiocholesterol and cholesterol were shown to occupy the same catalytic site in LCAT. Thus, thiocholesterol exhibits approx. 10% of the substrate efficiency of cholesterol when incubated with pure human LCAT. We conclude that LCAT can transacylate a fatty acyl moiety from the sn-2 position of lecithin to the 3 beta-SH group of thiocholesterol forming a cholesteryl thioester. Although the 3 beta-SH group is not as good a terminal acceptor as the 3 beta-OH group of cholesterol, LCAT is clearly capable of transacylating a fatty acid esterified via an oxyester linkage to one containing a thioester.

  2. Familial very long chain acyl-CoA dehydrogenase deficiency as a cause of neonatal sudden infant death: improved survival by prompt diagnosis.

    PubMed

    Scalais, Emmanuel; Bottu, Jean; Wanders, Ronald J A; Ferdinandusse, Sacha; Waterham, Hans R; De Meirleir, Linda

    2015-01-01

    In neonates, very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is often characterized by cardiomyopathy, hepatic encephalopathy, or severe hypoketotic hypoglycemia, or a combination thereof. The purpose of this study was to further elucidate a familial VLCAD deficiency in three patients, two of whom died in the neonatal period. We report on a family with VLCAD deficiency. Acyl-carnitine profiles were obtained from dried blood spot and/or from oxidation of (13) C-palmitate by cultured skin fibroblasts. In the index patient, VLCAD deficiency was ascertained by enzyme activity measurement in fibroblasts and by molecular analysis of ACADVL. At 30 hr of life, the proband was diagnosed with hypoglycemia (1.77 mmol/L), rhabdomyolysis (CK: 12966 IU/L) and hyperlactacidemia (10.6 mmol/L). Acylcarnitine profile performed at 31 hr of life was consistent with VLCAD deficiency and confirmed by cultured skin fibroblast enzyme activity measurement. Molecular analysis of ACADVL revealed a homozygous splice-site mutation (1077 + 2T>C). The acyl-carnitine profile obtained from the sibling's original newborn screening cards demonstrated a similar, but less pronounced abnormal profile. In the proband, the initial metabolic crisis was controlled with 10% dextrose solution and oral riboflavin followed by specific diet (Basic-F and medium chain triglyceride (MCT). This clinical report demonstrates a familial history of repeated neonatal deaths explained by VLCAD deficiency, and the clinical evolution of the latest affected, surviving sibling. It shows that very early metabolic screening is an effective approach to avoid sudden unexpected death.

  3. Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions

    SciTech Connect

    Lewis, R.N.A.H.; Mannock, D.A.; McElhaney, R.N.; Turner, D.C.; Gruner, S.M. )

    1989-01-24

    The lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transitions of aqueous dispersions of a number of synthetic phosphatidylethanolamines containing linear saturated, branched chain, and alicyclic fatty acyl chains of varying length were studied by differential scanning calorimetry, {sup 31}P nuclear magnetic resonance spectroscopy, and X-ray diffraction. For any given homologous series of phosphatidylethanolamines containing a single chemical class of fatty acids, the lamellar gel/liquid-crystalline phase transition temperature increases and the lamellar liquid-crystalline/reversed hexagonal phase transition temperature decreases with increases in hydrocarbon chain length. For a series of phosphatidylethanolamines of the same hydrocarbon chain length but with different chemical structures, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary markedly and in the same direction. These results suggest that for any given hydrocarbon chain length, there may be a critical thickness at which the liquid-crystalline phosphatidylethanolamine bilayer becomes unstable with respect to inverted nonbilayer phases such as the H{sub II} phase and that the temperature at which this critical thickness is reached is dependent upon that bilayers proximity to the hydrocarbon chain-melting phase transition temperature.

  4. Molecular cloning and nutrient regulation analysis of long chain acyl-CoA synthetase 1 gene in grass carp, Ctenopharyngodon idella L.

    PubMed

    Cheng, Han-Liang; Chen, Shuai; Xu, Jian-He; Yi, Le-Fei; Peng, Yong-Xing; Pan, Qian; Shen, Xin; Dong, Zhi-Guo; Zhang, Xia-Qing; Wang, Wen-Xiang

    2017-02-01

    Long chain acyl-CoA synthetase 1 (ACSL1), a key regulatory enzyme of fatty acid metabolism, catalyzes the conversion of long-chain fatty acids to acyl-coenzyme A. The full-length cDNAs of ACSL1a and ACSL1b were cloned from the liver of a grass carp. Both cDNAs contained a 2094bp open reading frame encoding 697 amino acids. Amino acid sequence alignment showed that ACSL1a shared 73.5% sequence identity with ACSL1b. Each of the two ACSL1s proteins had a transmembrane domain, a P-loop domain, and L-, A-, and G-motifs, which were relatively conserved in comparison to other vertebrates. Relative expression profile of ACSL1 mRNAs in different tissues indicated that ACSL1a is highly expressed in heart, mesenteric adipose, and brain tissues, whereas ACSL1b is highly expressed in heart, white muscle, foregut, and liver tissues. Nutrient regulation research showed that the expression levels of ACSL1a and ACSL1b were significantly down-regulated when 3, 6, and 9% fish oil were added in diet of grass carp as compared to the control group. However, no significant difference in the levels of ACSL1 mRNA was observed between the experimental groups. This study demonstrated the relationship between ACSL1a and ACSL1b genes in grass carp and laid a foundation for further research on ACSL family members in other species.

  5. Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

    PubMed

    Khayatt, Barzan I; Overmars, Lex; Siezen, Roland J; Francke, Christof

    2013-01-01

    There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

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

    PubMed

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

    2000-07-01

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

  7. 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.

  8. Characterization of trans-monounsaturated alkenyl chains in total plasmalogens (1-O-alk-1'-enyl-2-acyl glycerophospholipids) from sheep heart.

    PubMed

    Wolff, Robert L

    2002-08-01

    In the present study, we investigated the alkenyl chains from sheep heart plasmalogens (1-O-alk-1'-enyl-2-acyl glycerophospholipids) after their conversion into trimethylene dioxyalkanyl (TMDOA) derivatives. Particular attention was given to monounsaturated alkenyl chains (C18 mainly). For this purpose, a combination of silver ion TLC and GLC on highly polar, very long capillary columns was applied to TMDOA derivatives. Approximately 30 different alkenyl chains could be separated, and the main observation was that the component previously reported as a cis-9 18:1 alkenyl chain in plasmalogens embraces in fact a wide range of trans and cis isomers, in amounts equal to 7.9 and 5.6%, respectively, of total alkenyl chains. Concerning the trans-monoenoate fraction, isomers with their ethylenic bond spanning from delta6-delta8 to delta16 were tentatively identified on the basis of their distribution profile, which was similar to that of trans-18:1 acids prepared and isolated from sheep adipose tissue. The main trans-monoenoic C18 alkenyl chain in sheep heart plasmalogens would thus have its double bond in position 11, which seems logical, as alkenyl chains are derived from the corresponding alcohols, themselves issued from the corresponding FA, and in this particular case, vaccenic (trans-11 18:1) acid. cis-Monoenoic C18 alkenyl chains also appear more complex than realized earlier, showing in particular isomers with their ethylenic bond farther than the delta9 position, in addition to the main isomer derived from oleic acid. Several trans-16:1 alkenyl chains could be observed (totaling ca. 1%), but cis-16:1 isomers were present in trace amounts only.

  9. Chemical Reporters for Exploring Protein Acylation

    PubMed Central

    Thinon, Emmanuelle; Hang, Howard C.

    2015-01-01

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

  10. Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A.

    PubMed

    Dunn, Michael F; Araíza, Gisela; Mora, Jaime

    2004-02-01

    Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K(m) and V(max) values of 0.064 mM and 2885 nmol min(-1) (mg protein)(-1), respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K(m) values of 0.392 and 0.144 mM, respectively, and V(max) values of 423 and 268 nmol min(-1) (mg protein)(-1), respectively. K(+), or Cs(+) markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing alpha subunit and a 45 kDa biotin-free beta subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.

  11. 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.

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

    PubMed

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

    2013-01-01

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

  13. The vertical location of α-tocopherol in phosphatidylcholine membranes is not altered as a function of the degree of unsaturation of the fatty acyl chains.

    PubMed

    Ausili, Alessio; de Godos, Ana M; Torrecillas, Alejandro; Aranda, Francisco J; Corbalán-García, Senena; Gómez-Fernández, Juan C

    2017-03-01

    α-Tocopherol is a natural preservative that prevents free radical chain oxidations in biomembranes. We have studied the location of α-tocopherol in model membranes formed by different unsaturated phosphatidylcholines, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC). Small angle X-ray diffraction revealed that α-tocopherol was well mixed with all the phospholipids. In all the cases only one lamellar phase was detected. Very modest changes occasioned by α-tocopherol were observed in the electron density profiles. The results obtained from quenching of α-tocopherol intrinsic fluorescence by acrylamide showed that this vitamin was inefficiently quenched in the four types of membranes, indicating that the fluorescent chromanol ring was poorly accessible for this hydrophilic quencher. Compatible with that, quenching by doxyl derivatives of phosphatidylcholines indicated that the chromanol ring was close in the four membranes to the nitroxide probe located at position 5. Quenching by doxyl-phosphatidylcholines also indicated that the efficiency of quenching was higher in POPC than in the other unsaturated phospholipids. (1)H-MAS-NMR showed that α-tocopherol induced chemical shifts of protons from the phospholipids, especially of those bonded to carbons 2 and 3 of the acyl chains of the four phospholipids studied. The (1)H-MAS-NMR NOESY results suggested that the lower part of the chromanol ring was located between the C3 of the fatty acyl chains and the centre of the hydrophobic monolayer for the four phospholipid membranes studied. Taken together, these results suggest that α-tocopherol is located, in all the membranes studied, with the chromanol ring within the hydrophobic palisade but not far away from the lipid-water interface.

  14. Kinetic and Structural Basis for Acyl-Group Selectivity and NAD(+) Dependence in Sirtuin-Catalyzed Deacylation.

    PubMed

    Feldman, Jessica L; Dittenhafer-Reed, Kristin E; Kudo, Norio; Thelen, Julie N; Ito, Akihiro; Yoshida, Minoru; Denu, John M

    2015-05-19

    Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long chain deacylation, in addition to the well-known NAD(+)-dependent deacetylation activity [Feldman, J. L., Baeza, J., and Denu, J. M. (2013) J. Biol. Chem. 288, 31350-31356]. Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD(+)-binding and acyl-group selectivity for a diverse series of human Sirtuins, SIRT1-SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD(+) saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl substrate chain length. Though the rate of nucleophilic attack of the 2'-hydroxyl on the C1'-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however, for SIRT6, this step is no longer rate-limiting for long chain substrates. Cocrystallization of SIRT2 with myristoylated peptide and NAD(+) yielded a co-complex structure with reaction product 2'-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately determined co-complex structures containing either a myristoylated peptide or 2'-O-myristoyl-ADP-ribose indicates there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl substrate preferences and how different reaction kinetics influence NAD(+) dependence. The biological implications are discussed.

  15. Compound heterozygous mutations of ACADS gene in newborn with short chain acyl-CoA dehydrogenase deficiency: case report and literatures review

    PubMed Central

    An, Se Jin; Kim, Sook Za; Kim, Gu Hwan; Yoo, Han Wook

    2016-01-01

    Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive mitochondrial disorder of fatty acid β-oxidation, and is associated with mutations in the acyl-CoA dehydrogenase (ACADS) gene. Recent advances in spectrometric screening for inborn errors of metabolism have helped detect several metabolic disorders, including SCADD, without symptoms in the neonate period. This allows immediate initiation of treatment and monitoring, so they remain largely symptomless metabolic disease. Here, we report a 15-month-old asymptomatic male, who was diagnosed with SCADD by newborn screening. Spectrometric screening for inborn errors of metabolism 72 hours after birth revealed an elevated butyrylcarnitine (C4) concentration of 2.25 µmol/L (normal, <0.99 µmol/L). Urinary excretion of ethylmalonic acid was also elevated, as detected by urine organic acid analysis. To confirm the diagnosis of SCADD, direct sequencing analysis of 10 coding exons and the exon-intron boundaries of the ACADS gene were performed. Subsequent sequence analysis revealed compound heterozygous missense mutations c.164C>T (p.Pro55Leu) and c.1031A>G (p.Glu344Gly) on exons 2 and 9, respectively. The patient is now growing up, unretarded by symptoms such as seizure and developmental delay. PMID:28018444

  16. Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

    PubMed Central

    Liu, Chao; Liu, Weixiao; Ye, Yihong; Li, Wei

    2017-01-01

    Ubiquitination of a subset of proteins by ubiquitin chain elongation factors (E4), represented by Ufd2p in Saccharomyces cerevisiae, is a pivotal regulator for many biological processes. However, the mechanism of Ufd2p-mediated ubiquitination is largely unclear. Here, we show that Ufd2p catalyses K48-linked multi-monoubiquitination on K29-linked ubiquitin chains assembled by the ubiquitin ligase (Ufd4p), resulting in branched ubiquitin chains. This reaction depends on the interaction of K29-linked ubiquitin chains with two N-terminal loops of Ufd2p. Only following the addition of K48-linked ubiquitin to substrates modified with K29-linked ubiquitin chains, can the substrates be escorted to the proteasome for degradation. We demonstrate that this ubiquitin chain linkage switching reaction is essential for ERAD, oleic acid and acid pH resistance in yeast. Thus, our results suggest that Ufd2p functions by switching ubiquitin chain linkages to allow the degradation of proteins modified with a ubiquitin linkage, which is normally not targeted to the proteasome. PMID:28165462

  17. Recognition of medium-chain acyl-CoA dehydrogenase deficiency in asymptomatic siblings of children dying of sudden infant death or Reye-like syndromes.

    PubMed

    Roe, C R; Millington, D S; Maltby, D A; Kinnebrew, P

    1986-01-01

    The medium-chain acyl-CoA dehydrogenase (MCAD) deficiency of mitochondrial beta oxidation has been identified in two asymptomatic siblings in a family in which two previous deaths had been recorded, one attributed to sudden infant death syndrome and the other to Reye syndrome. Recognition of this disorder in one of the deceased and in the surviving siblings was accomplished by detection of a diagnostic metabolite, octanoylcarnitine, using a new mass spectrometric technique. This resulted in early treatment with L-carnitine supplement in the survivors, which should prevent metabolic deterioration. Further studies suggest that breast-feeding may be protective for infants with MCAD deficiency. Families with children who have had Reye syndrome or in which sudden infant death has occurred are at risk for MCAD deficiency. We suggest that survivors and asymptomatic siblings should be tested for this treatable disorder.

  18. Biochemical engineering of the N-acyl side chain of sialic acids alters the kinetics of a glycosylated potassium channel Kv3.1.

    PubMed

    Hall, M Kristen; Reutter, Werner; Lindhorst, Thisbe; Schwalbe, Ruth A

    2011-10-20

    The sialic acid of complex N-glycans can be biochemically engineered by substituting the physiological precursor N-acetylmannosamine with non-natural N-acylmannosamines. The Kv3.1 glycoprotein, a neuronal voltage-gated potassium channel, contains sialic acid. Western blots of the Kv3.1 glycoprotein isolated from transfected B35 neuroblastoma cells incubated with N-acylmannosamines verified sialylated N-glycans attached to the Kv3.1 glycoprotein. Outward ionic currents of Kv3.1 transfected B35 cells treated with N-pentanoylmannosamine or N-propanoylmannosamine had slower activation and inactivation rates than those of untreated cells. Therefore, the N-acyl side chain of sialic acid is intimately connected with the activation and inactivation rates of this glycosylated potassium channel.

  19. Long chain acyl-CoA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells.

    PubMed

    Yao, Hongbing; Ye, Jin

    2008-01-11

    Hepatocytes play a crucial role in regulating lipid metabolism by exporting cholesterol and triglyceride into plasma through secretion of very low density lipoproteins (VLDL). VLDL production is also required for release of hepatitis C virus (HCV) from infected hepatocytes. Here, we show that long chain acyl-CoA synthetase 3 (ACSL3) plays a crucial role in secretion of VLDL and HCV from hepatocytes. In cultured human hepatoma Huh7 cells, ACSL3 is specifically required for incorporation of fatty acids into phosphatidylcholine. In cells receiving small interfering RNA targeting ACSL3, secretion of apolipoprotein B, the major protein component of VLDL, was inhibited and the lipoprotein was rapidly degraded. This inhibition in secretion was completely eliminated when these cells were treated with phosphatidylcholine. Treatment of cells with small interfering RNA targeting ACSL3 also inhibited secretion of HCV from Huh7-derived cells. These results identify ACSL3 as a new enzymatic target to limit VLDL secretion and HCV infection.

  20. Commentary on a Delphi clinical practice protocol for the diagnosis and management of very long chain acyl-CoA dehydrogenase deficiency by Arnold et al.

    PubMed

    Marsden, Deborah Louise

    2009-03-01

    Very long chain acyl-CoA dehydrogenase deficiency (VLCAD) can now be detected by newborn screening by tandem mass spectrometry. The incidence is higher than previously estimated because of the identification of potentially milder later onset variants by screening. Although there is little information in the literature on the optimal management of rare inborn errors, there is a need for management guidelines, especially for non-specialist providers in the community. In the accompanying article, Arnold et al. present a diagnostic and management guideline for VLCAD, developed by the Delphi method for gaining consensus from a panel of 14 metabolic specialists. While consensus was gained for some issues, there was no clear consensus for several important management issues, particularly for the later onset variants. Clearly, there is an urgent need for multinational collaborative protocol driven outcomes studies that will provide the data necessary to establish robust guidelines for inborn errors of metabolism.

  1. Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

    PubMed Central

    Sugimoto, Masayuki; Wakabayashi, Masato; Shimizu, Yoichi; Yoshioka, Takeshi; Higashino, Kenichi; Numata, Yoshito; Okuda, Tomohiko; Zhao, Songji; Sakai, Shota; Igarashi, Yasuyuki; Kuge, Yuji

    2016-01-01

    Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys. PMID:27010944

  2. Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice.

    PubMed

    Sugimoto, Masayuki; Wakabayashi, Masato; Shimizu, Yoichi; Yoshioka, Takeshi; Higashino, Kenichi; Numata, Yoshito; Okuda, Tomohiko; Zhao, Songji; Sakai, Shota; Igarashi, Yasuyuki; Kuge, Yuji

    2016-01-01

    Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.

  3. Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism.

    PubMed

    Xie, Li-Juan; Yu, Lu-Jun; Chen, Qin-Fang; Wang, Feng-Zhu; Huang, Li; Xia, Fan-Nv; Zhu, Tian-Ren; Wu, Jian-Xin; Yin, Jian; Liao, Bin; Yao, Nan; Shu, Wensheng; Xiao, Shi

    2015-01-01

    In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.

  4. Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

    PubMed Central

    Mayer, Kimberly M; Shanklin, John

    2007-01-01

    Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented. PMID:17201914

  5. Plant fatty acyl reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications.

    PubMed

    Rowland, Owen; Domergue, Frédéric

    2012-09-01

    Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols.

  6. Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators

    PubMed Central

    Mathias, Rommel A.; Greco, Todd M.; Cristea, Ileana M.

    2016-01-01

    Recent studies have highlighted the three mitochondrial human sirtuins (SIRT3, SIRT4, and SIRT5) as critical regulators of a wide range of cellular metabolic pathways. A key factor to understanding their impact on metabolism has been the discovery that, in addition to their ability to deacetylate substrates, mitochondrial sirtuins can have other prominent enzymatic activities. SIRT4, one of the least characterized mitochondrial sirtuins, was shown to be the first known cellular lipoamidase, removing lipoyl modifications from lysine residues of substrates. Specifically, SIRT4 was found to delipoylate and modulate the activity of the pyruvate dehydrogenase complex (PDH), a protein complex critical for the production of acetyl-CoA. Furthermore, SIRT4 is well known to have ADP-ribosyltransferase activity and to regulate the activity of the glutamate dehydrogenase complex (GDH). Adding to its impressive range of enzymatic activities are its ability to deacetylate malonyl-CoA decarboxylase (MCD) to regulate lipid catabolism, and its newly recognized ability to remove biotinyl groups from substrates that remain to be defined. Given the wide range of enzymatic activities and the still limited knowledge of its substrates, further studies are needed to characterize its protein interactions and its impact on metabolic pathways. Here, we present several proven protocols for identifying SIRT4 protein interaction networks within the mitochondria. Specifically, we describe methods for generating human cell lines expressing SIRT4, purifying mitochondria from crude organelles, and effectively capturing SIRT4 with its interactions and substrates. PMID:27246218

  7. Effect of Acylglycerol Composition and Fatty Acyl Chain Length on Lipid Digestion in pH-Stat Digestion Model and Simulated In Vitro Digestion Model.

    PubMed

    Qi, Jin F; Jia, Cai H; Shin, Jung A; Woo, Jeong M; Wang, Xiang Y; Park, Jong T; Hong, Soon T; Lee, K-T

    2016-02-01

    In this study, a pH-stat digestion model and a simulated in vitro digestion model were employed to evaluate the digestion degree of lipids depending on different acylglycerols and acyl chain length (that is, diacylglycerol [DAG] compared with soybean oil representing long-chain triacylglycerol compared with medium-chain triacylglycerol [MCT]). In the pH-stat digestion model, differences were observed among the digestion degrees of 3 oils using digestion rate (k), digestion half-time (t1/2 ), and digestion extent (Φmax). The results showed the digestion rate order was MCT > soybean oil > DAG. Accordingly, the order of digestion half-times was MCT < soybean oil < DAG. In simulated in vitro digestion model, digestion rates (k') and digestion half-times (t'1/2 ) were also obtained and the results showed a digestion rate order of MCT (k' = 0.068 min(-1) ) > soybean oil (k' = 0.037 min(-1) ) > DAG (k' = 0.024 min(-1) ). Consequently, the order of digestion half-times was MCT (t'1/2 = 10.20 min) < soybean oil (t'1/2 = 18.74 min) < DAG (t'1/2 = 29.08 min). The parameters obtained using the 2 models showed MCT was digested faster than soybean oil, and that soybean oil was digested faster than DAG.

  8. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

    PubMed Central

    Vance, Steven; Tkachenko, Olga; Thomas, Ben; Bassuni, Mona; Hong, Hui; Nietlispach, Daniel; Broadhurst, William

    2016-01-01

    Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4′-phosphopantetheine (Ppant) to create the holo form, 15N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. PMID:26920023

  9. Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase.

    PubMed

    Dodt, Johannes; Reichwein, Jörg

    2003-09-01

    The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.

  10. 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

  11. 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

  12. 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.

  13. Different effects of fibrates on the microsomal fatty acid chain elongation and the acyl composition of phospholipids in guinea-pigs.

    PubMed

    Vázquez, M; Alegret, M; López, M; Rodríguez, C; Adzet, T; Merlos, M; Laguna, J C

    1995-12-01

    1. The effects in vitro and in vivo of three fibric acid derivatives, clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on some enzyme activities related to fatty acid biosynthesis, namely palmitoyl-CoA synthetase and hydrolases (microsomal and cytosolic), NADH and NADPH cytochrome c reductases and acyl-CoA elongases were investigated in guinea-pigs. 2. The three fibrates inhibited acyl-CoA elongation in vitro, irrespective of the substrate of elongation used (saturated, monounsaturated, polyunsaturated) and with an order of potency GFB > BFB > CFB. In the case of GFB, inhibition occurred at concentrations that can be reached in vivo. 3. Microsomal palmitoyl-CoA hydrolase and synthetase were also inhibited in vitro (GFB > or = BFB > CFB), whereas NADH cytochrome c reductase activity was increased by GFB. Nevertheless, the magnitude of changes were lower than those observed in elongation activities. 4. Treatment with fibrates did not produce peroxisomal proliferation in guinea-pigs, as measured by peroxisomal beta-oxidation activity and liver weight/body weight ratio. Nevertheless, fibrates provoked a reduction in plasma cholesterol and triglycerides, at least in GFB- and BFB-treated animals. 5. Fatty acid elongation was significantly modified by GFB treatment in vivo. The remaining enzyme activities studied were only slightly changed by fibrate treatment. 6. Treatment with BFB and to a lesser extent with CFB, increased the relative proportion of MUFA (palmitoleic and oleic acids) in microsomal phospholipids, whereas PUFA (mainly linoleic acid) decreased. GFB behaved differently, increasing palmitic and linoleic acids and decreasing stearic and oleic acids. The latter changes are attributable to an inhibition of elongation activity by GFB. 7. The changes observed after fibrate treatment in both rats and guinea-pigs, as they are not directly related to peroxisome proliferation, could be more reliably extrapolated to man than those observed only in rats.

  14. Different effects of fibrates on the microsomal fatty acid chain elongation and the acyl composition of phospholipids in guinea-pigs.

    PubMed Central

    Vázquez, M.; Alegret, M.; López, M.; Rodríguez, C.; Adzet, T.; Merlos, M.; Laguna, J. C.

    1995-01-01

    1. The effects in vitro and in vivo of three fibric acid derivatives, clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on some enzyme activities related to fatty acid biosynthesis, namely palmitoyl-CoA synthetase and hydrolases (microsomal and cytosolic), NADH and NADPH cytochrome c reductases and acyl-CoA elongases were investigated in guinea-pigs. 2. The three fibrates inhibited acyl-CoA elongation in vitro, irrespective of the substrate of elongation used (saturated, monounsaturated, polyunsaturated) and with an order of potency GFB > BFB > CFB. In the case of GFB, inhibition occurred at concentrations that can be reached in vivo. 3. Microsomal palmitoyl-CoA hydrolase and synthetase were also inhibited in vitro (GFB > or = BFB > CFB), whereas NADH cytochrome c reductase activity was increased by GFB. Nevertheless, the magnitude of changes were lower than those observed in elongation activities. 4. Treatment with fibrates did not produce peroxisomal proliferation in guinea-pigs, as measured by peroxisomal beta-oxidation activity and liver weight/body weight ratio. Nevertheless, fibrates provoked a reduction in plasma cholesterol and triglycerides, at least in GFB- and BFB-treated animals. 5. Fatty acid elongation was significantly modified by GFB treatment in vivo. The remaining enzyme activities studied were only slightly changed by fibrate treatment. 6. Treatment with BFB and to a lesser extent with CFB, increased the relative proportion of MUFA (palmitoleic and oleic acids) in microsomal phospholipids, whereas PUFA (mainly linoleic acid) decreased. GFB behaved differently, increasing palmitic and linoleic acids and decreasing stearic and oleic acids. The latter changes are attributable to an inhibition of elongation activity by GFB. 7. The changes observed after fibrate treatment in both rats and guinea-pigs, as they are not directly related to peroxisome proliferation, could be more reliably extrapolated to man than those observed only in rats. PMID

  15. Distinct transcriptional regulation of long-chain acyl-CoA synthetase isoforms and cytosolic thioesterase 1 in the rodent heart by fatty acids and insulin.

    PubMed

    Durgan, David J; Smith, Justin K; Hotze, Margaret A; Egbejimi, Oluwaseun; Cuthbert, Karalyn D; Zaha, Vlad G; Dyck, Jason R B; Abel, E Dale; Young, Martin E

    2006-06-01

    The molecular mechanism(s) responsible for channeling long-chain fatty acids (LCFAs) into oxidative versus nonoxidative pathways is (are) poorly understood in the heart. Intracellular LCFAs are converted to long-chain fatty acyl-CoAs (LCFA-CoAs) by a family of long-chain acyl-CoA synthetases (ACSLs). Cytosolic thioesterase 1 (CTE1) hydrolyzes cytosolic LCFA-CoAs to LCFAs, generating a potential futile cycle at the expense of ATP utilization. We hypothesized that ACSL isoforms and CTE1 are differentially regulated in the heart during physiological and pathophysiological conditions. Using quantitative RT-PCR, we report that the five known acsl isoforms (acsl1, acsl3, acsl4, acsl5, and acsl6) and cte1 are expressed in whole rat and mouse hearts, as well as adult rat cardiomyocytes (ARCs). Streptozotocin-induced insulin-dependent diabetes (4 wk) and fasting (

  16. Effect of small chain N acyl homoserine lactone quorum sensing signals on biofilms of food-borne pathogens.

    PubMed

    A, Jamuna Bai; V, Ravishankar Rai

    2016-09-01

    Quorum sensing or cell to cell communication which includes inter- and intra-cellular communication has been implicated in the production of virulence factor and formation of biofilm in food-borne pathogens. In the present study, the effect of quorum sensing signals on the biofilms of food-borne pathogens has been elucidated. N-butryl homoserine lactone and N-hexanoyl homoserine lactone belonging to acyl homoserine lactone (AHL) family of signaling molecules were investigated for their effect on the biofilm formation (attachment and exopolymeric substance production) in the food-borne pathogens Escherichia coli, Salmonella enterica serovar Typhimurium and Vibrio parahemolyticus. The signaling molecules at a concentration of 1 µM were capable of increasing biofilm formation in all the tested pathogens. There was an increase in the attachment of the bacterial cells and biomass as observed by microtiter plate assay and exopolymeric substances production in the biofilms in presence of the AHLs. Further, it needs to be elucidated if the effect of AHLS on the biofilms of E. coli and S. enterica serovar Typhimurium is SdiA dependent.

  17. The Effect of Temperature, Cations, and Number of Acyl Chains on the Lamellar to Non-Lamellar Transition in Lipid-A Membranes: A Microscopic View

    SciTech Connect

    Pontes, Frederico J.; Rusu, Victor H.; Soares, Thereza A.; Lins, Roberto D.

    2012-05-24

    Lipopolysaccharides (LPS) are the main constituent of the outer bacterial membrane of Gram-negative bacteria. Lipid-A is the structural region of LPS that interacts with the innate immune system and induces inflammatory responses. It is formed by a phosphorylated β-d-glucosaminyl-(1→6)-α-N-glucosamine disaccharide backbone containing ester-linked and amide-linked long-chain fatty acids, which may vary in length and number depending on the bacterial strains and the environment. Phenotypical variation (i.e., number of acyl chains), cation type, and temperature influence the phase transition, aggregate structure, and endotoxic activity of Lipid-A. We have applied an extension of the GROMOS force field 45a4 carbohydrate parameter set to investigate the behavior of hexa- and pentaacylated Lipid-A of Pseudomonas aeruginosa at two temperatures (300 and 328 K) and in the presence of mono- and divalent cations (represented by Ca2+ and Na+, respectively) through molecular dynamics simulations. The distinct phase of Lipid-A aggregates was characterized by structural properties, deuterium order parameters, the molecular shape of the lipid units (conical versus cylindrical), and molecular packing. Our results show that Na+ ions induce a transition from the lamellar to nonlamellar phase. In contrast, the bilayer integrity is maintained in the presence of Ca2+ ions. Through these findings, we present microscopic insights on the influence of different cations on the molecular behavior of Lipid-A associated with the lamellar to nonlamellar transition.

  18. Evidence that the major metabolites accumulating in medium-chain acyl-CoA dehydrogenase deficiency disturb mitochondrial energy homeostasis in rat brain.

    PubMed

    Schuck, Patrícia Fernanda; Ferreira, Gustavo da Costa; Tonin, Anelise Miotti; Viegas, Carolina Maso; Busanello, Estela Natacha Brandt; Moura, Alana Pimentel; Zanatta, Angela; Klamt, Fábio; Wajner, Moacir

    2009-11-03

    Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an inherited metabolic disorder of fatty acid oxidation in which the affected patients predominantly present high levels of octanoic (OA) and decanoic (DA) acids and their glycine and carnitine by-products in tissues and body fluids. It is clinically characterized by episodic encephalopathic crises with coma and seizures, as well as by progressive neurological involvement, whose pathophysiology is poorly known. In the present work, we investigated the in vitro effects of OA and DA on various parameters of energy homeostasis in mitochondrial preparations from brain of young rats. We found that OA and DA markedly increased state 4 respiration and diminished state 3 respiration as well as the respiratory control ratio, the mitochondrial membrane potential and the matrix NAD(P)H levels. In addition, DA-elicited increase in oxygen consumption in state 4 respiration was partially prevented by atractyloside, indicating the involvement of the adenine nucleotide translocator. OA and DA also reduced ADP/O ratio, CCCP-stimulated respiration and the activities of respiratory chain complexes. The data indicate that the major accumulating fatty acids in MCADD act as uncouplers of oxidative phosphorylation and as metabolic inhibitors. Furthermore, DA, but not OA, provoked a marked mitochondrial swelling and cytochrome c release from mitochondria, reflecting a permeabilization of the inner mitochondrial membrane. Taken together, these data suggest that OA and DA impair brain mitochondrial energy homeostasis that could underlie at least in part the neuropathology of MCADD.

  19. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    SciTech Connect

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S.

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  20. Chain conformation near the substrate interface in nanoparticle stabilized polymer thin films

    NASA Astrophysics Data System (ADS)

    Barkley, Deborah; Sen, Mani; Jiang, Naisheng; Endoh, Maya; Koga, Tadanori; Yuan, Guangcui; Satija, Sushil; Zhang, Yugang; Gang, Oleg; Karim, Alamgir

    When nanoparticles (NPs) are added to polymer thin films, they often migrate to the film-substrate interface and form a ``diffused immobile interfacial layer'', which serves to screen the polymer-substrate interaction and suppress dewetting. The fundamental, but unsolved question is how the conformations of the polymer chains in the layer are affected by the NPs and how that impacts the enhancement of film stability. To address the question, we used dodecane thiol-functionalized gold NPs (2.4 nm diameter) and polystyrene (PS, Mw =30kDa). We found that the critical concentration of the Au NPs to induce complete dewetting suppression of 20 nm-thick PS/Au thin films on cleaned Si substrates is 5 wt% (wt of particle/wt of polymer). To investigate the interfacial structures at the polymer-solid interface, we rinsed the annealed PS/Au thin films with toluene and characterized the residual interfacial layers by using various x-ray and neutron scattering techniques. The results indicate that the conformation of the polymer chains closer to the substrate becomes less flattened with the addition of gold NPs, allowing chains at the substrate to entangle more effectively with free chains comprising the bulk film. The detailed mechanism will be discussed. T.K. acknowledges funding from NSF Grant (CMMI-1332499).

  1. 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

  2. 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

  3. Formation of substrate-based gold nanocage chains through dealloying with nitric acid

    PubMed Central

    Yan, Ziren; Wu, Ying

    2015-01-01

    Summary Metal nanocages have raised great interest because of their new properties and wide applications. Here, we report on the use of galvanic replacement reactions to synthesize substrate-supported Ag–Au nanocages from silver templates electrodeposited on transparent indium tin oxide (ITO) film coated glass. The residual Ag in the composition was dealloyed with 10% nitric acid. It was found that chains of Au nanocages were formed on the substrate surface during dealloying. When the concentration of HNO3 increased to 20%, the structures of nanocages were damaged and formed crescent or semi-circular shapes. The transfer process on the substrate surface was discussed. PMID:26199839

  4. Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

    PubMed Central

    Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

    2017-01-01

    Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

  5. 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.

  6. Structure of a Specialized Acyl Carrier Protein Essential for Lipid A Biosynthesis with Very Long-chain Fatty Acids in Open and Closed Conformations

    SciTech Connect

    Ramelot, Theresa A.; Rossi, Paolo M.; Forouhar, Farhad; Lee, Hsiau-Wei; Yang, Yunhuang; Ni, Shuisong; Unser, Sarah; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Acton, Thomas; Everett, John K.; Prestegard, James H.; Hunt, John F.; Montelione, Gaetano; Kennedy, Michael A.

    2012-09-18

    The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.

  7. Rat long-chain acyl-CoA synthetase mRNA, protein, and activity vary in tissue distribution and in response to diet.

    PubMed

    Mashek, Douglas G; Li, Lei O; Coleman, Rosalind A

    2006-09-01

    Distinct isoforms of long-chain acyl-CoA synthetases (ACSLs) may partition fatty acids toward specific metabolic cellular pathways. For each of the five members of the rat ACSL family, we analyzed tissue mRNA distributions, and we correlated the mRNA, protein, and activity of ACSL1 and ACSL4 after fasting and refeeding a 69% sucrose diet. Not only did quantitative real-time PCR analyses reveal unique tissue expression patterns for each ACSL isoform, but expression varied markedly in different adipose depots. Fasting increased ACSL4 mRNA abundance in liver, muscle, and gonadal and inguinal adipose tissues, and refeeding decreased ACSL4 mRNA. A similar pattern was observed for ACSL1, but both fasting and refeeding decreased ACSL1 mRNA in gonadal adipose. Fasting also decreased ACSL3 and ACSL5 mRNAs in liver and ACSL6 mRNA in muscle. Surprisingly, in nearly every tissue measured, the effects of fasting and refeeding on the mRNA abundance of ACSL1 and ACSL4 were discordant with changes in protein abundance. These data suggest that the individual ACSL isoforms are distinctly regulated across tissues and show that mRNA expression may not provide useful information about isoform function. They further suggest that translational or posttranslational modifications are likely to contribute to the regulation of ACSL isoforms.

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

    PubMed Central

    2012-01-01

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

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

    PubMed

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

    2012-10-01

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

  10. Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy.

    PubMed

    Gobin-Limballe, S; Djouadi, F; Aubey, F; Olpin, S; Andresen, B S; Yamaguchi, S; Mandel, H; Fukao, T; Ruiter, J P N; Wanders, R J A; McAndrew, R; Kim, J J; Bastin, J

    2007-12-01

    Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and

  11. Long Chain Fatty Acid Acylated Derivatives of Quercetin-3-O-Glucoside as Antioxidants to Prevent Lipid Oxidation

    PubMed Central

    Warnakulasuriya, Sumudu N.; Ziaullah; Rupasinghe, H.P. Vasantha

    2014-01-01

    Flavonoids have shown promise as natural plant-based antioxidants for protecting lipids from oxidation. It was hypothesized that their applications in lipophilic food systems can be further enhanced by esterification of flavonoids with fatty acids. Quercetin-3-O-glucoside (Q3G) was esterified individually with six selected long chain fatty acids: stearic acid (STA), oleic acid (OLA), linoleic acid (LNA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA), using Candida antarctica B lipase as the biocatalyst. The antioxidant activity of esterified flavonoids was evaluated using lipid oxidation model systems of poly-unsaturated fatty acids-rich fish oil and human low density lipoprotein (LDL), in vitro. In the oil-in-water emulsion, Q3G esters exhibited 50% to 100% inhibition in primary oxidation and 30% to 75% inhibition in secondary oxidation. In bulk oil, Q3G esters did not provide considerable protection from lipid oxidation; however, Q3G demonstrated more than 50% inhibition in primary oxidation. EPA, DHA and ALA esters of Q3G showed significantly higher inhibition in Cu2+- and peroxyl radical-induced LDL oxidation in comparison to Q3G. PMID:25384198

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

    PubMed

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

    2011-08-15

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

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

    PubMed

    Reddy, G Shilpa; Sujatha, M

    2011-07-01

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

  14. Data mining methods for classification of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD) using non-derivatized tandem MS neonatal screening data.

    PubMed

    Van den Bulcke, Tim; Vanden Broucke, Paul; Van Hoof, Viviane; Wouters, Kristien; Vanden Broucke, Seppe; Smits, Geert; Smits, Elke; Proesmans, Sam; Van Genechten, Toon; Eyskens, François

    2011-04-01

    Newborn screening programs for severe metabolic disorders using tandem mass spectrometry are widely used. Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD) is the most prevalent mitochondrial fatty acid oxidation defect (1:15,000 newborns) and it has been proven that early detection of this metabolic disease decreases mortality and improves the outcome. In previous studies, data mining methods on derivatized tandem MS datasets have shown high classification accuracies. However, no machine learning methods currently have been applied to datasets based on non-derivatized screening methods. A dataset with 44,159 blood samples was collected using a non-derivatized screening method as part of a systematic newborn screening by the PCMA screening center (Belgium). Twelve MCADD cases were present in this partially MCADD-enriched dataset. We extended three data mining methods, namely C4.5 decision trees, logistic regression and ridge logistic regression, with a parameter and threshold optimization method and evaluated their applicability as a diagnostic support tool. Within a stratified cross-validation setting, a grid search was performed for each model for a wide range of model parameters, included variables and classification thresholds. The best performing model used ridge logistic regression and achieved a sensitivity of 100%, a specificity of 99.987% and a positive predictive value of 32% (recalibrated for a real population), obtained in a stratified cross-validation setting. These results were further validated on an independent test set. Using a method that combines ridge logistic regression with variable selection and threshold optimization, a significantly improved performance was achieved compared to the current state-of-the-art for derivatized data, while retaining more interpretability and requiring less variables. The results indicate the potential value of data mining methods as a diagnostic support tool.

  15. Long chain acyl-CoA synthetase-3 is a molecular target for peroxisome proliferator-activated receptor delta in HepG2 hepatoma cells.

    PubMed

    Cao, Aiqin; Li, Hai; Zhou, Yue; Wu, Minhao; Liu, Jingwen

    2010-05-28

    ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

  16. Reduction of serum free fatty acids and triglycerides by liver-targeted expression of long chain acyl-CoA synthetase 3.

    PubMed

    Wu, Minhao; Cao, Aiqin; Dong, Bin; Liu, Jingwen

    2011-05-01

    ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that consists of 5 isozymes responsible for cellular fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies have demonstrated that expression of ACSL3 mRNA and protein in liver was specifically increased after feeding hamsters with a fat- and cholesterol-enriched diet, providing the first in vivo evidence for the regulated expression of ACSL3 in liver tissue. The aim of the current study was to further investigate the role of ACSL3 in regulating hepatic lipid metabolism in vitro and in vivo. We utilized an adenoviral-mediated gene delivery approach to exogenously express hamster ACSL3 in hamster liver as well as in HepG2 cells. Transduction of HepG2 cells with Ad-hamACSL3 adenovirus elevated total cellular ACSL enzyme activity, which was accompanied by a significant reduction of cellular contents of triglycerides and total phospholipids. Immunostaining and confocal microscopy studies revealed that ACSL3 was localized to endoplasmic reticulum and mitochondria. In vivo, infection of hamsters with Ad-hamACSL3 led to sustained expression of ACSL3 mRNA and protein in liver two weeks after infection. Importantly, compared with Ad-GFP control virus infected hamsters, we observed significantly lower free fatty acids and triglycerides plus modest reduction of phospholipids in the serum of Ad-hamACSL3 infected animals. Furthermore, triglyceride levels were significantly reduced in Ad-hamACSL3 infected hamster liver. Altogether, these results provide important and physiologically relevant evidence that strengthens the link between ACSL3 expression and hepatic reduction of triglycerides and fatty acids.

  17. High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway.

    PubMed

    Dong, Bin; Kan, Chin Fung Kelvin; Singh, Amar B; Liu, Jingwen

    2013-05-01

    Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.

  18. Acylcarnitine profiles during carnitine loading and fasting tests in a Japanese patient with medium-chain acyl-CoA dehydrogenase deficiency.

    PubMed

    Yokoi, Kyoko; Ito, Tetsuya; Maeda, Yasuhiro; Nakajima, Yoko; Ueta, Akihito; Nomura, Takayasu; Koyama, Norihisa; Kato, Ineko; Suzuki, Satoshi; Kurono, Yukihisa; Sugiyama, Naruji; Togari, Hajime

    2007-12-01

    Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is rare among Asian individuals, and the clinical course and biochemical findings remain unclear. We report herein a 3-year-old Japanese girl with MCADD. The diagnosis was suggested by acylcarnitine profiles and confirmed by enzyme activity and genetic analysis after clinical presentation. Our described method with high-performance liquid chromatography/tandem mass spectrometry allows quantification of levels of n-octanoylcarnitine (C8-N) and other isomers (e.g. valproylcarnitine). We examined the patient's acylcarnitine profiles in serum and urine samples during carnitine loading and 14-hr fasting tests with/without carnitine supplementation. Under hypocarnitinemia, serum level of C8-N was 0.16 micromol/l and C8-N/decanoylcarnitine (C10) ratio was 1.8, which did not correspond to the diagnostic criteria for MCADD. However, intravenous carnitine loading test (100 mg/kg/day for 3 days and 50 mg/kg/day for 1 day) led to increased serum C8-N levels and urinary excretion was obvious, strongly suggesting MCADD. In the fasting test with carnitine supplementation, marked production of acylcarnitines (C8-N > C2 > C6 > C10) was found, compared to the fasting test without carnitine supplementation. These results indicate that carnitine supplementation may be useful for detoxification of accumulated acylcarnitines even in an asymptomatic state. Moreover, the one-point examination for serum C8-N level and/or C8-N/C10 ratio may make the diagnosis of MCADD difficult, particularly in the presence of significant hypocarnitinemia. To avoid this pitfall, attention should be given to serum levels of free carnitine, and carnitine loading may be demanded in hypocarnitinemia.

  19. Probing the role of the ceramide acyl chain length and sphingosine unsaturation in model skin barrier lipid mixtures by (2)H solid-state NMR spectroscopy.

    PubMed

    Stahlberg, Sören; Školová, Barbora; Madhu, Perunthiruthy K; Vogel, Alexander; Vávrová, Kateřina; Huster, Daniel

    2015-05-05

    We investigated equimolar mixtures of ceramides with lignoceric acid and cholesterol as models for the human stratum corneum by differential scanning calorimetry and (2)H solid-state NMR spectroscopy. Our reference system consisted of lignoceroyl sphingosine (Cer[NS24]), which represents one of the ceramides in the human stratum corneum. Furthermore, the effect of ceramide acyl chain truncation to 16 carbons as in Cer[NS16] and the loss of the C4 trans double bond as in dihydroceramide Cer[NDS24] were studied. Fully relaxed (2)H NMR spectra were acquired for each deuterated component of each mixture separately, allowing the quantitative determination of the individual lipid phases. At skin temperature, the reference system containing Cer[NS24] is characterized by large portions of each component of the mixture in a crystalline phase, which largely restricts the permeability of the skin lipid barrier. The loss of the C4 trans double bond in Cer[NDS24] leads to the replacement of more than 25% of the crystalline phase by an isotropic phase of the dihydroceramide that shows the importance of dihydroceramide desaturation in the formation of the skin lipid barrier. The truncated Cer[NS16] is mostly found in the gel phase at skin temperature, which may explain its negative effect on the transepidermal water loss in atopic dermatitis patients. These significant alterations in the phase behavior of all lipids are further reflected at elevated temperatures. The molecular insights of our study may help us to understand the importance of the structural parameters of ceramides in healthy and compromised skin barriers.

  20. Chemoenzymatic synthesis of bifunctional polyubiquitin substrates for monitoring ubiquitin chain remodeling.

    PubMed

    Trang, Vivian H; Rodgers, Margaret L; Boyle, Kevin J; Hoskins, Aaron A; Strieter, Eric R

    2014-07-21

    Covalent attachment of ubiquitin to target proteins is one of the most pervasive post-translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C-terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site-specifically modified polyubiquitin chains produced by thiol-ene chemistry. We have used this chemoenzymatic approach to prepare dual-functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual-functionalized ubiquitin chains enabled the first real-time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.

  1. Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: identification of a novel variant of isoform 6.

    PubMed

    Van Horn, Cynthia G; Caviglia, Jorge M; Li, Lei O; Wang, Shuli; Granger, Deborah A; Coleman, Rosalind A

    2005-02-08

    The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.

  2. Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

    PubMed Central

    Vandavasi, Venu Gopal; Cooper, Jonathan B.; Ginell, Stephan L.

    2016-01-01

    ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed. PMID:27795378

  3. Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii

    PubMed Central

    Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P.; Datta, Santanu; Venkatraman, Janani

    2017-01-01

    Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved. PMID:28107498

  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. Unique Substrate Recognition Mechanism of the Botulinum Neurotoxin D Light Chain*

    PubMed Central

    Guo, Jiubiao; Chen, Sheng

    2013-01-01

    Botulinum neurotoxins are the most potent protein toxins in nature. Despite the potential to block neurotransmitter release at the neuromuscular junction and cause human botulism, they are widely used in protein therapies. Among the seven botulinum neurotoxin serotypes, mechanisms of substrate recognition and specificity are known to a certain extent in the A, B, E, and F light chains, but not in the D light chain (LC/D). In this study, we addressed the unique substrate recognition mechanism of LC/D and showed that this serotype underwent hydrophobic interactions with VAMP-2 at its V1 motif. The LC/D B3, B4, and B5 binding sites specifically recognize the hydrophobic residues in the V1 motif of VAMP-2. Interestingly, we identified a novel dual recognition mechanism employed by LC/D in recognition of VAMP-2 sites at both the active site and distal binding sites, in which one site of VAMP-2 was recognized by two independent, but functionally similar LC/D sites that were complementary to each other. The dual recognition strategy increases the tolerance of LC/D to mutations and renders it a good candidate for engineering to improve its therapeutic properties. In conclusion, in this study, we identified a unique multistep substrate recognition mechanism by LC/D and provide insights for LC/D engineering and antitoxin development. PMID:23963459

  6. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer

    PubMed Central

    Chen, Wei-Ching; Wang, Chih-Yang; Hung, Yu-Hsuan; Weng, Tzu-Yang; Yen, Meng-Chi; Lai, Ming-Derg

    2016-01-01

    Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in

  7. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

    PubMed

    Chen, Wei-Ching; Wang, Chih-Yang; Hung, Yu-Hsuan; Weng, Tzu-Yang; Yen, Meng-Chi; Lai, Ming-Derg

    2016-01-01

    Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in

  8. The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins. Medium chain acyl-CoA dehydrogenase and mitochondrial malate dehydrogenase.

    PubMed

    Kelly, D P; Gordon, J I; Alpers, R; Strauss, A W

    1989-11-15

    To study the regulation of nuclear genes which encode mitochondrial enzymes involved in oxidative metabolism, absolute levels of mRNA encoding rat medium chain acyl-CoA dehydrogenase (MCAD) and rat mitochondrial malate dehydrogenase (mMDH) were determined in developing and adult male rat tissues. MCAD mRNA is expressed in a variety of adult male tissues with highest steady state levels in heart, adrenal, and skeletal muscle and lowest levels in brain, lung, and testes. In comparison, steady state levels of mMDH mRNA in adult male rat tissues were similar to those of MCAD mRNA in heart, small intestine, adrenal, and skeletal muscle but markedly different in brain, stomach, and testes. Thus, the steady-state levels of MCAD and mMDH mRNA are highest in adult tissues with high energy requirements. Dot blot analysis of RNA prepared from late fetal, suckling, and weaning rat heart, liver, and brain demonstrated the presence of MCAD and mMDH mRNA during the fetal period in all three tissues. Both MCAD and mMDH mRNA levels increased 2-2.5-fold at birth followed by a decline during the first postnatal week in heart and liver. The patterns of accumulation of these mRNAs in heart and liver during the weaning and early adult periods were also similar, although the absolute levels were significantly different. Brain MCAD mRNA levels were consistently low (less than 0.1 pg/micrograms total cellular RNA) throughout the developmental stages. However, brain mMDH mRNA levels exhibited a marked increase during the weaning period, reaching a peak concentration which is higher than the level of mMDH mRNA in heart and liver at any point during development. These results indicate that the level of expression of the nuclear genes encoding MCAD and mMDH is tissue-specific and developmentally regulated. The patterns of MCAD and mMDH mRNA accumulation parallel the changes in energy metabolism which occur during development and among adult tissues.

  9. Electrospray Ionization Tandem Mass Spectrometry (Esi-Ms/Ms) Analysis of the Lipid Molecular Species Composition of Yeast Subcellular Membranes Reveals Acyl Chain-Based Sorting/Remodeling of Distinct Molecular Species En Route to the Plasma Membrane

    PubMed Central

    Schneiter, Roger; Brügger, Britta; Sandhoff, Roger; Zellnig, Günther; Leber, Andrea; Lampl, Manfred; Athenstaedt, Karin; Hrastnik, Claudia; Eder, Sandra; Daum, Günther; Paltauf, Fritz; Wieland, Felix T.; Kohlwein, Sepp D.

    1999-01-01

    Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of ∼± 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Δ mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane. PMID:10459010

  10. The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A sup 2 H and sup 31 P NMR study

    SciTech Connect

    Jordi, W.; de Kroon, A.I.P.M.; Killian, A.; de Kruijff, B. )

    1990-03-06

    Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-(2-{sup 2}H{sub 1})serine) and acyl chain deuterated (1,2-(11,11-{sup 2}H{sub 2})dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. {sup 2}H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.

  11. Detachment of semiflexible polymer chains from a substrate: A molecular dynamics investigation

    NASA Astrophysics Data System (ADS)

    Paturej, J.; Erbas, A.; Milchev, A.; Rostiashvili, V. G.

    2014-12-01

    Using Molecular Dynamics simulations, we study the force-induced detachment of a coarse-grained model polymer chain from an adhesive substrate. One of the chain ends is thereby pulled at constant speed off the attractive substrate and the resulting saw-tooth profile of the measured mean force ⟨f⟩ vs height D of the end-segment over the plane is analyzed for a broad variety of parameters. It is shown that the observed characteristic oscillations in the ⟨f⟩-D profile depend on the bending and not on the torsional stiffness of the detached chains. Allowing for the presence of hydrodynamic interactions (HI) in a setup with explicit solvent and dissipative particle dynamics-thermostat, rather than the case of Langevin thermostat, one finds that HI have little effect on the ⟨f⟩-D profile. Also the change of substrate affinity with respect to the solvent from solvophilic to solvophobic is found to play negligible role in the desorption process. In contrast, a changing ratio ɛ _s^B / ɛ _s^A of the binding energies of A- and B-segments in the detachment of an AB-copolymer from adhesive surface strongly changes the ⟨f⟩-D profile whereby the B-spikes vanish when ɛ _s^B / ɛ _s^A < 0.15. Eventually, performing an atomistic simulation of (bio)-polymers, we demonstrate that the simulation results, derived from our coarse-grained model, comply favorably with those from the all-atom simulation.

  12. Detachment of semiflexible polymer chains from a substrate: A molecular dynamics investigation

    SciTech Connect

    Paturej, J.; Erbas, A.; Milchev, A.; Rostiashvili, V. G.

    2014-12-07

    Using Molecular Dynamics simulations, we study the force-induced detachment of a coarse-grained model polymer chain from an adhesive substrate. One of the chain ends is thereby pulled at constant speed off the attractive substrate and the resulting saw-tooth profile of the measured mean force 〈f〉 vs height D of the end-segment over the plane is analyzed for a broad variety of parameters. It is shown that the observed characteristic oscillations in the 〈f〉-D profile depend on the bending and not on the torsional stiffness of the detached chains. Allowing for the presence of hydrodynamic interactions (HI) in a setup with explicit solvent and dissipative particle dynamics-thermostat, rather than the case of Langevin thermostat, one finds that HI have little effect on the 〈f〉-D profile. Also the change of substrate affinity with respect to the solvent from solvophilic to solvophobic is found to play negligible role in the desorption process. In contrast, a changing ratio ε{sub s}{sup B}/ε{sub s}{sup A} of the binding energies of A- and B-segments in the detachment of an AB-copolymer from adhesive surface strongly changes the 〈f〉-D profile whereby the B-spikes vanish when ε{sub s}{sup B}/ε{sub s}{sup A}<0.15. Eventually, performing an atomistic simulation of (bio)-polymers, we demonstrate that the simulation results, derived from our coarse-grained model, comply favorably with those from the all-atom simulation.

  13. Novel Fluorinated Polymers Containing Short Perfluorobutyl Side Chains and Their Super Wetting Performance on Diverse Substrates.

    PubMed

    Jiang, Jingxian; Zhang, Guangfa; Wang, Qiongyan; Zhang, Qinghua; Zhan, Xiaoli; Chen, Fengqiu

    2016-04-27

    Because the emission of perfluorooctanoic acid (PFOA) was completely prohibited in 2015, the widely used poly- and perfluoroalkyl substances with long perfluoroalkyl groups must be substituted by environmentally friendly alternatives. In this study, one kind of potential alternative (i.e., fluorinated polymers with short perfluorobutyl side chains) has been synthesized from the prepared monomers {i.e., (perfluorobutyl)ethyl acrylate (C4A), (perfluorobutyl)ethyl methacrylate (C4MA), 2-[[[[2-(perfluorobutyl)]sulfonyl]methyl]amino]ethyl acrylate (C4SA), and methacrylate (C4SMA)}, and the microstructure, super wetting performance, and applications of the synthesized fluorinated polymers were systematically investigated. The thermal and crystallization behaviors of the fluoropolymer films were characterized by differential scanning calorimetry and wide-angle X-ray diffraction analysis, respectively. Dynamic water-repellent models were constructed. The stable low surface energy and dynamic water- and oil-repellent properties of these synthesized fluorinated polymers with short perfluorobutyl side chains were attributed to the synergetic effect of amorphous fluorinated side chains in perfluoroalkyl acrylate and crystalline hydrocarbon pendant groups in stearyl acrylate. Outstanding water- and oil-repellent properties of fabrics and any other substrates could be achieved by a facile dip-coating treatment using a fluorinated copolymer dispersion. As a result, we believe that our prepared fluorinated copolymers are potential candidates to replace the fluoroalkylated polymers with long perfluorinated chains in nonstick and self-cleaning applications in our daily life.

  14. 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

  15. Ricin A-chain structural determinant for binding substrate analogues: a molecular dynamics simulation analysis.

    PubMed

    Olson, M A

    1997-01-01

    Ricin A-chain is a cytotoxic protein that attacks ribosomes by hydrolyzing a specific adenine base from a highly conserved, single-stranded rRNA hairpin containing the tetraloop sequence GAGA. Molecular-dynamics simulation methods are used to analyze the structural determinant for three substrate analogues bound to the ricin A-chain molecule. Simulations were applied to the binding of the dinucleotide adenyl-3',5'-guanosine employing the x-ray crystal structure of the ricin complex and a modeled CGAGAG hexanucleotide loop taken from the NMR solution structure of a 29-mer oligonucleotide hairpin. A third simulation model is also presented describing a conformational search of the docked 29-mer structure by using a simulated-annealing method. Analysis of the structural interaction energies for each model shows the overall binding dominated by nonspecific interactions, which are mediated by specific arginine contracts from the highly basic region on the protein surface. The tetraloop conformation of the 29-mer was found to make specific interactions with conserved protein residues, in a manner that favored the GAGA sequence. A comparison of the two docked loop conformations with the NMR structure revealed significant positional deviations, suggesting that ricin may use an induced fit mechanism to recognize and bind the rRNA substrate. The conserved Tyr-80 may play an important conformational entropic role in the binding and release of the target adenine in the active site.

  16. Xanthommatin formation in rat liver mitochondria and its inhibition by respiratory chain substrates.

    PubMed

    Ogawa, H; Nagamura, Y; Ishiguro, I

    1983-11-01

    3-Hydroxykynurenine is condensed to xanthommatin by cytochrome c and cytochrome oxidase in rat liver mitochondria. In intact mitochondria the reaction is inhibited by respiratory chain substrates. However, this was not the case with preincubated mitochondria or with isolated cytochrome c and cytochrome oxidase. The inhibition of xanthommatin formation in native mitochondria by succinate was abolished by addition of antimycin A or malonate, whereas the inhibition by citrate, glutamate or fumarate was not impaired by antimycin A or amobarbital. However, after preincubation of mitochondria at 37 degrees C for 30 min the inhibition disappeared in these cases too. It is suggested that the inhibition by succinate is due to the supply of reduced cytochrome b which competes with 3-hydroxykynurenine for ferricytochrome c, while the other respiratory chain substrates inhibit xanthommatin formation only in the case of intact mitochondria by a yet unknown mechanism. These inhibition mechanisms prevent xanthommatin formation in rat liver mitochondria, even though 3-hydroxykynurenine is synthesized in the outer mitochondrial membrane.

  17. Effect of headgroup-substrate interactions on the thermal behavior of long-chain amphiphiles

    NASA Astrophysics Data System (ADS)

    Singla, Saranshu; Zhu, He; Dhinojwala, Ali

    The structure of amphiphilic molecules at liquid/solid and solid/solid interfaces is relevant in understanding lubrication, colloid stabilization, chromatography, and nucleation. Here, we characterize the interfacial structures of long chain amphiphilic molecules with different head groups (OH, COOH, NH2) using interface-sensitive sum frequency generation (SFG) spectroscopy. The behavior of these self-assembled monolayers (SAMs) on sapphire substrate is recorded in situ as a function of temperature (above and below bulk Tm) using SFG. Previous studies using synchrotron X-ray reflectivity and SFG show that the melting point of an ordered hexadecanol monolayer is around 30°C above its bulk Tm. The thermal stability of the monolayer is explained due to strong hydrogen bonding interactions between the head-group and the sapphire substrate. The strength of these hydrogen-bonding interactions between substrate and different head groups is calculated using the Badger-Bauer equation. Below Tm, the ordered monolayer influenced the structure of the interfacial crystalline layer, and the transition from monolayer to the bulk crystalline phases. The results with different head groups will be presented.

  18. Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time.

    PubMed

    Teng, Kuo-Hsun; Chen, Annie P-C; Kuo, Chih-Jung; Li, Yu-Chin; Liu, Hon-Ge; Chen, Chao-Tsen; Liang, Po-Huang

    2011-10-01

    Farnesyl pyrophosphate (FPP) is a common substrate for a variety of prenyltransferases for synthesizing isoprenoid compounds. In this study, (2E,6E)-8-O-(N-methyl-2-aminobenzoyl)-3,7-dimethyl-2,6-octandien-1-pyrophosphate (MANT-O-GPP), a fluorescent analog of FPP, was synthesized and demonstrated as a satisfactory substrate for Escherichia coli undecaprenyl pyrophosphate synthase (UPPS) with a K(m) of 1.5 μM and a k(cat) of 1.2s(-1) based on [(14)C]IPP consumption. Interesting, we found that its emission fluorescence intensity at 420 nm increased remarkably during chain elongation, thereby useful for real-time monitoring kinetics of UPPS to yield a K(m) of 1.1 μM and a k(cat) of 1.0 s(-1), consistent with those measured using radiolabeled substrate. Using this assay, the IC(50) of a known UPPS inhibitor farnesyl thiopyrophosphate (FsPP) was confirmed. Our studies provide a convenient and environmentally friendly alternative for kinetics and inhibition studies on UPPS drug target.

  19. Crystal Structure of Botulinum Neurotoxin Type G Light Chain - Serotype Divergence in Substrate Recognition†,‡

    PubMed Central

    Arndt, Joseph W.; Yu, Wayne; Bi, Fay; Stevens, Raymond C.

    2008-01-01

    The seven serotypes (A–G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). In order to understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 Å resolution. The structure of BoNT/G-LC reveals a C-terminal β-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1’ residue. In addition, structural and sequence analysis have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP. PMID:16008342

  20. Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition.

    PubMed

    Arndt, Joseph W; Yu, Wayne; Bi, Fay; Stevens, Raymond C

    2005-07-19

    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). To understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 A resolution. The structure of BoNT/G-LC reveals a C-terminal beta-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1' residue. In addition, structural and sequence analyses have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP.

  1. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGES

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; ...

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  2. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxygenase with a bacterial type-I fatty acid synthase in E. coli.

    PubMed

    Coursolle, Dan; Lian, Jiazhang; Shanklin, John; Zhao, Huimin

    2015-09-01

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg L(-1) long chain alcohol/alkane products including a 57 mg L(-1) titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  3. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    SciTech Connect

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  5. Engineering a disulfide bond in the lid hinge region of Rhizopus chinensis lipase: increased thermostability and altered acyl chain length specificity.

    PubMed

    Yu, Xiao-Wei; Tan, Nian-Jiang; Xiao, Rong; Xu, Yan

    2012-01-01

    The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for "interfacial activation" is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the "Disulfide by Design" algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t(1/2) value at 60°C and a 7°C increase of T(m) compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k(cat)) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.

  6. Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver[S

    PubMed Central

    Moffat, Cynthia; Bhatia, Lavesh; Nguyen, Teresa; Lynch, Peter; Wang, Miao; Wang, Dongning; Ilkayeva, Olga R.; Han, Xianlin; Hirschey, Matthew D.; Claypool, Steven M.; Seifert, Erin L.

    2014-01-01

    Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi­dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O2 consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix. PMID:25114170

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

    PubMed

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

    1996-09-01

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

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

    PubMed

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

    2015-03-31

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

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

    PubMed Central

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

    2015-01-01

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

  10. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter.

    PubMed

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-03-04

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript.

  11. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter*

    PubMed Central

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5′-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript. PMID:26728456

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

    PubMed Central

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

    2014-01-01

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

  13. 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

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

    PubMed Central

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

    2014-01-01

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

  15. 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

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

    PubMed

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

    2016-09-06

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

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

    PubMed Central

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

    2016-01-01

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

  18. Recognition of Acyl Carrier Proteins by Ketoreductases in Assembly Line Polyketide Synthases

    PubMed Central

    Ostrowski, Matthew P.; Cane, David E.; Khosla, Chaitan

    2016-01-01

    Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

  19. dAcsl, the Drosophila ortholog of acyl-CoA synthetase long-chain family member 3 and 4, inhibits synapse growth by attenuating bone morphogenetic protein signaling via endocytic recycling.

    PubMed

    Liu, Zhihua; Huang, Yan; Hu, Wen; Huang, Sheng; Wang, Qifu; Han, Junhai; Zhang, Yong Q

    2014-02-19

    Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. We report here that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, our results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation.

  20. Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing cis-monounsaturated acyl chain homologues of oleic acid: differential scanning calorimetric and /sup 31/P NMR spectroscopic studies

    SciTech Connect

    Lewis, R.N.A.H.; Sykes, B.D.; McElhaney, R.N.

    1988-02-09

    The thermotropic phase behavior of dioleoylphosphatidylcholine and six of its longer chain homologues was studied by differential scanning calorimetry and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy. Aqueous dispersions of these compounds all exhibit a single endotherm upon heating but upon cooling exhibit at least two exotherms, both of which occur at temperatures lower than those of their heating endotherm. The single transition observed upon heating was shown by /sup 31/P NMR spectroscopy to be a net conversion from a condensed, subgel-like phase (L/sub c/ phase) to the liquid-crystalline state. Aqueous ethylene glycol dispersions of these compounds also exhibit single endotherms upon heating and cooling exotherms centered at temperatures lower than those of their corresponding heating endotherm. However, the behavior of the aqueous ethylene glycol dispersions differs with respect to their transition temperatures and enthalpies as well as the extent of undercooling observed, and there is some evidence of discontinuities in the cooling behavior of the odd- and even-numbered members of the homologous series. Like the aqueous dispersions, /sup 31/P NMR spectroscopy also shows that the calorimetric events observed in aqueous ethylene glycol involve net interconversions between an L/sub c/-like phase and the liquid-crystalline state. These results demonstrate that although the presence of a cis double bond can perturb the solid-state packing of the acyl chains, its presence does not preclude the formation of highly ordered subgel-like phases in lipid bilayers. In the particular case of these unsaturated phosphatidylcholines, the formation of the subgel phases is more kinetically favorable than is the case with their saturated n-acyl counterparts.

  1. N-terminal fatty acylation of the alpha-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation.

    PubMed

    Galbiati, F; Guzzi, F; Magee, A I; Milligan, G; Parenti, M

    1994-11-01

    The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

  2. Synthesis of fluorescent C24-ceramide: evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin.

    PubMed

    Novotný, Jakub; Pospechová, Katerina; Hrabálek, Alexandr; Cáp, Robert; Vávrová, Katerina

    2009-12-15

    Topical skin lipid supplementation may provide opportunities for controlling ceramide (Cer) deficiency in skin diseases such as atopic dermatitis or psoriasis. Here we describe the synthesis of a long-chain 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD)-labeled Cer and its different penetration through human skin compared to widely used short-chain fluorescent Cer tools.

  3. Influence of the side chain and substrate on polythiophene thin film surface, bulk, and buried interfacial structures.

    PubMed

    Xiao, Minyu; Jasensky, Joshua; Zhang, Xiaoxian; Li, Yaoxin; Pichan, Cayla; Lu, Xiaolin; Chen, Zhan

    2016-08-10

    The molecular structures of organic semiconducting thin films mediate the performance of various devices composed of such materials. To fully understand how the structures of organic semiconductors alter on substrates due to different polymer side chains and different interfacial interactions, thin films of two kinds of polythiophene derivatives with different side-chains, poly(3-hexylthiophene) (P3HT) and poly(3-potassium-6-hexanoate thiophene) (P3KHT), were deposited and compared on various surfaces. A combination of analytical tools was applied in this research: contact angle goniometry and X-ray photoelectron spectroscopy (XPS) were used to characterize substrate dielectric surfaces with varied hydrophobicity for polymer film deposition; X-ray diffraction and UV-vis spectroscopy were used to examine the polythiophene film bulk structure; sum frequency generation (SFG) vibrational spectroscopy was utilized to probe the molecular structures of polymer film surfaces in air and buried solid/solid interfaces. Both side-chain hydrophobicity and substrate hydrophobicity were found to mediate the crystallinity of the polythiophene film, as well as the orientation of the thiophene ring within the polymer backbone at the buried polymer/substrate interface and the polymer thin film surface in air. For the same type of polythiophene film deposited on different substrates, a more hydrophobic substrate surface induced thiophene ring alignment with the surface normal at both the buried interface and on the surface in air. For different films (P3HT vs. P3KHT) deposited on the same dielectric substrate, a more hydrophobic polythiophene side chain caused the thiophene ring to align more towards the surface at the buried polymer/substrate interface and on the surface in air. We believe that the polythiophene surface, bulk, and buried interfacial molecular structures all influence the hole mobility within the polythiophene film. Successful characterization of an organic conducting

  4. A PLP-dependent polyketide chain releasing mechanism in the biosynthesis of mycotoxin fumonisins in Fusarium verticillioides.

    PubMed

    Gerber, Ryan; Lou, Lili; Du, Liangcheng

    2009-03-11

    Fumonisins are polyketide-derived mycotoxins produced by several plant pathogenic fungi. The toxins cause several fatal diseases in domestic animals and are associated with esophageal cancer and neural tube defects in humans. Fumonisins contain a highly reduced, acyclic 18-carbon chain, which is synthesized by an iterative polyketide synthase (PKS). This PKS does not contain a thioesterase or cyclase domain that is found in other PKSs for the release of the covalently linked polyketide chain. In this study, we expressed the acyl carrier protein (ACP) of FUM1 and in vitro loaded acyl chains to the ACP from acyl-CoA using a promiscuous 4'-phosphopantetheinyl transferase. We then expressed FUM8, which is homologous to 2-oxoamine synthase genes, in yeast and showed that the enzyme is able to offload the acyl chains from ACP. Products resulted from the decarboxylative condensation between l-alanine and acyl-S-ACP were detected by GC-MS. The enzyme activity was dependent on pyridoxal 5'-phosphate (PLP), and C18-S-ACP was the preferred substrate. The results revealed a novel polyketide chain-releasing mechanism, in which a PLP-dependent enzyme catalyzes the termination and offloading of the polyketide chain as well as the introduction of a new carbon-carbon bond and an amino group to the chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain release found in bacterial and other fungal polyketide biosyntheses.

  5. Ricin A-chain substrate specificity in RNA, DNA, and hybrid stem-loop structures.

    PubMed

    Amukele, Tim K; Schramm, Vern L

    2004-05-04

    Ricin toxin A-chain (RTA) is the catalytic subunit of ricin, a heterodimeric toxin from castor beans. Its ribosomal inactivating activity arises from depurination of a single adenine from position A(4324) in a GAGA tetraloop from 28S ribosomal RNA. Minimal substrate requirements are the GAGA tetraloop and stem of two or more base pairs. Depurination activity also occurs on stem-loop DNA with the same sequence, but with the k(cat) reduced 200-fold. Systematic variation of RNA 5'-G(1)C(2)G(3)C(4)[G(5)A(6)G(7)A(8)]G(9)C(10)G(11)C(12)-3' 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k(cat) for A(6) --> dA(6) but reduced k(cat) up to 300-fold for the other sites. Methylation of individual 2'-hydroxyls in a similar experiment reduced k(cat) by as much as 3 x 10(-3)-fold. In stem-loop DNA, replacement of d[G(5)A(6)G(7)A(8)] with individual ribonucleotides resulted in small kinetic changes, except for the dA(6) --> A(6) replacement for which k(cat) decreased 6-fold. Insertion of d[G(5)A(6)G(7)A(8)] into an RNA stem-loop or G(5)A(6)G(7)A(8) into a DNA stem-loop reduced k(cat) by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem-loops in one case (dG(5),dG(7)) decreased k(cat)/K(m) by 10(5)-fold, while a second change (dG(5),dA(8)) decreased k(cat) by 100-fold. Mapping these interactions on the structure of GAGA stem-loop RNA suggests that all the loop 2'-hydroxyl groups play a significant role in the action of ricin A-chain. Improved binding of RNA-DNA stem-loop hybrids provides a scaffold for inhibitor design. Replacing the adenosine of the RTA depurination site with deoxyadenosine in a small RNA stem-loop increased k(cat) 20-fold to 1660 min(-1), a value similar to RTA's k(cat) on intact ribosomes.

  6. 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.

  7. Endoglucanase Peripheral Loops Facilitate Complexation of Glucan Chains on Cellulose via Adaptive Coupling to the Emergent Substrate Structures

    SciTech Connect

    Lin, Yuchun; Beckham, Gregg T.; Himmel, Michael E.; Crowley, Michael F.; Chu, Jhih-wei

    2013-09-19

    We examine how the catalytic domain of a glycoside hydrolase family 7 endoglucanase catalytic domain (Cel7B CD) facilitates complexation of cellulose chains from a crystal surface. With direct relevance to the science of biofuel production, this problem also represents a model system of biopolymer processing by proteins in Nature. Interactions of Cel7B CD with a cellulose microfibril along different paths of complexation are characterized by mapping the atomistic fluctuations recorded in free-energy simulations onto the parameters of a coarse-grain model. The resulting patterns of protein-biopolymer couplings also uncover the sequence signatures of the enzyme in peeling off glucan chains from the microfibril substrate. We show that the semiopen active site of Cel7B CD exhibits similar barriers and free energies of complexation over two distinct routes; namely, scooping of a chain into the active-site cleft and threading from the chain end into the channel. On the other hand, the complexation energetics strongly depends on the surface packing of the targeted chain and the resulting interaction sites with the enzyme. A revealed principle is that Cel7B CD facilitates cellulose deconstruction via adaptive coupling to the emergent substrate. The flexible, peripheral segments of the protein outside of the active-site cleft are able to accommodate the varying features of cellulose along the simulated paths of complexation. The general strategy of linking physics-based molecular interactions to protein sequence could also be helpful in elucidating how other protein machines process biopolymers.

  8. Engineering Escherichia coli for odd straight medium chain free fatty acid production.

    PubMed

    Wu, Hui; San, Ka-Yiu

    2014-10-01

    Microbial biosynthesis of free fatty acids (FFAs) can be achieved by introducing an acyl-acyl carrier protein thioesterase gene into Escherichia coli. The engineered E. coli usually produced even chain FFAs. In this study, propionyl-CoA synthetase (prpE) from Salmonella enterica was overexpressed in two efficient even chain FFAs producers, ML103 (pXZM12) carrying the acyl-ACP thioesterase gene from Umbellularia californica and ML103 (pXZ18) carrying the acyl-ACP thioesterase gene from Ricinus communis combined with supplement of extracellular propionate. With these metabolically engineered E. coli, the odd straight chain FFAs, undecanoic acid (C11:0), tridecanoic acid (C13:0), and pentadecanoic acid (C15:0) were produced from glucose and propionate. The highest total odd straight chain FFAs produced by ML103 (pXZM12, pBAD-prpE) reached 276 mg/l with a ratio of 23.43 % of the total FFAs. In ML103 (pXZ18, pBAD-prpE), the highest total odd straight chain FFAs accumulated to 297 mg/l, and the ratio reached 17.68 % of the total FFAs. Due to the different substrate specificity of the acyl-ACP thioesterases, the major odd straight chain FFA components of ML103 (pXZM12, pBAD-prpE) were undecanoic acid and tridecanoic acid, while the ML103 (pXZ18, pBAD-prpE) preferred pentadecanoic acid.

  9. Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

    PubMed

    Nini, L; Sarda, L; Comeau, L C; Boitard, E; Dubès, J P; Chahinian, H

    2001-11-30

    We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific

  10. Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development.

    PubMed

    Böttcher, Christine; Boss, Paul K; Davies, Christopher

    2011-08-01

    Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.

  11. Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

    PubMed Central

    Böttcher, Christine; Boss, Paul K.; Davies, Christopher

    2011-01-01

    Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes. PMID:21543520

  12. The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

    PubMed

    Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

    1995-12-01

    Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

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

    PubMed

    Wältermann, Marc; Stöveken, Tim; Steinbüchel, Alexander

    2007-02-01

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

  14. Optimum alcohol concentration for chain elongation in mixed-culture fermentation of cellulosic substrate.

    PubMed

    Lonkar, Sagar; Fu, Zhihong; Holtzapple, Mark

    2016-12-01

    Medium-chain fatty acids (MCFA, e.g., caproic, heptanoic, caprylic acid) are more valuable than short-chain fatty acids (SCFA, e.g., acetic, propionic, butyric, valeric acid). SCFAs are major products in methane-inhibited mixed-culture anaerobic fermentation. By feeding ethanol to the fermentor, MCFA formation is enhanced through chain elongation. Microorganisms such as Clostridium kluyveri elongate short-chain acids by combining them with alcohol. Very low ethanol concentration reduces chain elongation rates, whereas very high ethanol concentrations inhibit microorganisms. To maximize MCFA production, different ethanol concentrations were investigated in the mixed-culture fermentation of office paper and chicken manure. At 10 g/L ethanol concentration, 10 g/L MCFA was formed. High ethanol concentrations (above 40 g/L) inhibit microorganisms resulting in no chain elongation. For chain elongation, propanol was found to be more inhibitory than ethanol. The data suggest that MCFA production will increase by continuously extracting MCFA and maintaining 5-10 g/L ethanol concentration by periodic addition. Biotechnol. Bioeng. 2016;113: 2597-2604. © 2016 Wiley Periodicals, Inc.

  15. 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

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

    PubMed Central

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

    2002-01-01

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

  17. Unusual long-chain N-acyl homoserine lactone production by and presence of quorum quenching activity in bacterial isolates from diseased tilapia fish.

    PubMed

    Chang, Chien-Yi; Koh, Chong-Lek; Sam, Choon-Kook; Chan, Xin-Yue; Yin, Wai Fong; Chan, Kok Gan

    2012-01-01

    Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long-chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long-chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules.

  18. Unusual Long-Chain N-Acyl Homoserine Lactone Production by and Presence of Quorum Quenching Activity in Bacterial Isolates from Diseased Tilapia Fish

    PubMed Central

    Chang, Chien-Yi; Koh, Chong-Lek; Sam, Choon-Kook; Chan, Xin-Yue; Yin, Wai Fong; Chan, Kok Gan

    2012-01-01

    Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long-chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long-chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules. PMID:22952864

  19. Substrate specificity of the sialic acid biosynthetic pathway

    SciTech Connect

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

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

    SciTech Connect

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

    1988-09-01

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

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

    PubMed

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

    2005-09-16

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

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

    PubMed

    Lee, C Y; Meighen, E A

    1997-04-04

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

  3. Z-Selective Olefin Metathesis on Peptides: Investigation of Side-Chain Influence, Preorganization, and Guidelines in Substrate Selection

    PubMed Central

    2015-01-01

    Olefin metathesis has emerged as a promising strategy for modulating the stability and activity of biologically relevant compounds; however, the ability to control olefin geometry in the product remains a challenge. Recent advances in the design of cyclometalated ruthenium catalysts has led to new strategies for achieving such control with high fidelity and Z selectivity, but the scope and limitations of these catalysts on substrates bearing multiple functionalities, including peptides, remained unexplored. Herein, we report an assessment of various factors that contribute to both productive and nonproductive Z-selective metathesis on peptides. The influence of sterics, side-chain identity, and preorganization through peptide secondary structure are explored by homodimerization, cross metathesis, and ring-closing metathesis. Our results indicate that the amino acid side chain and identity of the olefin profoundly influence the activity of cyclometalated ruthenium catalysts in Z-selective metathesis. The criteria set forth for achieving high conversion and Z selectivity are highlighted by cross metathesis and ring-closing metathesis on diverse peptide substrates. The principles outlined in this report are important not only for expanding the scope of Z-selective olefin metathesis to peptides but also for applying stereoselective olefin metathesis in general synthetic endeavors. PMID:25102124

  4. A facile route to reassemble titania nanoparticles into ordered chain-like networks on substrate.

    PubMed

    Cheng, Ya-Jun; Wolkenhauer, Markus; Bumbu, Gina-Gabriela; Gutmann, Jochen S

    2012-02-13

    A facile route to reassemble titania nanoparticles within the titania-block copolymer composite films has been developed. The titania nanoparticles templated by the amphiphilic block copolymer of poly(styrene)-block-poly (ethylene oxide) (PS-b-PEO) were frozen in the continuous PS matrix. Upon UV exposure, the PS matrix was partially degraded, allowing the titania nanoparticles to rearrange into chain-like networks exhibiting a closer packing. The local structures of the Titania chain-like networks were investigated by both AFM and SEM; the lateral structures and vertical structures of the films were studied by GISAXS and X-ray reflectivity respectively. Both the image analysis and X-ray scattering characterization prove the reassembly of the titania nanoparticles after UV exposure. The mechanism of the nanoparticle assembly is discussed.

  5. Novel inhibitory action of tunicamycin homologues suggests a role for dynamic protein fatty acylation in growth cone-mediated neurite extension

    PubMed Central

    1994-01-01

    In neuronal growth cones, the advancing tips of elongating axons and dendrites, specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by long-chain homologues of the antibiotic tunicamycin, a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system, indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons, under conditions in which protein palmitoylation is inhibited, produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic, even in the absence of protein synthesis, or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation, rather than its previously established effects on protein glycosylation and synthesis. (a) The abilities of different tunicamycin homologues to induce growth cone collapse very systematically with the length of the fatty acyl side- chain of tunicamycin, in a manner predicted and observed for the inhibition of protein palmitoylation. Homologues with fatty acyl moieties shorter than palmitic acid (16 hydrocarbons), including potent inhibitors of glycosylation, are poor inhibitors of growth cone function. (b) The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid, which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for

  6. Nonideal mixing and phase separation in phosphatidylcholine-phosphatidic acid mixtures as a function of acyl chain length and pH.

    PubMed Central

    Garidel, P; Johann, C; Blume, A

    1997-01-01

    The miscibilities of phosphatidic acids (PAs) and phosphatidylcholines (PCs) with different chain lengths (n = 14, 16) at pH 4, pH 7, and pH 12 were examined by differential scanning calorimetry. Simulation of heat capacity curves was performed using a new approach that incorporates changes of cooperativity of the transition in addition to nonideal mixing in the gel and the liquid-crystalline phase as a function of composition. From the simulations of the heat capacity curves, first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained, and phase diagrams were constructed using temperatures for onset and end of melting, which were corrected for the broadening effect caused by a decrease in cooperativity. In all cases the composition dependence of the nonideality parameters indicated nonsymmetrical mixing behavior. The phase diagrams were therefore further refined by simulations of the coexistence curves using a four-parameter approximation to account for nonideal and nonsymmetrical mixing in the gel and the liquid-crystalline phase. The mixing behavior was studied at three different pH values to investigate how changes in headgroup charge of the PA influences the miscibility. The experiments showed that at pH 7, where the PA component is negatively charged, the nonideality parameters are in most cases negative, indicating that electrostatic effects favor a mixing of the two components. Partial protonation of the PA component at pH 4 leads to strong changes in miscibility; the nonideality parameters for the liquid-crystalline phase are now in most cases positive, indicating clustering of like molecules. The phase diagram for 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid:1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine mixtures at pH 4 indicates that a fluid-fluid immiscibility is likely. The results show that a decrease in ionization of PAs can induce large changes in mixing behavior. This occurs because of a

  7. Unveiling the Pathogenic Molecular Mechanisms of the Most Common Variant (p.K329E) in Medium-Chain Acyl-CoA Dehydrogenase Deficiency by in Vitro and in Silico Approaches.

    PubMed

    Bonito, Cátia A; Nunes, Joana; Leandro, João; Louro, Filipa; Leandro, Paula; Ventura, Fátima V; Guedes, Rita C

    2016-12-27

    Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid β-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramer assembled as a dimer of dimers (monomers A/B and C/D). Each monomer binds a FAD cofactor, necessary for the enzyme's activity. The most frequent mutation in MCADD results from the substitution of a lysine with a glutamate in position 304 of mature hMCAD (p.K329E in the precursor protein). Here, we combined in vitro and in silico approaches to assess the impact of the p.K329E mutation on the protein's structure and function. Our in silico results demonstrated for the first time that the p.K329E mutation, despite lying at the dimer-dimer interface and being deeply buried inside the tetrameric core, seems to affect the tetramer surface, especially the β-domain that forms part of the catalytic pocket wall. Additionally, the molecular dynamics data indicate a stronger impact of the mutation on the protein's motions in dimer A/B, while dimer C/D remains similar to the wild type. For dimer A/B, severe disruptions in the architecture of the pockets and in the FAD and octanoyl-CoA binding affinities were also observed. The presence of unaffected pockets (C/D) in the in silico studies may explain the decreased enzymatic activity determined for the variant protein (46% residual activity). Moreover, the in silico structural changes observed for the p.K329E variant protein provide an explanation for the structural instability observed experimentally, namely, the disturbed oligomeric profile, thermal stability, and conformational flexibility, with respect to the wild-type.

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

    SciTech Connect

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

    1990-03-25

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

  9. In Vivo Evidence that S-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases

    PubMed Central

    Val, Dale L.; Cronan, John E.

    1998-01-01

    Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine. PMID:9573148

  10. [Inhibition by IFN-(Asp)4-Lys-HIV chimeric protein of hydrolysis of the low molecular substrate by the enteropeptidase light chain].

    PubMed

    Shibanova, E D; Grishina, Iu B; Rumsh, L D

    2002-01-01

    The full length enteropeptidase or it's light chain have often used for the limited proteolysis of recombinant chimeric proteins incorporating the linker-(Asp)4Lys- to obtain the target protein. Any chimeric proteins were not cleaved by the full length enteropeptidase efficiently. The resistant to the hydrolysis chimeric protein IFN-(Asp)4Lys-HIV earlier was shown to be the competitive inhibitor (Ki = 3,4 x 10(-6) M) in relation to the low molecular substrate. In present study we were determined this chimeric protein competitive inhibited the same substrate hydrolysis by enteropeptidase light chain (Ki = 2,7 x 10(-5) M). Comparison the Ki values for the substrate hydrolysis by full length enzyme and its light chain suggests that the enteropeptidase heavy chain may participate in chimeric protein binding.

  11. 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

  12. High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

    PubMed

    Figueroa-Teran, Rubi; Pak, Heidi; Blomquist, Gary J; Tittiger, Claus

    2016-09-01

    Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

  13. Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes.

    PubMed

    Sirobhushanam, Sirisha; Galva, Charitha; Saunders, Lauren P; Sen, Suranjana; Jayaswal, Radheshyam; Wilkinson, Brian J; Gatto, Craig

    2017-03-01

    Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.

  14. Silver conductive features on flexible substrates from a thermally accelerated chain reaction at low sintering temperatures.

    PubMed

    Chen, Shih-Pin; Kao, Zhen-Kai; Lin, Jeng-Lung; Liao, Ying-Chih

    2012-12-01

    In this study, a simple and effective silver ink formulation was developed to generate silver tracks with high electrical conductivity on flexible substrates at low sintering temperatures. Diethanolamine (DEA), a self-oxidizing compound at moderate temperatures, was mixed with a silver ammonia solution to form a clear and stable solution. After inkjet-printed or pen-written on plastic sheets, DEA in the silver ink decomposes at temperatures higher than 50 °C and generates formaldehyde, which reacts spontaneously with silver ammonia ions to form silver thin films. The electrical conductivity of the inkjet-printed silver films can be 26% of the bulk silver after heating at 75 °C for 20 min and show great adhesion on plastic sheets.

  15. Simulation analysis of formycin 5?-monophosphate analog substrates in the ricin A-chain active site

    NASA Astrophysics Data System (ADS)

    Olson, Mark A.; Scovill, John P.; Hack, Dallas C.

    1995-06-01

    Ricin is an RNA N-glycosidase that hydrolyzes a single adenine base from a conserved loop of 28S ribosomal RNA, thus inactivating protein synthesis. Molecular-dynamics simulation methods are used to analyze the structural interactions and thermodynamics that govern the binding of formycin 5'-monophosphate (FMP) and several of its analogs to the active site of ricin A-chain. Simulations are carried out initiated from the X-ray crystal structure of the ricin-FMP complex with the ligand modeled as a dianion, monoanion and zwitterion. Relative changes in binding free energies are estimated for FMP analogs constructed from amino substitutions at the 2- and 2'-positions, and from hydroxyl substitution at the 2'-position.

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

    PubMed

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

    1997-04-01

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

  17. Branched-chain amino acid administration in surgical patients. Effects on amino acid and fuel substrate profiles.

    PubMed

    Desai, S P; Bistrian, B R; Palombo, J D; Moldawer, L L; Blackburn, G L

    1987-07-01

    During the first five days following gastric bypass surgery, 15 patients received near isotonic amino acid solutions that varied in their branched-chain amino acid (BCAA) content and amino acid profiles (15.6%, 50%, or 100% BCAA solutions). Plasma valine concentrations were elevated in patients receiving 50% and 100% BCAA solutions. Plasma alanine concentrations were highest in patients receiving 50% BCAA. Plasma free fatty acids and blood lactate concentrations were unchanged by either the operation or BCAA administration. Serum glucose concentration was unaffected by the different amino acid administrations and followed the pattern induced by stress initially and later by starvation. beta-Hydroxybutyrate concentrations increased as starvation proceeded and were highest in patients receiving the 15.6% BCAA solution. Branched-chain amino acid-enriched solutions without additional energy may be administered safely to patients recovering from operative trauma. Plasma amino acid concentrations and fuel substrate profiles appear to follow metabolic patterns determined by the physiologic response to stress and starvation and can be affected by large quantities of BCAAs.

  18. Glycerine and levulinic acid: renewable co-substrates for the fermentative synthesis of short-chain poly(hydroxyalkanoate) biopolymers.

    PubMed

    Ashby, Richard D; Solaiman, Daniel K Y; Strahan, Gary D; Zhu, Chengjun; Tappel, Ryan C; Nomura, Christopher T

    2012-08-01

    Glycerine (a biodiesel co-product) and levulinic acid (a pulp and paper co-product) were used as co-substrates for the fermentative synthesis of short-chain polyhydroxyalkanoate (sc-PHA) biopolymers with tunable monomer and molecular weight characteristics. Pseudomonas oleovorans NRRL B-14682 utilized glycerine alone to produce poly(3-hydroxybutyrate) (PHB). When levulinic acid was added to the media at shake-flask scale in concentrations ≤0.6 wt.%, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) copolymers were produced with 3-HV contents ranging from 37 to 97 mol%; a glycerine:levulinic acid ratio of 0.2%:0.8% (w/v) resulted in poly(3-hydroxyvalerate) (PHV). Ten-liter batch fermentations using glycerine:levulinic acid ratios of 1%:0, 0.75%:0.25%, 0.5%:0.5% and 0.25%:0.75% (w/v) resulted in PHB, P(73%-3HB-co-27%-3HV), P(30%-3HB-co-70%-3HV) and PHV with increasing number average molecular weights (×10(3) g/mol) of 328, 511, 728 and 1330, respectively, owing to glycerine-based chain termination. These results provide a novel means by which glycerine and levulinic acid can be used collectively to produce an array of distinct sc-PHA biopolymers.

  19. Influence of Belousov-Zhabotinsky substrate concentrations on autonomous oscillation of polymer chains with Fe(bpy)3 catalyst.

    PubMed

    Hara, Yusuke; Mayama, Hiroyuki; Fujimoto, Kenji

    2014-06-19

    We studied the effect of initial substrate concentrations in the Belousov-Zhabotinsky (BZ) reaction on the optical transmittance self-oscillation behavior of a polymer chain consisting of N-isopropylacrylamide (NIPAAm) and a Fe catalyst ([Fe(bpy)3]). The driving force of this transmittance self-oscillation was the solubility difference between the reduced and oxidized states of the [Fe(bpy)3] moiety in the polymer chain. The amplitude of the soluble-insoluble self-oscillation of poly(NIPAAm-co-[Fe(bpy)3]) was significantly smaller than that of poly(NIPAAm-co-[Ru(bpy)3]). Theoretical simulation results attributed this behavior to the small difference in the solvent qualities, C*, of the reduced and oxidized states. Furthermore, we clarified that poly(NIPAAm-co-[Fe(bpy)3]) required a narrower concentration range of HNO3 to exhibit self-oscillation than poly(NIPAAm-co-[Ru(bpy)3]), since transmittance self-oscillation occurred only for [HNO3] = 0.3 M. The period of self-oscillation of poly(NIPAAm-co-[Fe(bpy)3]) in solution was controlled mainly by NaBrO3 concentration and was hardly influenced by the initial concentration of malonic acid.

  20. Endogenous N-acyl taurines regulate skin wound healing

    PubMed Central

    Sasso, Oscar; Pontis, Silvia; Armirotti, Andrea; Cardinali, Giorgia; Kovacs, Daniela; Migliore, Marco; Summa, Maria; Moreno-Sanz, Guillermo; Picardo, Mauro; Piomelli, Daniele

    2016-01-01

    The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs—N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]—as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy. PMID:27412859

  1. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    SciTech Connect

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

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

    PubMed

    Juárez, M Patricia

    2004-08-01

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

  3. Solid-phase synthesis of arginine-containing peptides and fluorogenic substrates using a side-chain anchoring approach.

    PubMed

    Hamzé, Abdallah; Martinez, Jean; Hernandez, Jean-François

    2004-11-26

    Attachment of an amino acid to a solid support by its side chain is sometimes necessary to take advantage of an alpha-carboxylic group available for diverse modifications, including the incorporation of a fluorophore for the preparation of fluorogenic substrates. In contrast to most other amino acids, anchoring the guanidinium group of an arginine to a resin requires the use of a supplementary linker. To avoid the usually multistep synthesis of such a linker as well as its difficult attachment to the guanidine group, we developed a simple method where the guanidine group is built on a Rink amide resin. Our strategy followed the steps of guanidine formation: (i) addition of an isothiocyanate derivative of ornithine to the amino group of a solid support, yielding Nomega-linked thiocitrulline; (ii) S-methylation of thiourea; (iii) guanidinylation using ammonium acetate. Cleavage of the resin generated the arginine-containing compound, the amine group of the resin becoming part of the guanidine. We have demonstrated the usefulness of this method by the synthesis of a series of fluorogenic substrates for trypsin-like serine proteases, which were obtained in high yield and purity. Then, our strategy also allowed generation from the same precursor differentially substituted arginine derivatives, including Nomega-methyl- and Nomega-ethylarginines. The ability to prepare such analogues together with the intermediates thiocitrulline and S-methylisothiocitrulline from a unique precursor while the alpha-amine and carboxylic groups remain available for modification also makes this method a powerful tool for combinatorial solid-phase synthesis of NO synthase inhibitors.

  4. Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis.

    PubMed Central

    Pratt, R F; Krishnaraj, R; Xu, H

    1992-01-01

    Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues. This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site. Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis. The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either. PMID:1417747

  5. Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis.

    PubMed

    Pratt, R F; Krishnaraj, R; Xu, H

    1992-09-15

    Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues. This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site. Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis. The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either.

  6. The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase.

    PubMed

    Stöveken, Tim; Kalscheuer, Rainer; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2005-02-01

    The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.

  7. Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin.

    PubMed

    Adachi, H; Ohta, T; Matsuzawa, H

    1991-02-15

    Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate. For the Asp mutant, the complex decayed slowly and the hydrolytic activity was slightly retained both in vivo and in vitro. In contrast, the other mutants lost the hydrolytic activity completely and their complexes were stable. These results indicate that the side-chain carboxylate of Glu166 acts as a special catalyst for deacylation. Residues for deacylation have not been identified in other acyl enzymes, such as serine proteases and class C beta-lactamases. Furthermore, the acyl-enzyme intermediates obtained are so stable that they are considered to be ideal materials for crystallographic studies for elucidating the catalytic mechanism in more detail. In addition, the mutants can more easily form inclusion bodies than the wild-type, when they are produced in a large amount, suggesting that the residue also plays an important role in proper folding of the enzyme.

  8. A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution.

    PubMed

    Yang, Weili; Simpson, Jeffrey P; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B

    2012-10-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.

  9. Structural basis of substrate selectivity of Δ(1)-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): semialdehyde chain length.

    PubMed

    Pemberton, Travis A; Tanner, John J

    2013-10-01

    The enzyme Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase (aka P5CDH and ALDH4A1) is an aldehyde dehydrogenase that catalyzes the oxidation of γ-glutamate semialdehyde to l-glutamate. The crystal structures of mouse P5CDH complexed with glutarate, succinate, malonate, glyoxylate, and acetate are reported. The structures are used to build a structure-activity relationship that describes the semialdehyde carbon chain length and the position of the aldehyde group in relation to the cysteine nucleophile and oxyanion hole. Efficient 4- and 5-carbon substrates share the common feature of being long enough to span the distance between the anchor loop at the bottom of the active site and the oxyanion hole at the top of the active site. The inactive 2- and 3-carbon semialdehydes bind the anchor loop but are too short to reach the oxyanion hole. Inhibition of P5CDH by glyoxylate, malonate, succinate, glutarate, and l-glutamate is also examined. The Ki values are 0.27 mM for glyoxylate, 58 mM for succinate, 30 mM for glutarate, and 12 mM for l-glutamate. Curiously, malonate is not an inhibitor. The trends in Ki likely reflect a trade-off between the penalty for desolvating the carboxylates of the free inhibitor and the number of compensating hydrogen bonds formed in the enzyme-inhibitor complex.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Long-Chain Fatty Acid Oxidation Disorders (LC-FAOD) Extension Study for Subjects Previously Enrolled in Triheptanoin Studies.

    ClinicalTrials.gov

    2016-09-15

    Carnitine Palmitoyltransferase (CPT I or CPT II) Deficiency; Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency; Long-chain 3-hydroxy-acyl-CoA Dehydrogenase (LCHAD) Deficiency; Trifunctional Protein (TFP) Deficiency; Carnitine-acylcarnitine Translocase (CACT) Deficiency

  12. Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

    PubMed

    Luttgeharm, Kyle D; Cahoon, Edgar B; Markham, Jonathan E

    2016-03-01

    Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism.

  13. Inhibition of rat liver microsomal fatty acid chain elongation by gemfibrozil in vitro.

    PubMed

    Sánchez, R M; Viñals, M; Alegret, M; Vázquez, M; Adzet, T; Merlos, M; Laguna, J C

    1992-03-23

    Gemfibrozil, a hypolipidemic drug mainly used in the treatment of hypertriglyceridemic states, strongly inhibits the rat hepatic microsomal fatty acid chain elongation system in vitro. The inhibition is independent on the reducing cofactor used in the assay. Furthermore, gemfibrozil seems to act by inhibiting the rate-limiting step of the elongation process, the condensing reaction, without discriminating among the proposed three different condensing enzymes, devoted to condensation of saturated, mono-unsaturated and polyunsaturated acyl-CoA substrates.

  14. Synthesis of acyl arbutin by an immobilized lipase and its suppressive ability against lipid oxidation in a bulk system and O/W emulsion.

    PubMed

    Nagai, Mizuka; Watanabe, Yoshiyuki; Nomura, Masato

    2009-11-01

    Acyl arbutin was synthesized through the condensation of arbutin with a saturated fatty acid (C6-18) by the immobilized lipase in a batch reaction. The conversion at 10 and 20 g/l-solvent of immobilized lipase reached 45% over 2 d, but the initial reaction rate per amount of immobilized lipase decreased at 20 g/l-solvent. The radical scavenging activity of acyl arbutin in an ethanol solution was independent of the acyl chain length, although the rate constant, k, estimated for the oxidation of methyl linoleate in a bulk system with acyl arbutin by using the Weibull equation, decreased as the acyl chain length increased. This indicates the antioxidative ability of acyl arbutin with a long acyl chain to be due to its lipophilicity. Furthermore, it is suggested that dodecanoyl arbutin mainly acted on the interface between the oil and water phases in an O/W emulsion, and effectively suppressed the oxidation induced at the interface.

  15. Enzymatic Synthesis of Sorboyl-Polydatin Prodrug in Biomass-Derived 2-Methyltetrahydrofuran and Antiradical Activity of the Unsaturated Acylated Derivatives

    PubMed Central

    Yang, Rongling; Zhao, Xiangjie; Jiang, Ling; Zhu, Chun; Zhao, Yuping; Jia, Jianbo

    2016-01-01

    Efficient and highly regioselective synthesis of the potential 6′′-O-sorboyl-polydatin prodrug in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was achieved using Candida antarctica lipase B for the first time. Under the optimal conditions, the initial reaction rate, maximum substrate conversion, and 6′′-regioselectivity were as high as 8.65 mM/h, 100%, and 100%, respectively. Kinetic and operational stability investigations evidently demonstrated excellent enzyme compatibility of the 2-MeTHF compared to the traditional organic solvents. With respect to the antioxidant properties, three unsaturated ester derivatives showed slightly lower DPPH radical scavenging activities than the parent agent. Interestingly, further studies also revealed that the antiradical capacities of the acylates decreased with the elongation of the unsaturated aliphatic chain length from C4 to C11. The reason might be attributed to the increased steric hindrance derived from the acyl residues in derivatives. PMID:27668253

  16. Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

    PubMed Central

    Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

    2014-01-01

    The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

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

    PubMed

    Sachdeva, Sarbjot; Reynolds, Kevin A

    2008-01-01

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

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

    PubMed

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

    2009-07-01

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

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

    PubMed

    Bizzozero, O A; Lees, M B

    1986-02-01

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

  20. Selective acylation of plasma membrane proteins of Mycoplasma agalactiae: the causal agent of agalactia.

    PubMed

    Le Hénaff, M; Guéguen, M M; Fontenelle, C

    2000-01-01

    Revealed by in vivo labeling with (14)C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) approximately palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria.

  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. Structural limits of specificity of methylcholanthrene-repressible nitrosamine N-dealkylases. Inhibition by analog substrates.

    PubMed

    Arcos, J C; Bryant, G M; Pastor, K M; Argus, M F

    1976-06-15

    The dealkylation of dimethyl-, diethyl- and dipropylnitrosamine by hepatic microsomes of Sprague-Dawley rats is repressed by pretreatment of the animals with 3-methylcholanthrene (MC), and this repression progressively decreases with the increase of alkyl chain length. In contrast to its effect on the demethylation of dimethylnitrosamine (DMN), in vivo phenobarbital induces rather than represses the deethylation of diethylnitrosamine. The rates of demethylation of the DMN analog substrates (dimethylformamide, dimethylacetamide, dimethylpropionamide, and dimethylbutyramide), although low as compared to DMN, increase with the acyl chain length. These analogs are potent in vitro inhibitors of Dmn demethylation when used in combination with DMN as substrates, and the inhibition decreases with the length of the acyl chain. Dimethylaminoacetone, which corresponds to the insertion of a CH2 group between the N atom and the carbonyl group in dimethylacetamide, is not an in vitro inhibitor of DMN demethylation; the demethylation rates are additive when theis compound is used as substrate in combination with DMN. The rate of demethylation of dimethylaminoacetone is substantially higher than the rates of the dimethylacylamides, and is significantly repressed by MC-pretreatment. The rate of demethylation of methylphenylnitrosamine is not influenced by MC-pretreatment; the compound is, however, a potent inhibitor of demethylation when used as substrate in combination with DMN. The demethylation rates of 1,1-dimethylhydrazine (the reduction product of DMN) and dimethylaniline are not influenced by MC-pretreatment; neither do they affect the overall rate of demethylation when used as substrate in combination with DMN.

  3. A major substrate for MPF: cDNA cloning and expression of polypeptide chain elongation factor 1 gamma from goldfish (Carassius auratus).

    PubMed

    Tokumoto, Mika; Nagahama, Yoshitaka; Tokumoto, Toshinobu

    2002-02-01

    One of the eukaryotic polypeptide chain elongation factors, EF-1 beta gamma delta complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 alpha. In the complex, EF-1 gamma has been reported to be a major substrate for maturation promoting factor (MPF). Here, we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, EF-1 gamma from the goldfish ovary. The cloned cDNA was 1490 bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 gamma from other species. Although, the phosphorylation site identified in Xenopus EF-1 gamma was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 gamma was phosphorylated by MPF. We concluded that EF-1 gamma is a substrate for MPF during oocyte maturation in goldfish.

  4. 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.

  5. 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

  6. Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

    PubMed Central

    Knutzon, Deborah S.; Bleibaum, Janice L.; Nelsen, Janet; Kridl, Jean C.; Thompson, Gregory A.

    1992-01-01

    Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34- and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50- to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16- and 18-carbon acyl-ACP substrates. Images Figure 1 Figure 4 PMID:16653193

  7. 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

  8. Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA

    PubMed Central

    Finzel, Kara; Nguyen, Chi; Jackson, David R.; Gupta, Aarushi; Tsai, Shiou-Chuan; Burkart, Michael D.

    2015-01-01

    Summary Microbial fatty acid biosynthetic enzymes are important targets for areas as diverse as antibiotic development to biofuel production. Elucidating the molecular basis of chain length control during fatty acid biosynthesis is crucial for the understanding of regulatory processes of this fundamental metabolic pathway. In Escherichia coli, the acyl carrier protein (AcpP) plays a central role by sequestering and shuttling the growing acyl chain between fatty acid biosynthetic enzymes. FabA, a β-hydroxylacyl-AcpP dehydratase, is an important enzyme in controlling fatty acid chain length and saturation levels. FabA-AcpP interactions are transient in nature and thus difficult to visualize. In this study, four mechanistic crosslinking probes mimicking varying acyl chain lengths were synthesized to systematically probe for modified chain length specificity of fourteen FabA mutants. These studies provide evidence for the AcpP interacting “positive patch,” FabA mutations that altered substrate specificity, and the roles that the FabA “gating residues” play in chain-length selection. PMID:26526101

  9. Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli*

    PubMed Central

    Lin, Yibin; Bogdanov, Mikhail; Tong, Shuilong; Guan, Ziqiang; Zheng, Lei

    2016-01-01

    Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a “sideways sliding” mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane. PMID:26613781

  10. A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

    PubMed Central

    Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

    2012-01-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

  11. Effects of higher dietary protein intake on energy balance and metabolic control in children with long-chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deWciency

    PubMed Central

    Gillingham, Melanie B.; Purnell, Jonathan Q.; Jordan, Julia; Stadler, Diane; Haqq, Andrea M.; Harding, Cary O.

    2009-01-01

    The incidence of overweight and obesity is increasing among children with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) or mitochondrial trifunctional (TFP) deficiency. Traditional treatment includes fasting avoidance and consumption of a low-fat, high-carbohydrate diet. A diet higher in protein and lower in carbohydrate may help to lower total energy intake while maintaining good metabolic control. To determine the short-term safety and eYcacy of a high protein diet, subjects were admitted to the General Clinical Research Center and fed an ad-libitum high-protein diet and a high-carbohydrate diet for 6 days each using a randomized, crossover design. Nine subjects with LCHAD or TFP deficiency, age 7–14 were enrolled. Body composition was determined by DEXA. Total energy intake was evaluated daily. Resting energy expenditure and substrate utilization were determined by indirect calorimetry. Post-prandial metabolic responses of plasma glucose, insulin, leptin, ghrelin, acylcarnitines, and triglyceride were determined in response to a liquid meal. Subjects had a higher fat mass, lower lean mass and higher plasma leptin levels compared to reference values. While on the high protein diet energy consumption was an average of 50 kcals/day lower (p=0.02) and resting energy expenditure was an average of 170 kcals/day higher (p=0.05) compared to the high carbohydrate diet. Short-term higher protein diets were safe, well tolerated, and resulted in lowered energy intake and increased energy expenditure than the standard high-carbohydrate diet. Long-term studies are needed to determine whether higher protein diets will reduce the risk of overweight and obesity in children with LCHAD or TFP deficiency. PMID:16996288

  12. An Open-label Phase 2 Study of UX007 (Triheptanoin) in Subjects With Long-Chain Fatty Acid Oxidation Disorders (LC-FAOD)

    ClinicalTrials.gov

    2016-11-23

    Long-chain Fatty Acid Oxidation Disorders (LC-FAOD); Carnitine Palmitoyltransferase (CPT II) Deficiency; Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency; Longchain 3-hydroxy-acyl-CoA Dehydrogenase (LCHAD) Deficiency; Trifunctional Protein (TFP) Deficiency

  13. RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa.

    PubMed

    Zhu, Kun; Rock, Charles O

    2008-05-01

    Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily beta-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for beta-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

  14. Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains.

    PubMed

    Zhou, Zi-Ren; Zhang, Yu-Hang; Liu, Shuai; Song, Ai-Xin; Hu, Hong-Yu

    2012-01-01

    UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.

  15. Monolignol acylation and lignin structure in some nonwoody plants: a 2D NMR study.

    PubMed

    Martínez, Angel T; Rencoret, Jorge; Marques, Gisela; Gutiérrez, Ana; Ibarra, David; Jiménez-Barbero, Jesús; del Río, José C

    2008-11-01

    Lignins from three nonwoody angiosperms were analyzed by 2D NMR revealing important differences in their molecular structures. The Musa textilis milled-wood-lignin (MWL), with a syringyl-to-guaiacyl (S/G) ratio of 9, was strongly acylated (near 85% of side-chains) at the gamma-carbon by both acetates and p-coumarates, as estimated from (1)H-(13)C correlations in C(gamma)-esterified and C(gamma)-OH units. The p-coumarate H(3,5)-C(3,5) correlation signal was completely displaced by acetylation, and disappeared after alkali treatment, indicating that p-coumaric acid was esterified maintaining its free phenolic group. By contrast, the Cannabis sativa MWL (S/G approximately 0.8) was free of acylating groups, and the Agave sisalana MWL (S/G approximately 4) showed high acylation degree (near 80%) but exclusively with acetates. Extensive C(gamma)-acylation results in the absence (in M. textilis lignin) or low abundance (4% in A. sisalana lignin) of beta-beta' resinol linkages, which require free C(gamma)-OH to form the double tetrahydrofuran ring. However, minor signals revealed unusual acylated beta-beta' structures confirming that acylation is produced at the monolignol level, in agreement with chromatographic identification of gamma-acetylated sinapyl alcohol among the plant extractives. In contrast, resinol substructures involved 22% side-chains in the C.sativa MWL. The ratio between beta-beta' and beta-O-4' side-chains in these and other MWL varied from 0.32 in C.sativa MWL to 0.02 in M. textilis MWL, and was inversely correlated with the degree of acylation. The opposite was observed for the S/G ratio that was directly correlated with the acylation degree. Monolignol acylation is discussed as a mechanism potentially involved in the control of lignin structure.

  16. 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

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

    PubMed

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

    2003-01-01

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

  18. The Role of Mitochondrial Non-Enzymatic Protein Acylation in Ageing

    PubMed Central

    Hong, Shin Yee; Ng, Li Theng; Ng, Li Fang; Inoue, Takao; Tolwinski, Nicholas S.; Hagen, Thilo; Gruber, Jan

    2016-01-01

    In recent years, various large-scale proteomic studies have demonstrated that mitochondrial proteins are highly acylated, most commonly by addition of acetyl and succinyl groups. These acyl modifications may be enzyme catalysed but can also be driven non-enzymatically. The latter mechanism is promoted in mitochondria due to the nature of the mitochondrial microenvironment, which is alkaline and contains high concentrations of acyl-CoA species. Protein acylation may modify enzyme activity, typically inhibiting it. We posited that organismal ageing might be accompanied by an accumulation of acylated proteins, especially in mitochondria, and that this might compromise mitochondrial function and contribute to ageing. In this study, we used R. norvegicus, C. elegans and D. melanogaster to compare the acylation status of mitochondrial proteins between young and old animals. We observed a specific age-dependent increase in protein succinylation in worms and flies but not in rat. Rats have two substrate-specific mitochondrial deacylases, SIRT3 and SIRT5 while both flies and worms lack these enzymes. We propose that accumulation of mitochondrial protein acylation contributes to age-dependent mitochondrial functional decline and that SIRT3 and SIRT5 enzymes may promote longevity through regulation of mitochondrial protein acylation during ageing. PMID:28033361

  19. Acyl tunichlorins: a new class of nickel chlorins isolated from the Caribbean tunicate Trididemnum solidum.

    PubMed Central

    Sings, H L; Bible, K C; Rinehart, K L

    1996-01-01

    A new class of nickel-containing chlorins (acyl tunichlorins) has been isolated from the Caribbean tunicate Trididemnum solidum. The structures of 28 of these nickel (II) hydroporphyrins were elucidated using mass spectrometry, one- and two-dimensional NMR spectroscopy, and chemical degradation/derivatization. Unique structural features of these compounds include the diversity of aliphatic side chains, which are derived from C14:0 to C22:6 fatty acids, and their location at an unprecedented position at C-2a on the hydroporphyrin nucleus. No chlorins with ester-linked acyl side chains at C-2a have been reported previously. Although the exact biological role that these compounds play in T. solidum remains unknown, acyl tunichlorins represent the only nickel-containing chlorins to be isolated from a living system and are the C-2a acyl derivatives of tunichlorin, a nickel chlorin reported by this laboratory in 1988. PMID:8855217

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

    PubMed

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

    2017-03-14

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

  1. 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

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

    PubMed Central

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

    2016-01-01

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

  3. Metabolism of Acyl-Homoserine Lactone Quorum-Sensing Signals by Variovorax paradoxus

    PubMed Central

    Leadbetter, Jared R.; Greenberg, E. P.

    2000-01-01

    Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-l-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus. PMID:11092851

  4. Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus.

    PubMed

    Leadbetter, J R; Greenberg, E P

    2000-12-01

    Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus.

  5. Reduction of the 20-Carbonyl Group of C-21 Steroids by Spores of Fusarium solani and Other Microorganisms. I. Side-Chain Degradation, Epoxide Cleavage, and Substrate Specificity

    PubMed Central

    Plourde, Rosaire; El-Tayeb, Ossama M.; Hafez-Zedan, Hamdallah

    1972-01-01

    The spores of Fusarium solani reduced the C2-carbonyl group, 1-dehydrogenated ring „A” and cleaved the side chain of 16α, 17α-oxidopregn-4-ene-3, 20-dione (16α, 17α-oxidoprogesterone)(I) to give the following products: 20α-hydroxy-16α, 17α-oxidopregn-4-en-3-one(II); 20α-hydroxy-16α, 17α-oxidopregna-1, 4-dien-3-one(III); 16α-hydroxy-17a-oxa-androsta-1, 4-diene-3, 17-dione (16α-hydroxy-1-dehydrotestololactone)(IV); and 16α, 17β-dihydroxy-androsta-1, 4-dien-3-one (16α-hydroxy-1-dehydrotestosterone)(V). When II was used as a substrate, it was metabolized into III, IV, and V at a slower rate than I. Furthermore, 16α-hydroxy-androst-4-ene-3, 17-dione (16α-hydroxyandrostenedione)(X) was transformed into IV and V. Pregn-4-ene-3, 20-dione (progesterone)(XII) was transformed into androsta-1, 4-diene-3, 17-dione (androstadienedione)(VIII) and 17a-oxa-androsta-1, 4-diene-3, 17-dione (1-dehydrotestololactone)(IX), while 17α-hydroxy-pregn-4-ene-3, 20-dione (17α-hydroxyprogesterone)(VI) was converted into its 1-dehydro analogue (VII) without accumulation of any 20-dihydro compounds. Substrate specificity in the 20-reductase system of F. solani, Cylindrocarpon radicicola, Septomyxa affinis, Bacillus lentus, and three strains of B. sphaericus are demonstrated. The 20-reductase is active only on steroids having the 16α, 17α-oxido, and Δ4-3-keto functions. Evidence of competition between side-chain degrading enzymes and the 20-reductase for the steroid molecule and evidence of side-chain degradation followed by epoxide cleavage (and not the reverse) are presented. A mechanism for the epoxide opening by nongerminating spores of F. solani is postulated. PMID:5021973

  6. beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin.

    PubMed

    Fisher, J; Belasco, J G; Khosla, S; Knowles, J R

    1980-06-24

    The use of cefoxitin, a poor substrate of the RTEM beta-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction. The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly identical with the observed Kcat. Burst kinetics are observed with this substrate, consistent with the rate-limiting deacylation of the cefoxitinoyl-enzyme. That the reaction intermediate involves a covalent link between enzyme and substrate was shown by gel filtration after rapid denaturation of an enzyme-[14C]cefoxitin reaction at the steady state. Fourier transform infrared measurements indicate that the intermediate is an acyl-enzyme involving a hydroxyl group of the beta-lactamase. The evident relationship between the acylation-deacylation sequence of the beta-lactamases and the acylation reaction suffered by the D-Ala-D-Ala-carboxypeptidases is discussed.

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

    PubMed Central

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

    1970-01-01

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

  8. Broad Substrate Specificity of the Loading Didomain of the Lipomycin Polyketide Synthase

    SciTech Connect

    Yuzawa, S; Eng, CH; Katz, L; Keasling, JD

    2013-06-04

    LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic alpha-lipomycin in Streptomyces aureofaciens Tu117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.

  9. Acyl-coenzyme A: cholesterol acyltransferase inhibitor Avasimibe affect survival and proliferation of glioma tumor cell lines.

    PubMed

    Bemlih, Sana; Poirier, Marie-Denise; El Andaloussi, Abdeljabar

    2010-06-15

    Glioblastoma is the most common primary brain tumor in adults and one of its hallmarks is resistance to apoptosis. Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular membrane-bound enzyme that uses cholesterol and long chain fatty acyl-CoA as substrates to produce cholesteryl esters. The presence of cholesteryl esters in glioblastoma may be related to vascular and/or cell neoplastic proliferation in the tumor mass, two prerequisites for tumor cell growth. ACAT activity has been detected in glioblastoma cell homogenates. The present study is the first report on the effect of Avasimibe, a specific inhibitor of ACAT, on glioma cell lines (U87, A172 and GL261). Our results showed that Avasimibe inhibited ACAT-1 expression and cholesterol ester synthesis in glioma cell lines. Moreover, Avasimibe inhibited the growth of the cells by inducing cell cycle arrest and induced apoptosis as a result of caspase-8 and caspase-3 activation. Also, Our findings provide proof of principle that targeting ACAT-1 with the inhibitor Avasimibe could be an efficient therapy in the treatment of glioblastoma.

  10. ECERIFERUM2-LIKE Proteins Have Unique Biochemical and Physiological Functions in Very-Long-Chain Fatty Acid Elongation1[OPEN

    PubMed Central

    Haslam, Tegan M.; Haslam, Richard; Thoraval, Didier; Pascal, Stéphanie; Delude, Camille; Domergue, Frédéric; Fernández, Aurora Mañas; Beaudoin, Frédéric; Napier, Johnathan A.; Kunst, Ljerka; Joubès, Jérôme

    2015-01-01

    The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function. PMID:25596184

  11. By-products from the biodiesel chain as a substrate to citric acid production by solid-state fermentation.

    PubMed

    Schneider, Manuella; Zimmer, Gabriela F; Cremonese, Ezequiel B; de C de S Schneider, Rosana; Corbellini, Valeriano A

    2014-07-01

    In this study, we propose the use of tung cake for the production of organic acids, with an emphasis on citric acid by solid-state fermentation. We evaluated the conditions of production and the by-products from the biodiesel chain as raw materials involved in this bioprocess. First, we standardized the conditions of solid-state fermentation in tung cake with and without residual fat and with different concentrations of glycerine using the fungus Aspergillus niger The solid-state fermentation process was monitored for 7 days considering the biomass growth and pH level. Citric acid production was determined by high-performance liquid chromatography. Fungal development was better in the crude tung cake, consisting of 20% glycerine. The highest citric acid yield was 350 g kg(-1) of biomass. Therefore, the solid-state fermentation of the tung cake with glycerine led to citric acid production using the Aspergillus niger fungus.

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

    PubMed

    McFie, Pamela J; Stone, Scot J

    2011-09-01

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

  13. Linear ion-trap MSn with high resolution mass spectrometry reveals structural diversity of epidermal 1-O-acyl ceramide family in mouse epidermis.

    PubMed

    Lin, Meei-Hua; Miner, Jeffery; Turk, John; Hsu, Fong-Fu

    2017-02-02

    1-O-acylceramide is a new class of epidermal ceramide found in humans and mice. Here, we report ESI linear ion-trap (LIT) multiple stage mass spectrometric (MSn) approach with high resolution towards structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions was subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H - H2O - (1-O-fatty acid)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on ([M + H - H2O - (1-O-fatty acid)]+) ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 26:0-O-acyl) and N-acyl chains (from 20:0- to 26:0-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1- 17:1-, 18:2-, and 19:1-sphingosine LCB, with odd fatty acyl chain, or with monounsaturated N- or O- fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers are present, underscoring the complexity of this ceramide family.

  14. 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 ...

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

    PubMed Central

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

    2016-01-01

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

  16. An insight on acyl migration in solvent-free ethanolysis of model triglycerides using Novozym 435.

    PubMed

    Sánchez, Daniel Alberto; Tonetto, Gabriela Marta; Ferreira, María Luján

    2016-02-20

    In this work, the ethanolysis of triglycerides catalyzed by immobilized lipase was studied, focusing on the secondary reaction of acyl migration. The catalytic tests were performed in a solvent-free reaction medium using Novozym 435 as biocatalyst. The selected experimental variables were biocatalyst loading (5-20mg), reaction time (30-90min), and chain length of the fatty acids in triglycerides with and without unsaturation (short (triacetin), medium (tricaprylin) and long (tripalmitin/triolein)). The formation of 2-monoglyceride by ethanolysis of triglycerides was favored by long reaction times and large biocatalyst loading with saturated short- to medium-chain triglycerides. In the case of long-chain triglycerides, the formation of this monoglyceride was widely limited by acyl migration. In turn, acyl migration increased the yield of ethyl esters and minimized the content of monoglycerides and diglycerides. Thus, the enzymatic synthesis of biodiesel was favored by long-chain triglycerides (which favor the acyl migration), long reaction times and large biocatalyst loading. The conversion of acylglycerides made from long-chain fatty acids with unsaturation was relatively low due to limitations in their access to the active site of the lipase.

  17. Biogas production within the bioethanol production chain: Use of co-substrates for anaerobic digestion of sugar beet vinasse.

    PubMed

    Moraes, B S; Triolo, J M; Lecona, V P; Zaiat, M; Sommer, S G

    2015-08-01

    Bioethanol production generates large amounts of vinasse, which is suitable for biogas production. In this study, the anaerobic digestion of sugar beet vinasse was optimised using continuous stirred-tank reactors (CSTR) supplemented either with lime fertiliser or with 3% cow manure. In both reactors, the C/N ratio was adjusted by adding straw. The biochemical methane potential (BMP) of vinasse was 267.4±4.5LCH4kgVS(-1). Due to the low content of macro- and micronutrients and low C/N ratio of vinasse, biogas production failed when vinasse alone was fed to the reactor. When co-substrate was added, biogas production achieved very close to the BMP of vinasse, being 235.7±32.2LCH4kgVS(-1) from the fertiliser supplied reactor and 265.2±26.8LCH4kgVS(-1) in manure supplied reactor at steady state. Anaerobic digestion was the most stable when cow manure was supplied to digestion of vinasse.

  18. From nucleotides to DNA analysis by a SERS substrate of a self similar chain of silver nanospheres

    NASA Astrophysics Data System (ADS)

    Coluccio, M. L.; Gentile, F.; Das, G.; Perozziello, G.; Malara, N.; Alrasheed, S.; Candeloro, P.; Di Fabrizio, E.

    2015-11-01

    In this work we realized a device of silver nanostructures designed so that they have a great ability to sustain the surface-enhanced Raman scattering effect. The nanostructures were silver self-similar chains of three nanospheres, having constant ratios between their diameters and between their reciprocal distances. They were realized by electron beam lithography, to write the pattern, and by silver electroless deposition technique, to fill it with the metal. The obtained device showed the capability to increase the Raman signal coming from the gap between the two smallest nanospheres (whose size is around 10 nm) and so it allows the detection of biomolecules fallen into this hot spot. In particular, oligonucleotides with 6 DNA bases, deposited on these devices with a drop coating method, gave a Raman spectrum characterized by a clear fingerprint coming from the hot spot and, with the help of a fitting method, also oligonucleotides of 9 bases, which are less than 3 nm long, were resolved. In conclusion the silver nanolens results in a SERS device able to measure all the molecules, or part of them, held into the hot spot of the nanolenses, and thus it could be a future instrument with which to analyze DNA portions.

  19. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.

    PubMed

    Chang, C C; Lee, C Y; Chang, E T; Cruz, J C; Levesque, M C; Chang, T Y

    1998-12-25

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol.

  20. Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

    PubMed

    Ansari, Israr-Ul H; Longacre, Melissa J; Stoker, Scott W; Kendrick, Mindy A; O'Neill, Lucas M; Zitur, Laura J; Fernandez, Luis A; Ntambi, James M; MacDonald, Michael J

    2017-03-15

    Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion.

  1. Synthesis of medium-chain length capsinoids from coconut oil catalyzed by Candida rugosa lipases.

    PubMed

    Trbojević Ivić, Jovana; Milosavić, Nenad; Dimitrijević, Aleksandra; Gavrović Jankulović, Marija; Bezbradica, Dejan; Kolarski, Dušan; Veličković, Dušan

    2017-03-01

    A commercial preparation of Candida rugosa lipases (CRL) was tested for the production of capsinoids by esterification of vanillyl alcohol (VA) with free fatty acids (FA) and coconut oil (CO) as acyl donors. Screening of FA chain length indicated that C8-C12 FA (the most common FA found in CO triglycerides) are the best acyl-donors, yielding 80-85% of their specific capsinoids. Hence, when CO, which is rich in these FA, was used as the substrate, a mixture of capsinoids (vanillyl caprylate, vanillyl decanoate and vanillyl laurate) was obtained. The findings presented here suggest that our experimental method can be applied for the enrichment of CO with capsinoids, thus giving it additional health promoting properties.

  2. Cryptosporidium parvum Long-Chain Fatty Acid Elongase▿ †

    PubMed Central

    Fritzler, Jason M.; Millership, Jason J.; Zhu, Guan

    2007-01-01

    We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C14:0) and palmitoyl-CoA (C16:0) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C16:0 and C14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1. PMID:17827345

  3. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  4. Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition.

    PubMed

    Moreno-Pérez, Antonio J; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2011-01-01

    The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results.

  5. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  6. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-06

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

  7. Natural variability in acyl moieties of sugar esters produced by certain tobacco and other Solanaceae species.

    PubMed

    Kroumova, Antoaneta B M; Zaitlin, Dave; Wagner, George J

    2016-10-01

    A unique feature of glandular trichomes of plants in the botanical family Solanaceae is that they produce sugar esters (SE), chemicals that have been shown to possess insecticidal, antifungal, and antibacterial properties. Sugar esters of tobacco (Nicotiana tabacum) provide pest resistance, and are important flavor precursors in oriental tobacco cultivars. Acyl moieties of SEs in Nicotiana spp., petunia, and tomato are shown to vary with respect to carbon length and isomer structure (2-12 carbon chain length; anteiso-, iso-, and straight-chain). Sugar esters and their acyl groups could serve as a model to explore the basis of phenotypic diversity and adaptation to natural and agricultural environments. However, information on the diversity of acyl composition among species, cultivars, and accessions is lacking. Herein, described is the analysis of SE acyl groups found in 21 accessions of Nicotiana obtusifolia (desert tobacco), six of Nicotiana occidentalis subsp. hesperis, three of Nicotiana alata, two of N. occidentalis, four modern tobacco cultivars, five petunia hybrids, and one accession each of a primitive potato (Solanum berthaultii) and tomato (Solanum pennellii). A total of 20 different acyl groups was observed that were represented differently among cultivars, species, and accessions. In Nicotiana species, acetate and iso- and anteiso-branched acids prevailed. Straight-chain groups (2-8 carbons) were prominent in petunias, while octanoic acid was prominent in N. alata and N. × sanderae. Two unexpected acyl groups, 8-methyl nonanoate and decanoate were found in N. occidentalis subsp. hesperis. Longer chain groups were found in the petunia, tomato, and potato species studied.

  8. A new role for penicillin acylases: degradation of acyl homoserine lactone quorum sensing signals by Kluyvera citrophila penicillin G acylase.

    PubMed

    Mukherji, Ruchira; Varshney, Nishant Kumar; Panigrahi, Priyabrata; Suresh, C G; Prabhune, Asmita

    2014-03-05

    Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 °C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V(max) and K(m) values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V(max) and K(m) value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site.

  9. Production of medium chain length fatty alcohols from glucose in Escherichia coli

    PubMed Central

    Youngquist, J. Tyler; Schumacher, Martin H.; Rose, Joshua P.; Raines, Thomas C.; Politz, Mark C.; Copeland, Matthew F.; Pfleger, Brian F.

    2013-01-01

    Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol / g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli. PMID:24141053

  10. Production of medium chain length fatty alcohols from glucose in Escherichia coli.

    PubMed

    Youngquist, J Tyler; Schumacher, Martin H; Rose, Joshua P; Raines, Thomas C; Politz, Mark C; Copeland, Matthew F; Pfleger, Brian F

    2013-11-01

    Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol/g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli.

  11. Arthrobacter strain VAI-A utilizes acyl-homoserine lactone inactivation products and stimulates quorum signal biodegradation by Variovorax paradoxus.

    PubMed

    Flagan, Suvi; Ching, Weng-Ki; Leadbetter, Jared R

    2003-02-01

    Many Proteobacteria produce acyl-homoserine lactones (acyl-HSLs) and employ them as dedicated cell-to-cell signals in a process known as quorum sensing. Previously, Variovorax paradoxus VAI-C was shown to utilize diverse acyl-HSLs as sole sources of energy and nitrogen. We describe here the properties of a second isolate, Arthrobacter strain VAI-A, obtained from the same enrichment culture that yielded V. paradoxus VAI-C. Although strain VAI-A grew rapidly and exponentially on a number of substrates, it grew only slowly and aberrantly (i.e., linearly) in media amended with oxohexanoyl-HSL as the sole energy source. Increasing the culture pH markedly improved the growth rate in media containing this substrate but did not abolish the aberrant kinetics. The observed growth was remarkably similar to the known kinetics of the pH-influenced half-life of acyl-HSLs, which decay chemically to yield the corresponding acyl-homoserines. Strain VAI-A grew rapidly and exponentially when provided with an acyl-homoserine as the sole energy or nitrogen source. The isolate was also able to utilize HSL as a sole source of nitrogen but not as energy for growth. V. paradoxus, known to release HSL as a product of quorum signal degradation, was examined for the ability to support the growth of Arthrobacter strain VAI-A in defined cocultures. It did. Moreover, the acyl-HSL-dependent growth rate and yield of the coculture were dramatically superior to those of the monocultures. This suggested that the original coenrichment of these two organisms from the same soil sample was not coincidental and that consortia may play a role in quorum signal turnover and mineralization. The fact that Arthrobacter strain VAI-A utilizes the two known nitrogenous degradation products of acyl-HSLs, acyl-homoserine and HSL, begins to explain why none of the three compounds are known to accumulate in the environment.

  12. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells.

    PubMed

    Pedroza, Diego A; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J; Mito, Shizue

    2014-01-15

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death.

  13. Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25

    SciTech Connect

    Chang, C.C.Y.; Chang, W.; Chang, T.Y. ); Noll, W.W.; Nutile-McMenemy, N. ); Lindsay, E.A.; Baldini, A. )

    1994-01-01

    Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently the authors' laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. They have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human-hamster somatic cell hybrid panels.

  14. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells

    PubMed Central

    Pedroza, Diego A.; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J.; Mito, Shizue

    2014-01-01

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death. PMID:24368029

  15. A thermostat chip of indium tin oxide glass substrate for static polymerase chain reaction and in situ real time fluorescence monitoring.

    PubMed

    Wu, Zhi-Yong; Chen, Kun; Qu, Bai-Yan; Tian, Xiao-Xi; Wang, Xiao-Jie; Fang, Fang

    2008-03-03

    A thermostat chip of indium-tin oxide glass substrate for static chip polymerase chain reaction (PCR) is, for the first time, introduced in this paper. The transparent conductive layer was used as an electro-heating element. Pulse width modulation and fuzzy proportional integration-differentiation algorithm were adopted in the temperature programming of the chip. The temperature distribution was investigated, and a dynamic control precision within +/-2 degrees C was achieved. The highest ramping rates were 37 degrees Cs(-1) for heating and 8 degrees Cs(-1) for cooling with an electric fan. The PCR reaction vials were constructed with polyethylene tubes or poly(dimethylsiloxane) directly on the thermostat chip; the chip had a typical size of 25 mm x 25 mm and a thickness of 1.1mm. Static chip PCR was successfully demonstrated either in a single vial or in an up to 8-parallel array vials. In situ real time fluorescence monitoring during PCR of a lambda DNA fragments (236bp) with SYBR Green I was demonstrated using a blue light emission diode as a light source and a photomultiplier as a detector. The method proposed here is characterized by open access, easy fabrication and low cost. This work could be the basis for developing a portable real time PCR system with disposable chips for point of care tests.

  16. 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.

  17. Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

    PubMed

    Schaefer, A L; Val, D L; Hanzelka, B L; Cronan, J E; Greenberg, E P

    1996-09-03

    Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

  18. Long chain acyl-CoA synthetases and other acyl activating enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proper synthesis and breakdown of molecules containing carboxylic acids is a vital part of metabolism in all living organisms. Given the relatively inert chemical nature of many carboxylic acids, activation is a necessary step prior to use in the various anabolic and catabolic pathways that utilize...

  19. A special acyl carrier protein for transferring long hydroxylated fatty acids to lipid A in Rhizobium.

    PubMed

    Brozek, K A; Carlson, R W; Raetz, C R

    1996-12-13

    Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4'. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4'-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24

  20. Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI

    SciTech Connect

    Watson, W.T.; Murphy IV, Frank V.; Gould, Ty A.; Jambeck, Per; Val, Dale L.; Cronan, Jr., John E.; Beck von Bodman, Susan; Churchill, Mair E.A.

    2009-04-22

    Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 66.40, c = 47.33 {angstrom}. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 {angstrom} and the perrhenate ion binding sites and liganding residues have been identified.

  1. Synthesis, Surface Active Properties and Cytotoxicity of Sodium N-Acyl Prolines.

    PubMed

    Sreenu, Madhumanchi; Narayana Prasad, Rachapudi Badari; Sujitha, Pombala; Kumar, Chityal Ganesh

    2015-01-01

    Sodium N-acyl prolines (NaNAPro) were synthesized using mixture of fatty acids obtained from coconut, palm, karanja, Sterculia foetida and high oleic sunflower oils via Schotten-Baumann reaction in 58-75% yields to study the synergetic effect of mixture of hydrophobic fatty acyl functionalities like saturation, unsaturation and cyclopropene fatty acids with different chain lengths and aliphatic hetero cyclic proline head group on their surface and cytotoxicity activities. The products were characterized by chromatographic and spectral techniques. The synthesized products were evaluated for their surface active properties such as surface tension, wetting power, foaming characteristics, emulsion stability, calcium tolerance, critical micelle concentration (CMC) and thermodynamic properties. The results revealed that all the products exhibited superior surface active properties like CMC, calcium tolerance and emulsion stability as compared to the standard surfactant, sodium lauryl sulphate (SLS). In addition, palm, Sterculia foetida and high oleic sunflower fatty N-acyl prolines exhibited promising cytotoxicity against different tumor cell lines.

  2. Study of Triheptanoin for Treatment of Long-Chain Fatty Acid Oxidation Disorder

    ClinicalTrials.gov

    2017-03-21

    Very Long-chain acylCoA Dehydrogenase (VLCAD) Deficiency; Carnitine Palmitoyltransferase 2 (CPT2) Deficiency; Mitochondrial Trifunctional Protein (TFP) Deficiency; Long-chain 3 hydroxyacylCoA Dehydrogenase (LCHAD) Deficiency

  3. Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate.

    PubMed

    Amara, Sawsan; Lafont, Dominique; Fiorentino, Brice; Boullanger, Paul; Carrière, Frédéric; De Caro, Alain

    2009-10-01

    Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0-9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10(-2) M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000+/-500 and 2800+/-60 micromol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240+/-17 and 432+/-62 U/mg, respectively) on MGDG.

  4. 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.

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

    SciTech Connect

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

    1992-01-21

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

  6. Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

    SciTech Connect

    Carey, L.M.; Rodriguez, A.; Meighen, E.

    1984-08-25

    The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of > 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.

  7. 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.

  8. Computational modeling analysis of mitochondrial superoxide production under varying substrate conditions and upon inhibition of different segments of the electron transport chain.

    PubMed

    Markevich, Nikolai I; Hoek, Jan B

    2015-01-01

    A computational mechanistic model of superoxide (O2•-) formation in the mitochondrial electron transport chain (ETC) was developed to facilitate the quantitative analysis of factors controlling mitochondrial O2•- production and assist in the interpretation of experimental studies. The model takes into account all individual electron transfer reactions in Complexes I and III. The model accounts for multiple, often seemingly contradictory observations on the effects of ΔΨ and ΔpH, and for the effects of multiple substrate and inhibitor conditions, including differential effects of Complex III inhibitors antimycin A, myxothiazol and stigmatellin. Simulation results confirm that, in addition to O2•- formation in Complex III and at the flavin site of Complex I, the quinone binding site of Complex I is an additional superoxide generating site that accounts for experimental observations on O2•- production during reverse electron transfer. However, our simulation results predict that, when cytochrome c oxidase is inhibited during oxidation of succinate, ROS production at this site is eliminated and almost all superoxide in Complex I is generated by reduced FMN, even when the redox pressure for reverse electron transfer from succinate is strong. In addition, the model indicates that conflicting literature data on the kinetics of electron transfer in Complex III involving the iron-sulfur protein-cytochrome bL complex can be resolved in favor of a dissociation of the protein only after electron transfer to cytochrome bH. The model predictions can be helpful in understanding factors driving mitochondrial superoxide formation in intact cells and tissues.

  9. Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

    PubMed

    Monroig, Oscar; Navarro, Juan C; Dick, James R; Alemany, Frederic; Tocher, Douglas R

    2012-08-01

    Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

  10. Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds.

    PubMed

    Kim, Hae Jin; Silva, Jillian E; Iskandarov, Umidjon; Andersson, Mariette; Cahoon, Rebecca E; Mockaitis, Keithanne; Cahoon, Edgar B

    2015-12-01

    Lysophosphatidic acid acyltransferase (LPAT) catalyzes acylation of the sn-2 position on lysophosphatidic acid by an acyl CoA substrate to produce the phosphatidic acid precursor of polar glycerolipids and triacylglycerols (TAGs). In the case of TAGs, this reaction is typically catalyzed by an LPAT2 from microsomal LPAT class A that has high specificity for C18 fatty acids containing Δ9 unsaturation. Because of this specificity, the occurrence of saturated fatty acids in the TAG sn-2 position is infrequent in seed oils. To identify LPATs with variant substrate specificities, deep transcriptomic mining was performed on seeds of two Cuphea species producing TAGs that are highly enriched in saturated C8 and C10 fatty acids. From these analyses, cDNAs for seven previously unreported LPATs were identified, including cDNAs from Cuphea viscosissima (CvLPAT2) and Cuphea avigera var. pulcherrima (CpuLPAT2a) encoding microsomal, seed-specific class A LPAT2s and a cDNA from C. avigera var. pulcherrima (CpuLPATB) encoding a microsomal, seed-specific LPAT from the bacterial-type class B. The activities of these enzymes were characterized in Camelina sativa by seed-specific co-expression with cDNAs for various Cuphea FatB acyl-acyl carrier protein thioesterases (FatB) that produce a variety of saturated medium-chain fatty acids. CvLPAT2 and CpuLPAT2a expression resulted in accumulation of 10:0 fatty acids in the Camelina sativa TAG sn-2 position, indicating a 10:0 CoA specificity that has not been previously described for plant LPATs. CpuLPATB expression generated TAGs with 14:0 at the sn-2 position, but not 10:0. Identification of these LPATs provides tools for understanding the structural basis of LPAT substrate specificity and for generating altered oil functionalities.

  11. Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder

    PubMed Central

    Frye, R E; Melnyk, S; MacFabe, D F

    2013-01-01

    Autism spectrum disorder (ASD) has been associated with mitochondrial disease (MD). Interestingly, most individuals with ASD and MD do not have a specific genetic mutation to explain the MD, raising the possibility of that MD may be acquired, at least in a subgroup of children with ASD. Acquired MD has been demonstrated in a rodent ASD model in which propionic acid (PPA), an enteric bacterial fermentation product of ASD-associated gut bacteria, is infused intracerebroventricularly. This animal model shows validity as it demonstrates many behavioral, metabolic, neuropathologic and neurophysiologic abnormalities associated with ASD. This animal model also demonstrates a unique pattern of elevations in short-chain and long-chain acyl-carnitines suggesting abnormalities in fatty-acid metabolism. To determine if the same pattern of biomarkers of abnormal fatty-acid metabolism are present in children with ASD, the laboratory results from a large cohort of children with ASD (n=213) who underwent screening for metabolic disorders, including mitochondrial and fatty-acid oxidation disorders, in a medically based autism clinic were reviewed. Acyl-carnitine panels were determined to be abnormal if three or more individual acyl-carnitine species were abnormal in the panel and these abnormalities were verified by repeated testing. Overall, 17% of individuals with ASD demonstrated consistently abnormal acyl-carnitine panels. Next, it was determined if specific acyl-carnitine species were consistently elevated across the individuals with consistently abnormal acyl-carnitine panels. Significant elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines were found in the ASD individuals with consistently abnormal acyl-carnitine panels—a pattern consistent with the PPA rodent ASD model. Examination of electron transport chain function in muscle and fibroblast culture, histological and electron microscopy examination of muscle and other biomarkers of

  12. Acyl chain composition and coexisting fluid phases in lipid bilayers

    NASA Astrophysics Data System (ADS)

    Gu, Yongwen; Bradley, Miranda; Mitchell, Drake

    2011-10-01

    At room temperature phospholipid bilayers enriched in sphingolipids and cholesterol may form a solid phase as well as two coexisting fluid phases. These are the standard fluid phase, or the liquid-disordered phase, ld, and the liquid-ordered phase, lo, which is commonly associated with lipid rafts. Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form coexisting lo, ld and solid phases over a wide range of molar ratios. We are examining the ability of two fluorescent probes to detect these 2 phases: NBD linked to di-16:0 PE which partitions strongly into the lo phase and NBD linked to di-18:1 PE which partitions strongly into the ld phase. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the ternary phase diagram of POPC/SPM/Chol with particular focus on the functionally important lo/ld coexistence region. We report on the fluorescence lifetime and anisotropy decay dynamics of these two fluorescent probes.

  13. Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation

    PubMed Central

    Hopkins, Anna L.; Nelson, Timothy A. S.; Guschina, Irina A.; Parsons, Lydia C.; Lewis, Charlotte L.; Brown, Richard C.; Christian, Helen C.; Davies, Jeffrey S.; Wells, Timothy

    2017-01-01

    Despite being unable to activate the cognate ghrelin receptor (GHS-R), unacylated ghrelin (UAG) possesses a unique activity spectrum that includes promoting bone marrow adipogenesis. Since a receptor mediating this action has not been identified, we re-appraised the potential interaction of UAG with GHS-R in the regulation of bone marrow adiposity. Surprisingly, the adipogenic effects of intra-bone marrow (ibm)-infused acylated ghrelin (AG) and UAG were abolished in male GHS-R-null mice. Gas chromatography showed that isolated tibial marrow adipocytes contain the medium-chain fatty acids utilised in the acylation of UAG, including octanoic acid. Additionally, immunohistochemistry and immunogold electron microscopy revealed that tibial marrow adipocytes show prominent expression of the UAG-activating enzyme ghrelin O-acyl transferase (GOAT), which is located in the membranes of lipid trafficking vesicles and in the plasma membrane. Finally, the adipogenic effect of ibm-infused UAG was completely abolished in GOAT-KO mice. Thus, the adipogenic action of exogenous UAG in tibial marrow is dependent upon acylation by GOAT and activation of GHS-R. This suggests that UAG is subject to target cell-mediated activation – a novel mechanism for manipulating hormone activity. PMID:28361877

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

    PubMed

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

    2017-02-01

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

  15. Short-chain ceramides decrease skin barrier properties.

    PubMed

    Novotný, J; Janůsová, B; Novotný, M; Hrabálek, A; Vávrová, K

    2009-01-01

    Stratum corneum ceramides are major determinants of skin barrier function. Although their physiological and pathological role has been widely investigated, to date no structure-activity relationships have been established. In this study, a series of short-chain ceramide analogues with polar head structure identical to ceramide NS, a sphingosine length of 12 carbons and an acyl chain length of 2-12 carbons was synthesized. Their effect on skin permeability was evaluated using porcine skin and two model drugs, theophylline and indomethacin, and compared to that of a physiological ceramide NS. The results showed that the ceramide chain length was crucial for their barrier properties. Ceramides with a 4- to 8-carbon acyl chain were able to increase skin permeability for both drugs up to 10.8 times with maximum effect at a 6-carbon acyl chain. No increase in permeability was found for ceramide analogues with 2- and 12-carbon acyl chains and ceramide NS. The same relationships were obtained for skin concentrations of the model drugs. The relationship between ceramide acyl chain length and its ability to perturb skin barrier showed striking similarity to the behavior of short-chain ceramides in sphingomyelin/phospholipid membranes and confirmed that short-chain ceramides do not act as natural ceramides and their use as experimental tools should be cautious.

  16. Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution.

    PubMed

    Sándor, Viktor; Dörnyei, Ágnes; Makszin, Lilla; Kilár, Ferenc; Péterfi, Zoltán; Kocsis, Béla; Kilár, Anikó

    2016-11-01

    We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.

  17. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation.

    PubMed

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred; Madsen, Andreas S; Olsen, Christian A

    2016-02-11

    Sirtuins are important regulators of lysine acylation, which is implicated in cellular metabolism and transcriptional control. This makes the sirtuin class of enzymes interesting targets for development of small molecule probes with pharmaceutical potential. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation, and two known inhibitors, suramin and SirReal2, were profiled against substrates containing ε-N-acyllysine modifications of varying length.

  18. A fatty acyl-CoA reductase highly expressed in the head of honey bee (Apis mellifera) involves biosynthesis of a wide range of aliphatic fatty alcohols.

    PubMed

    Teerawanichpan, Prapapan; Robertson, Albert J; Qiu, Xiao

    2010-09-01

    Honey bees (Apis mellifera) are social insects which have remarkable complexity in communication pheromones. These chemical signals comprise a mixture of hydrocarbons, wax esters, fatty acids, aldehydes and alcohols. In this study, we detected several long chain aliphatic alcohols ranging from C18-C32 in honey bees and the level of these alcohols varied in each body segment. C18:0Alc and C20:0Alc are more pronounced in the head, whereas C22:0Alc to C32Alc are abundant in the abdomen. One of the cDNAs coding for a fatty acyl-CoA reductase (AmFAR1) involved in the synthesis of fatty alcohols was isolated and characterized. AmFAR1 was ubiquitously expressed in all body segments with the predominance in the head of honey bees. Heterologous expression of AmFAR1 in yeast revealed that AmFAR1 could convert a wide range of fatty acids (14:0-22:0) to their corresponding alcohols, with stearic acid 18:0 as the most preferred substrate. The substrate preference and the expression pattern of AmFAR1 were correlated with the level of total fatty alcohols in bees. Reconstitution of the wax biosynthetic pathway by heterologous expression of AmFAR1, together with Euglena wax synthase led to the high level production of medium to long chain wax monoesters in yeast.

  19. ROMPgel beads in IRORI format: acylations revisited.

    PubMed

    Roberts, Richard S

    2005-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  1. 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

  2. Giardia fatty acyl-CoA synthetases as potential drug targets

    PubMed Central

    Guo, Fengguang; Ortega-Pierres, Guadalupe; Argüello-García, Raúl; Zhang, Haili; Zhu, Guan

    2015-01-01

    Giardiasis caused by Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the leading causes of diarrheal parasitic diseases worldwide. Although limited drugs to treat giardiasis are available, there are concerns regarding toxicity in some patients and the emerging drug resistance. By data-mining genome sequences, we observed that G. intestinalis is incapable of synthesizing fatty acids (FA) de novo. However, this parasite has five long-chain fatty acyl-CoA synthetases (GiACS1 to GiACS5) to activate FA scavenged from the host. ACS is an essential enzyme because FA need to be activated to form acyl-CoA thioesters before they can enter subsequent metabolism. In the present study, we performed experiments to explore whether some GiACS enzymes could serve as drug targets in Giardia. Based on the high-throughput datasets and protein modeling analyses, we initially studied the GiACS1 and GiACS2, because genes encoding these two enzymes were found to be more consistently expressed in varied parasite life cycle stages and when interacting with host cells based on previously reported transcriptome data. These two proteins were cloned and expressed as recombinant proteins. Biochemical analysis revealed that both had apparent substrate preference toward palmitic acid (C16:0) and myristic acid (C14:0), and allosteric or Michaelis–Menten kinetics on palmitic acid or ATP. The ACS inhibitor triacsin C inhibited the activity of both enzymes (IC50 = 1.56 μM, Ki = 0.18 μM for GiACS1, and IC50 = 2.28 μM, Ki = 0.23 μM for GiACS2, respectively) and the growth of G. intestinalis in vitro (IC50 = 0.8 μM). As expected from giardial evolutionary characteristics, both GiACSs displayed differences in overall folding structure as compared with their human counterparts. These observations support the notion that some of the GiACS enzymes may be explored as drug targets in this parasite. PMID:26257723

  3. Location and biosynthesis of monoterpenyl fatty acyl esters in rose petals.

    PubMed

    Dunphy, Patrick J

    2006-06-01

    The upper epidermal layer of cells and the epicuticular wax surface of Lady Seton rose petals are sites of biosynthesis and accumulation, respectively, of a family of terpenyl fatty acyl esters. These esters are based mainly on the acyclic monoterpene alcohol geraniol coupled primarily to fatty acids of chain lengths 16-20 and in mass terms represent from 14% to 64% of the total monoterpenes present in the petals. The lipophilic nature of these non-volatile esters of the monoterpene alcohols contrasts with that of the lipophilic volatile parent alcohols themselves and with the hydrophilic, non-volatile, glucoside derivative of the other principal petal fragrant compounds, the phenylpropanoids, beta-phenyl ethanol and benzyl alcohol. These latter compounds are also synthesised and are resident in the petal. Biosynthetic studies confirmed that the petal upper epidermal cell layer has the capacity to incorporate mevalonic acid into the monoterpene component of the fatty acyl ester. The biosynthesis of the monoterpene component of the fatty acyl ester occurs via the mevalonic acid pathway in Lady Seton as well as in the hybrid tea rose Fragrant Cloud. In the latter flower the biosynthesis of geraniol was biosynthetically trans as was the formation of nerol and citronellol. Both geraniol and nerol were shown to be precursors of citronellol via an NADPH dependent reductase reaction. Oleic acid is assimilated into the acyl moiety of the terpenyl ester in Lady Seton isolated petal discs. It is probable that the lipophilic non-volatile terpenyl fatty acyl esters represent a stable storage form of the corresponding alcohols from their residency within the epicuticular wax layer. These acyl esters may realise, on hydrolysis, additional aroma notes from the living flower and potentially commercially significant quantities of the fragrant terpenols during oil of rose essence production.

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

    PubMed

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

    2015-01-01

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

  5. Cloning, characterization, and expression analysis of acyl-acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis).

    PubMed

    Dong, Shubin; Huang, Jiacong; Li, Yannan; Zhang, Jing; Lin, Shanzhi; Zhang, Zhixiang

    2014-05-25

    Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)△FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.

  6. Structure, supramolecular organization and phase behavior of N-acyl-β-alanines: Structural homologues of mammalian brain constituents N-acylglycine and N-acyl-GABA.

    PubMed

    Sivaramakrishna, D; Swamy, Musti J

    2016-12-01

    N-Acyl-β-alanines (NABAs) are structural homologues of N-acylglycines (NAGs) and N-acyl-γ-aminobutyric acids (NAGABAs), and achiral isomers of N-acylalanines, which are all present in mammalian brain and other tissues and modulate activity of biological receptors with various functions. In the present study, we synthesized and characterized a homologous series of NABAs bearing saturated acyl chains (n=8-20) and investigated their supramolecular organization and thermotropic phase behavior. In differential scanning calorimetric (DSC) studies, most of the NABAs gave one or two minor transitions before the main chain-melting phase transition in the dry state as well as upon hydration with water, but gave only a single transition when hydrated with buffer (pH7.6). Transition enthalpies (ΔHt) and entropies (ΔSt), obtained from the DSC studies showed linear dependence on the chain length in the dry state and upon hydration with buffer, whereas odd-even alteration was observed when hydrated with water. The crystal structures of N-lauroyl-β-alanine (NLBA) and N-myristoyl-β-alanine (NMBA) were solved in monoclinic system in the P21/c space group. Both NLBA and NMBA were packed in tilted bilayers with head-to-head (and tail-to-tail) arrangement with tilt angles of 33.28° and 34.42°, respectively. Strong hydrogen bonding interactions between COOH groups of the molecules from opposite leaflets as well as NH⋯O hydrogen bonds between the amide groups from adjacent molecules in the same leaflet as well as dispersion interactions between the acyl chains stabilize the bilayer structure. The d-spacings calculated from powder X-ray diffraction studies showed odd-even alteration with odd-chain length compounds exhibiting higher values as compared to the even-chain length ones and the tilt angles calculated from the PXRD data are higher for the even chain NABAs. These observations are relevant to developing structure-activity relationships for these amphiphiles and understand

  7. Heterologous expression of the acyl-acyl carrier protein thioesterase gene from the plant Umbellularia californica mediates polyhydroxyalkanoate biosynthesis in recombinant Escherichia coli.

    PubMed

    Rehm, B H; Steinbüchel, A

    2001-03-01

    The acyl-acyl carrier protein (ACP) thioesterase cDNA from the plant Umbellularia californica was functionally expressed in various recombinant Escherichia coli strains in order to establish a new metabolic route toward medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis from non-related carbon sources. Coexpression of the PHA synthase genes from Ralstonia eutropha and Pseudomonas aeruginosa, or only the PHA synthase gene from P. aeruginosa, respectively, showed PHA(MCL) accumulation when the type II PHA synthase from P. aeruginosa was produced. Both wild-type E. coli and various fad mutants were investigated; and only when the beta-oxidation pathway was impaired PHA(MCL) accumulation from gluconate was observed, contributing to about 6% of cellular dry weight. Thus coexpression of type II PHA synthase gene with cDNA encoding the medium-chain acyl-ACP thioesterase from U. californica established a new PHA(MCL) biosynthesis pathway, connecting fatty acid de novo biosynthesis with fatty acid beta-oxidation, using a non-related carbon source.

  8. In silico prediction of acyl glucuronide reactivity

    NASA Astrophysics Data System (ADS)

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

    2011-11-01

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

  9. Intramolecular Friedel-Crafts Acylation Reaction Promoted by 1,1,1,3,3,3-Hexafluoro-2-propanol.

    PubMed

    Motiwala, Hashim F; Vekariya, Rakesh H; Aubé, Jeffrey

    2015-11-06

    Simple dissolution of an arylalkyl acid chloride in 1,1,1,3,3,3-hexafluoro-2-propanol promotes an intramolecular Friedel-Crafts acylation without additional catalysts or reagents. This reaction is operationally trivial in both execution and product isolation (only requiring concentration followed by purification) and accommodates a broad range of substrates. Preliminary studies that bear upon potential reaction mechanisms are reported.

  10. Exploration of an imide capture/N,N-acyl shift sequence for asparagine native peptide bond formation.

    PubMed

    Mhidia, Reda; Boll, Emmanuelle; Fécourt, Fabien; Ermolenko, Mikhail; Ollivier, Nathalie; Sasaki, Kaname; Crich, David; Delpech, Bernard; Melnyk, Oleg

    2013-06-15

    Imide capture of a C-terminal peptidylazide with a side-chain thioacid derivative of an N-terminally protected aspartyl peptide leads to the formation of an imide bond bringing the two peptide ends into close proximity. Unmasking of the N(α) protecting group and intramolecular acyl migration results in the formation of a native peptide bond to asparagine.

  11. Downregulation of carnitine acyl-carnitine translocase by miRNAs 132 and 212 amplifies glucose-stimulated insulin secretion.

    PubMed

    Soni, Mufaddal S; Rabaglia, Mary E; Bhatnagar, Sushant; Shang, Jin; Ilkayeva, Olga; Mynatt, Randall; Zhou, Yun-Ping; Schadt, Eric E; Thornberry, Nancy A; Muoio, Deborah M; Keller, Mark P; Attie, Alan D

    2014-11-01

    We previously demonstrated that micro-RNAs (miRNAs) 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of miRNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including nonfuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT; Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for β-oxidation. Small interfering RNA-mediated knockdown of CACT in β-cells led to the accumulation of fatty acyl-carnitines and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from rat insulinoma β-cells (INS-1) as well as primary mouse islets. The effect on INS-1 cells was augmented in response to suppression of CACT. A nonhydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that β-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine-mediated regulation of IS.

  12. Downregulation of Carnitine Acyl-Carnitine Translocase by miRNAs 132 and 212 Amplifies Glucose-Stimulated Insulin Secretion

    PubMed Central

    Soni, Mufaddal S.; Rabaglia, Mary E.; Bhatnagar, Sushant; Shang, Jin; Ilkayeva, Olga; Mynatt, Randall; Zhou, Yun-Ping; Schadt, Eric E.; Thornberry, Nancy A.; Muoio, Deborah M.; Keller, Mark P.

    2014-01-01

    We previously demonstrated that micro-RNAs (miRNAs) 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of miRNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including nonfuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT; Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for β-oxidation. Small interfering RNA–mediated knockdown of CACT in β-cells led to the accumulation of fatty acyl-carnitines and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from rat insulinoma β-cells (INS-1) as well as primary mouse islets. The effect on INS-1 cells was augmented in response to suppression of CACT. A nonhydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that β-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine–mediated regulation of IS. PMID:24969106

  13. Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis*

    PubMed Central

    Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

    2016-01-01

    Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe45 in helix II and Phe18 in the α1α2 loop and a hydrogen bonding between Ser15 in helix I and Ile20 in the α1α2 loop, resulting in its high thermal stability. Phe45-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser58 in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains. PMID:26631734

  14. Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis.

    PubMed

    Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

    2016-01-22

    Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3-17), helix II (residues 39-53), helix III (residues 60-64), and helix IV (residues 68-78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe(45) in helix II and Phe(18) in the α1α2 loop and a hydrogen bonding between Ser(15) in helix I and Ile(20) in the α1α2 loop, resulting in its high thermal stability. Phe(45)-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser(58) in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.

  15. Tunable Oleo-Furan Surfactants by Acylation of Renewable Furans

    PubMed Central

    2016-01-01

    An important advance in fluid surface control was the amphiphilic surfactant composed of coupled molecular structures (i.e., hydrophilic and hydrophobic) to reduce surface tension between two distinct fluid phases. However, implementation of simple surfactants has been hindered by the broad range of applications in water containing alkaline earth metals (i.e., hard water), which disrupt surfactant function and require extensive use of undesirable and expensive chelating additives. Here we show that sugar-derived furans can be linked with triglyceride-derived fatty acid chains via Friedel–Crafts acylation within single layer (SPP) zeolite catalysts. These alkylfuran surfactants independently suppress the effects of hard water while simultaneously permitting broad tunability of size, structure, and function, which can be optimized for superior capability for forming micelles and solubilizing in water. PMID:27924310

  16. 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.

  17. 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.

  18. Acyl anion free N-heterocyclic carbene organocatalysis.

    PubMed

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

    2013-06-21

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

  19. Highly acylated (acetylated and/or p-coumaroylated) native lignins from diverse herbaceous plants.

    PubMed

    del Río, José C; Rencoret, Jorge; Marques, Gisela; Gutiérrez, Ana; Ibarra, David; Santos, J Ignacio; Jiménez-Barbero, Jesús; Zhang, Liming; Martínez, Angel T

    2008-10-22

    The structure of lignins isolated from the herbaceous plants sisal ( Agave sisalana), kenaf ( Hibiscus cannabinus), abaca ( Musa textilis) and curaua ( Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage) methods. The analyses demonstrate that the structure of the lignins from these plants is highly remarkable, being extensively acylated at the gamma-carbon of the lignin side chain (up to 80% acylation) with acetate and/or p-coumarate groups and preferentially over syringyl units. Whereas the lignins from sisal and kenaf are gamma-acylated exclusively with acetate groups, the lignins from abaca and curaua are esterified with acetate and p-coumarate groups. The structures of all these highly acylated lignins are characterized by a very high syringyl/guaiacyl ratio, a large predominance of beta- O-4' linkages (up to 94% of all linkages), and a strikingly low proportion of traditional beta-beta' linkages, which indeed are completely absent in the lignins from abaca and curaua. The occurrence of beta-beta' homocoupling and cross-coupling products of sinapyl acetate in the lignins from sisal and kenaf indicates that sinapyl alcohol is acetylated at the monomer stage and that, therefore, sinapyl acetate should be considered as a real monolignol involved in the lignification reactions.

  20. High acyl gellan as an emulsion stabilizer.

    PubMed

    Vilela, Joice Aline Pires; da Cunha, Rosiane Lopes

    2016-03-30

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

  1. Acylated pregnane glycosides from Caralluma russeliana.

    PubMed

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

    2007-05-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    1999-11-01

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

  4. Impact of substrate contamination with mycotoxins, heavy metals and pesticides on growth performance and composition of black soldier fly larvae (Hermetia illucens) for use in the feed and food value chain.

    PubMed

    Purschke, Benedict; Scheibelberger, Rafaela; Axmann, Sonja; Adler, Andreas; Jäger, Henry

    2017-02-26

    Edible insects have emerged as an alternative and sustainable source of high-quality, animal-derived protein and fat for livestock production or direct human nutrition. During production of insects, substrate quality is a key parameter to assure optimal insect biomass gain as well as the safety of feed and food derived from commercially reared insects. Therefore, the influence of a realistic substrate contamination scenario on growth performance and accumulation behaviour of black soldier fly larvae (BSFL, Hermetia illucens L.) was investigated. Newly hatched larvae were fed on a corn-based substrate spiked with heavy metals (As, Cd, Cr, Hg, Ni, Pb), mycotoxins (aflatoxin B1/B2/G2, deoxynivalenol, ochratoxin A, zearalenone) and pesticides (chlorpyrifos, chlorpyrifos-methyl, pirimiphos-methyl) under defined breeding conditions (10 d, 28 °C, 67 % RH). The extent of contaminants bio-accumulation in the larval tissue as well as the effect on growing determinants was examined. The applied heavy metal substrate contamination was shown to impair larval growing indicated by significantly lower post-trial larval mass and feed conversion ratio (FCR). Cadmium and lead accumulation factors of 9 and 2, respectively, were determined, while concentration of other heavy metals in the larvae remained below the initial substrate concentration. In contrast, mycotoxins and pesticides have neither been accumulated in the larval tissue nor significantly affected the growing determinants in comparison to the control. The use of BSFL as livestock feed requires contaminant monitoring - especially for cadmium and lead - in the substrates as well as in feedstuff containing BSFL to ensure feed and food safety along the value chain.

  5. Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

    PubMed

    Christensen, H; Martin, M T; Waley, S G

    1990-03-15

    The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.

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

    PubMed Central

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

    2002-01-01

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

  7. 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.

  8. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  9. 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.

  10. Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival

    PubMed Central

    Boll, Joseph M.; Tucker, Ashley T.; Klein, Dustin R.; Beltran, Alexander M.; Brodbelt, Jennifer S.; Davies, Bryan W.

    2015-01-01

    ABSTRACT Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxLAb (A. baumannii LpxL) and LpxMAb (A. baumannii LpxM), which transfer one and two lauroyl (C12:0) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxMAb-dependent acylation of lipid A is essential for A. baumannii desiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxMAb-dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections. PMID:25991684

  11. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    PubMed Central

    Bansal, Sunil; Durrett, Timothy P.

    2016-01-01

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

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

    SciTech Connect

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

    2011-02-18

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

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

    SciTech Connect

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

    2011-12-31

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

  14. Modifications of the acyl-d-alanyl-d-alanine terminus affecting complex-formation with vancomycin

    PubMed Central

    Nieto, M.; Perkins, H. R.

    1971-01-01

    Vancomycin forms complexes with peptides terminating in d-alanyl-d-alanine that are analogous to the biosynthetic precursors of bacterial mucopeptides. The specificity of complex-formation has been studied by means of many synthetic peptides, prepared by both solid-phase and conventional methods. The following conclusions can be drawn: (a) three amide linkages are required to form a stable complex; (b) the terminal carboxyl group must be free; (c) the carboxyl terminal and subterminal residues must be either glycine or of the d-configuration; (d) the size of the side chain in these residues greatly influences the affinity for vancomycin, a methyl group being the optimum in each case; (e) the nature of the side chain in the third and fourth residues has a smaller effect on complex-formation, but an l-configuration was somewhat better than a d-configuration in the third position. In addition to acyl-d-alanyl-d-alanine, other peptides that occur in bacterial cell walls will combine with vancomycin, although less strongly, e.g. acyl-d-alanyl-d-α-amino acid (where the terminal d-residue may form the cross-link in mucopeptide structure) and acyl-l-alanyl-d-glutamylglycine (a sequence found in the mucopeptide of Micrococcus lysodeikticus and related organisms). These results throw some light on the specificity of the uptake of vancomycin by living bacteria. PMID:5124386

  15. The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

    PubMed

    Griffiths, G; Stobart, A K; Stymne, S

    1985-09-01

    Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate

  16. A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

    PubMed Central

    Mareš, Jan; Hájek, Jan; Urajová, Petra; Kopecký, Jiří; Hrouzek, Pavel

    2014-01-01

    A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides. PMID:25369527

  17. Synthesis and characterization of O-acylated-ω-hydroxy fatty acids as skin-protecting barrier lipids.

    PubMed

    Pérez, B; Dahlgaard, S E; Bulsara, P; Rawlings, A V; Jensen, M M; Dong, M; Glasius, M; Clarke, M J; Guo, Z

    2017-03-15

    A series of O-acylated-ω-hydroxy fatty acids (Acyl acids) of up to 34 carbons were synthesized and characterized through DSC, FTIR and Langmuir isotherm measurements to identify potential replacements to petrolatum, a highly used occlusive technology that if unrefined, it can potentially be classified as carcinogenic. Fourier transform infrared spectroscopy studies demonstrated that long acyl acids engender orthorhombic packing; packing behavior that is predominant in the lipid matrix of healthy stratum corneum, the outmost layer of the skin. In addition, Differential Scanning Calorimetry (DSC) and Langmuir isotherm studies suggested that the length of the hydrocarbon chain and the position of the ester bond influence the molecular organization of the acyl acids. For instance, 16-(tetradecanoyloxy)hexadecanoic acid (30 carbons) displayed a higher melting point (mp=68°C) than 10-(stearoyloxy)decanoic acid (28 carbons; mp=63°C) and 10-(tetradecanoyloxy)decanoic acid (24 carbons; mp=55°C) according to DSC. Moreover, Langmuir isotherm studies showed that mixtures of acyl acid with distearoylphosphatidylcholine improved packing behavior. Finally, Water Vapor Transmission Rate (WVTR) measurements showed that the compounds in fact decrease WVTR compared to untreated control (P<0.001) which demonstrates the potential of these ingredients as occlusive technologies to combat skin barrier diseases.

  18. Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis.

    PubMed

    Xue, Qiao; Cui, Ying-Lu; Zheng, Qing-Chuan; Zhang, Hong-Xing

    2016-05-01

    BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

  19. A Novel Fatty Acyl-CoA Synthetase Is Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis[C][W

    PubMed Central

    de Azevedo Souza, Clarice; Kim, Sung Soo; Koch, Stefanie; Kienow, Lucie; Schneider, Katja; McKim, Sarah M.; Haughn, George W.; Kombrink, Erich; Douglas, Carl J.

    2009-01-01

    Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by self-fertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein–ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes. PMID:19218397

  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