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Sample records for acyl chain substrates

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

    PubMed

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

    2013-04-01

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

  2. Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.

    PubMed

    Dridi, Kaouthar; Amara, Sawsan; Bezzine, Sofiane; Rodriguez, Jorge A; Carrière, Frédéric; Gaussier, Hélène

    2013-07-01

    Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions. PMID:24046870

  3. Lipid Acyl Chain Remodeling in Yeast

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2015-01-01

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

  6. Mammalian long-chain acyl-CoA synthetases.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2008-05-01

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

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

    PubMed

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

    2001-06-26

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

  8. Naphthalene Derivatives Induce Acyl Chain Interdigitation in Dipalmitoylphosphatidylcholine Bilayers.

    PubMed

    Kamal, Md Arif; Raghunathan, V A

    2016-01-14

    The interdigitated phase of the lipid bilayer results when acyl chains from opposing monolayers fully interpenetrate such that the terminal methyl groups of the respective lipid chains are located at the interfacial region on the opposite sides of the bilayer. Usually, chain interdigitation is not encountered in a symmetric chain phosphatidylcholine (PC) membrane but can be induced under certain special conditions. In this article, we elucidate the contribution of small amphiphatic molecules in altering the physical properties of a symmetric chain PC bilayer membrane, which results in acyl chain interdigitation. Using small-angle X-ray scattering (SAXS), we have carried out a systematic investigation of the physical interactions of three naphthalene derivatives containing hydroxyl groups: β-naphthol, 2,3-dihydroxynaphthalene, and 2,7-dihydroxynaphthalene, with dipalmitoylphosphatidylcholine (DPPC) bilayers. On the basis of the diffraction patterns, we have determined the temperature-composition phase diagrams of these binary mixtures. The present study not only enables us to gain insight into the role played by small molecules in altering the packing arrangement of the acyl chains of the constituting PC lipids of the bilayer but also brings to light some important features that have not yet been reported hitherto. One such feature is the stabilization of the enigmatic asymmetric ripple phase over a wide temperature and concentration range. The results presented here strongly point toward a clear correlation between chain interdigitation and the stability of the ripple phase.

  9. Diacylglycerol Kinase ϵ Is Selective for Both Acyl Chains of Phosphatidic Acid or Diacylglycerol*

    PubMed Central

    Lung, Michael; Shulga, Yulia V.; Ivanova, Pavlina T.; Myers, David S.; Milne, Stephen B.; Brown, H. Alex; Topham, Matthew K.; Epand, Richard M.

    2009-01-01

    The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase ϵ (DGKϵ) has an important role in this cycle. DGKϵ is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGKϵ by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGKϵ does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGKϵ regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGKϵ remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell. PMID:19744926

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

    PubMed

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

    2015-09-18

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

  11. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase

    PubMed Central

    Tran, Timothy H.; Hsiao, Yu-Shan; Jo, Jeanyoung; Chou, Chi-Yuan; Dietrich, Lars E.P.; Walz, Thomas; Tong, Liang

    2014-01-01

    Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds 1–6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase (ACC) is a target for drug discovery against diabetes, cancer and other diseases 7–9. We report here the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kD), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active toward short- and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase (LCC). The holoenzyme is a homo-hexamer with molecular weight of 720 kD. The 3.0 Å crystal structure of Mycobacterium avium subspecies paratuberculosis LCC (MapLCC) holoenzyme revealed an architecture that is strikingly different compared to those of related biotin-dependent carboxylases 10,11. In addition, the domains of each monomer have no direct contacts with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources. PMID:25383525

  12. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

    PubMed

    Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

    2014-09-26

    Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. PMID:25111069

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  15. Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.

    PubMed

    Voelker, T A; Jones, A; Cranmer, A M; Davies, H M; Knutzon, D S

    1997-06-01

    In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented. PMID:9193098

  16. 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.

  17. Thioesterase superfamily member 2 (Them2)/acyl-CoA thioesterase 13 (Acot13): a homotetrameric hotdog fold thioesterase with selectivity for long-chain fatty acyl-CoAs

    PubMed Central

    Wei, Jie; Kang, Hye Won; Cohen, David E.

    2011-01-01

    Them2 (thioesterase superfamily member 2) is a 140-aminoacid protein of unknown biological function that comprises a single hotdog fold thioesterase domain. On the basis of its putative association with mitochondria, accentuated expression in oxidative tissues and interaction with StarD2 (also known as phosphatidylcholine-transfer protein, PC-TP), a regulator of fatty acid metabolism, we explored whether Them2 functions as a physiologically relevant fatty acyl-CoA thioesterase. In solution, Them2 formed a stable homotetramer, which denatured in a single transition at 59.3 °C. Them2 exhibited thioesterase activity for medium- and long-chain acyl-CoAs, with Km values that decreased exponentially as a function of increasing acyl chain length. Steady-state kinetic parameters for Them2 were characteristic of long-chain mammalian acyl-CoA thioesterases, with minimal values of Km and maximal values of kcat/Km observed for myristoyl-CoA and palmitoyl-CoA. For these acyl-CoAs, substrate inhibition was observed when concentrations approached their critical micellar concentrations. The acyl-CoA thioesterase activity of Them2 was optimized at physiological temperature, ionic strength and pH. For both myristoyl-CoA and palmitoyl-CoA, the addition of StarD2 increased the kcat of Them2. Enzymatic activity was decreased by the addition of phosphatidic acid/phosphatidylcholine small unilamellar vesicles. Them2 expression, which was most pronounced in mouse heart, was associated with mitochondria and was induced by activation of PPARα (peroxisome-proliferator-activated receptor α). We conclude that, under biological conditions, Them2 probably functions as a homotetrameric long-chain acyl-CoA thioesterase. Accordingly, Them2 has been designated as the 13th member of the mammalian acyl-CoA thioesterase family, Acot13. PMID:19405909

  18. 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

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

    PubMed Central

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

    2014-01-01

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

  20. 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

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

    PubMed

    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.

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

    PubMed

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

    2000-05-01

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

  3. Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids*

    PubMed Central

    Vats, Archana; Singh, Anil Kumar; Mukherjee, Raju; Chopra, Tarun; Ravindran, Madhu Sudhan; Mohanty, Debasisa; Chatterji, Dipankar; Reyrat, Jean-Marc; Gokhale, Rajesh S.

    2012-01-01

    Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs. PMID:22798073

  4. Carbohydrate conformation and lipid condensation in monolayers containing glycosphingolipid Gb3: influence of acyl chain structure.

    PubMed

    Watkins, Erik B; Gao, Haifei; Dennison, Andrew J C; Chopin, Nathalie; Struth, Bernd; Arnold, Thomas; Florent, Jean-Claude; Johannes, Ludger

    2014-09-01

    Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3's influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3's capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment's impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.

  5. 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

  6. Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase.

    PubMed Central

    Fraisl, Peter; Forss-Petter, Sonja; Zigman, Mihaela; Berger, Johannes

    2004-01-01

    It has been suggested that a gene termed bubblegum (Bgm), encoding an acyl-CoA synthetase, could be involved in the pathogenesis of the inherited neurodegenerative disorder X-ALD (X-linked adrenoleukodystrophy). The precise function of the ALDP (ALD protein) still remains unclear. Aldp deficiency in mammals and Bgm deficiency in Drosophila led to accumulation of VLCFAs (very long-chain fatty acids). As a first step towards studying this interaction in wild-type versus Aldp-deficient mice, we analysed the expression pattern of the murine orthologue of the Bgm gene. In contrast with the ubiquitously expressed Ald gene, Bgm expression is restricted to the tissues that are affected by X-ALD such as brain, testis and adrenals. During mouse brain development, Bgm mRNA was first detected by Northern-blot analysis on embryonic day 18 and increased steadily towards adulthood, whereas the highest level of Ald mRNA was found on embryonic day 12 and decreased gradually during differentiation. Protein fractionation and confocal laser imaging of Bgm-green fluorescent protein fusion proteins revealed a microsomal localization that was different from peroxisomes (where Aldp is detected), endoplasmic reticulum and Golgi. Mouse Bgm showed acyl-CoA synthetase activity towards a VLCFA substrate in addition to LCFAs, and this activity was enriched in the microsomal compartment. Speculating that Bgm expression could be regulated by Ald deficiency, we compared the abundance of Bgm mRNA in wild-type and Ald knockout mice but observed no difference. Although mouse Bgm is capable of activating VLCFA, we conclude that a direct interaction between the mouse Bgm and the Aldp seems unlikely. PMID:14516277

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-11-22

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

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

    PubMed Central

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

    2013-01-01

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

  10. The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants.

    PubMed

    Sayanova, Olga; Ruiz-Lopez, Noemi; Haslam, Richard P; Napier, Johnathan A

    2012-02-01

    The role of acyl-CoA-dependent Δ6-desaturation in the heterologous synthesis of omega-3 long-chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl-CoA Δ6-desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6-desaturated acyl-CoAs, in contrast to the phospholipid-dependent Δ6-desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl-CoA Δ6-desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid-dependent Δ6-desaturase. The use of acyl-CoA-dependent Δ6-desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ-linolenic acid in total seed lipids. Expression of acyl-CoA Δ6-desaturases resulted in increased distribution of long-chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6-desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6-desaturated fatty acids. This study provides evidence for the efficacy of using acyl-CoA-dependent Δ6-desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega-3 LC-PUFAs. PMID:21902798

  11. An esterase on the outer membrane of Pseudomonas aeruginosa for the hydrolysis of long chain acyl esters.

    PubMed

    Ohkawa, I; Shiga, S; Kageyama, M

    1979-09-01

    A new esterase activity which hydrolyzes palmitoyl-CoA was found in the membrane fraction of Pseudomonas aeruginosa. All the 11 strains of P. aeruginosa tested possessed this esterase activity. The esterase was constitutive and was fully active on the intact cell bodies toward substrates in the medium. It was located on the outer membrane of the cell envelope, and was not released into the culture medium. This activity was designated as OM (outer membrane) esterase. OM esterase was solubilized from the cell envelope with EDTA-Triton X-100 and purified 690-fold. It was a minor component of the outer membrane. Its molecular weight was approximately 55,000. The activity was rather stable to heat, a wide range of pH, and treatment with detergents and organic solvents. No cofactors were required. The pH optimum of the reaction was 8.5. Among various acyl-CoAs, only long chain (C12--C18) thioesters were hydrolyzed. OM esterase also hydrolyzed some kinds of oxy-esters such as p-nitrophenyl acyl esters, monoacyl esters of sucrose and Tween 80 (polyoxyethylene sorbitan monooleate). On the other hand, triglycerides, phospholipids, or hydrophobic monoesters were not hydrolyzed at all. Thus, this enzyme seems to have specificity for long chain acyl esters with hydrophilic groups, whether thio- or oxy-ester. Mutants deficient in this esterase activity were isolated. These mutants were unable to grow on Tween 80 as a sole carbon source. This suggests a possible role of OM esterase in the utilization of acyl esters as carbon sources.

  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 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.

  14. 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.

  15. 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.

  16. Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

    PubMed

    Jordi, W; Nibbeling, R; de Kruijff, B

    1990-02-12

    The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.

  17. Evidence for the Intercalation of Lipid Acyl Chains into Polypropylene Fiber Matrices.

    PubMed

    Schadock-Hewitt, Abby J; Bruce, Terri F; Marcus, R Kenneth

    2015-09-29

    Headgroup-functionalized lipids are being developed as ligand tethers for high selectivity separations on polypropylene capillary-channeled polymer fiber stationary phases. Surface modification is affected under ambient conditions from aqueous solution. This basic methodology has promise in many areas where robust modifications are desired on hydrophobic surfaces. In order to understand the mode of adsorption of the lipid tail to the polypropylene surface, lipids labeled with the environmentally sensitive 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) fluorophore were used, with NBD covalently attached to the headgroup (NBD-PE) or the acyl chain (acyl NBD-PE) of the lipid. When modified with the acyl NBD-PE, fluorescence imaging of the fiber at excitation wavelengths increasing from 470 to 510 nm caused a 32 nm shift in emission toward the red edge of the absorption band, indicating that the NBD molecule (and thus the lipid tail) is motionally restricted. Fluorescence imaging on fibers modified with NBD-PE or the free NBD-Cl dye molecule yields no change in the emission response. The results of these imaging studies provide evidence that the acyl chain portions of the lipids intercalate into free volume of the polypropylene fiber structure, yielding a robust means of surface modification and the potential for high ligand densities. PMID:26381380

  18. Synthesis of rapeseed biodiesel using short-chained alkyl acetates as acyl acceptor.

    PubMed

    Jeong, Gwi-Taek; Park, Don-Hee

    2010-05-01

    In this study, we conducted experiments using a response surface methodology to determine the optimal reaction conditions for the enzymatic synthesis of biodiesel from rapeseed oil and short-chained alkyl acetates, such as methyl acetate or ethyl acetate, as the acyl acceptor at 40 degrees C. Based on our response surface methodology experiments, the optimal reaction conditions for the synthesis of biodiesel were as follows: methyl acetate as acyl acceptor, catalyst concentration of 16.50%, oil-to-methyl acetate molar ratio of 1:12.44, and reaction time of 19.70 h; ethyl acetate as acyl acceptor, catalyst concentration of 16.95%, oil-to-ethyl acetate molar ratio of 1:12.56, and reaction time of 19.73 h. The fatty acid ester content under the above conditions when methyl acetate and ethyl acetate were used as the acyl acceptor was 58.0% and 62.6%, respectively. The statistical method described in this study can be applied to effectively optimize the enzymatic conditions required for biodiesel production with short-chained alkyl acetates.

  19. Synthesis of rapeseed biodiesel using short-chained alkyl acetates as acyl acceptor.

    PubMed

    Jeong, Gwi-Taek; Park, Don-Hee

    2010-05-01

    In this study, we conducted experiments using a response surface methodology to determine the optimal reaction conditions for the enzymatic synthesis of biodiesel from rapeseed oil and short-chained alkyl acetates, such as methyl acetate or ethyl acetate, as the acyl acceptor at 40 degrees C. Based on our response surface methodology experiments, the optimal reaction conditions for the synthesis of biodiesel were as follows: methyl acetate as acyl acceptor, catalyst concentration of 16.50%, oil-to-methyl acetate molar ratio of 1:12.44, and reaction time of 19.70 h; ethyl acetate as acyl acceptor, catalyst concentration of 16.95%, oil-to-ethyl acetate molar ratio of 1:12.56, and reaction time of 19.73 h. The fatty acid ester content under the above conditions when methyl acetate and ethyl acetate were used as the acyl acceptor was 58.0% and 62.6%, respectively. The statistical method described in this study can be applied to effectively optimize the enzymatic conditions required for biodiesel production with short-chained alkyl acetates. PMID:19802734

  20. Lipid Gymnastics: Evidence of Complete Acyl Chain Reversal in Oxidized Phospholipids from Molecular Simulations

    PubMed Central

    Khandelia, Himanshu; Mouritsen, Ole G.

    2009-01-01

    In oxidative environments, biomembranes contain oxidized lipids with short, polar acyl chains. Two stable lipid oxidation products are PoxnoPC and PazePC. PoxnoPC has a carbonyl group, and PazePC has an anionic carboxyl group pendant at the end of the short, oxidized acyl chain. We have used MD simulations to explore the possibility of complete chain reversal in OXPLs in POPC-OXPL mixtures. The polar AZ chain of PazePC undergoes chain reversal without compromising the lipid bilayer integrity at concentrations up to 25% OXPL, and the carboxyl group points into the aqueous phase. Counterintuitively, the perturbation of overall membrane structural and dynamic properties is stronger for PoxnoPC than for PazePC. This is because of the overall condensing and ordering effect of sodium ions bound strongly to the lipids in the PazePC simulations. The reorientation of AZ chain is similar for two different lipid force fields. This work provides the first molecular evidence of the “extended lipid conformation” in phospholipid membranes. The chain reversal of PazePC lipids decorates the membrane interface with reactive, negatively charged functional groups. Such chain reversal is likely to exert a profound influence on the structure and dynamics of biological membranes, and on membrane-associated biological processes. PMID:19348756

  1. 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}.

  2. 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.

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

    DOE PAGES

    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 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

  4. A thiolate anion buried within the hydrocarbon ruler perturbs PagP lipid acyl chain selection.

    PubMed

    Khan, M Adil; Moktar, Joel; Mott, Patrick J; Bishop, Russell E

    2010-03-23

    The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP exhibits remarkable selectivity because its binding pocket for lipid acyl chains excludes those differing in length from palmitate by a solitary methylene unit. This narrow detergent-binding hydrophobic pocket buried within the eight-strand antiparallel beta-barrel is known as the hydrocarbon ruler. Gly88 lines the acyl chain binding pocket floor, and its substitution can raise the floor to correspondingly shorten the selected acyl chain. An aromatic exciton interaction between Tyr26 and Trp66 provides an intrinsic spectroscopic probe located immediately adjacent to Gly88. The Gly88Cys PagP enzyme was engineered to function as a dedicated myristoyltransferase, but the mutant enzyme instead selected both myristoyl and pentadecanoyl groups, was devoid of the exciton, and displayed a 21 degrees C reduction in thermal stability. We now demonstrate that the structural perturbation results from a buried thiolate anion attributed to suppression of the Cys sulfhydryl group pK(a) from 9.4 in aqueous solvent to 7.5 in the hydrocarbon ruler microenvironment. The Cys thiol is sandwiched at the interface between a nonpolar and a polar beta-barrel interior milieu, suggesting that local electrostatics near the otherwise hydrophobic hydrocarbon ruler pocket serve to perturb the thiol pK(a). Neutralization of the Cys thiolate anion by protonation restores wild-type exciton and thermal stability signatures to Gly88Cys PagP, which then functions as a dedicated myristoyltransferase at pH 7. Gly88Cys PagP assembled in bacterial membranes recapitulates lipid A myristoylation in vivo. Hydrocarbon ruler-exciton coupling in PagP thus reveals a thiol-thiolate ionization mechanism for modulating lipid acyl chain selection.

  5. Protease-catalyzed peptide synthesis using inverse substrates: the influence of reaction conditions on the trypsin acyl transfer efficiency.

    PubMed

    Schellenberger, V; Jakubke, H D; Zapevalova, N P; Mitin, Y V

    1991-06-01

    Benzyloxycarbonyl-L-alanine p-guanidinophenyl ester behaves as a trypsin "inverse substrate," i.e., a cationic center is included in the leaving group instead of being in the acyl moiety. Using this substrate as an acyl donor, trypsin catalyzes the synthesis of peptide bonds that cannot be split by this enzyme. An optimal acyl transfer efficiency was achieved between pH 8 and 9 at 30 degrees C.The addition of as much as 50% cosolvent was shown to be of minor influence on the acyl transfer efficiency, whereas the reaction velocity decreases by more than one order of magnitude. The efficiency of H-Leu-NH(2) and H-Val-NH(2) in deacylation is almost the same for "inverse" and normal type substrates. PMID:18600704

  6. 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

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

    PubMed

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

    2016-08-01

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

  8. 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

  9. 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

    2016-01-01

    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

  10. 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.

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

    PubMed

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

    2011-07-15

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

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

    PubMed

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

    1990-03-10

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

  13. 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

  14. 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.

  15. 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

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

    PubMed Central

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

    2016-01-01

    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 isooctyl 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

  17. Inhibition of Long Chain Fatty Acyl-CoA Synthetase (ACSL) and Ischemia Reperfusion Injury

    PubMed Central

    Prior, Allan M.; Zhang, Man; Blakeman, Nina; Datta, Palika; Pham, Hung; Young, Lindon H.; Weis, Margaret T.; Hua, Duy H.

    2014-01-01

    Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion. PMID:24480468

  18. Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model.

    PubMed

    Tai, Akihiro; Goto, Satomi; Ishiguro, Yutaka; Suzuki, Kazuko; Nitoda, Teruhiko; Yamamoto, Itaru

    2004-02-01

    A series of novel lipophilic vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids possessing a branched-acyl chain of varying length from C(8) to C(16) (6-bAcyl-AA-2G), were evaluated as topical prodrugs of ascorbic acid (AA) with transdermal activity in a human living skin equivalent model. The permeability of 6-bAcyl-AA-2G was compared with those of the derivatives having a straight-acyl chain (6-sAcyl-AA-2G). Out of 10 derivatives of 6-sAcyl-AA-2G and 6-bAcyl-AA-2G, 6-sDode-AA-2G and 6-bDode-AA-2G exhibited most excellent permeability in this model. Measurement of the metabolites permeated from the skin model suggested that 6-bDode-AA-2G was mainly hydrolyzed via 6-O-acyl AA to AA by tissue enzymes, while 6-sDode-AA-2G was hydrolyzed via 2-O-alpha-D-glucopyranosyl-L-ascorbic acid to AA. The former metabolic pathway seems to be advantageous for a readily available source of AA, because 6-O-acyl AA, as well as AA, is able to show vitamin C activity.

  19. Ontogenic development of the fatty acyl chain composition of the turkey (Meleagris gallopavo) pectoralis superficialis muscle membranes: an allometric approach.

    PubMed

    Szabó, A; Fébel, Hedvig; Horn, P; Andrássy-Baka, G; Bázár, Gy; Romvári, R

    2006-06-01

    The growth-associated development of the m. pectoralis superficialis (MPS) phospholipid (PL) and triacylglycerol (TAG) fatty acyl (FA) chain composition was determined in BUT8 meat-type turkeys. Samples (3 d, 8, 12, 16 and 20 wk) of each 6 males were analysed by lipid fractionation and subsequent gas chromatography. Results were interpreted on an allometric basis. The MPS mass increased linearly (MPS weight = 0.2787 BW- 123.67; R2 = 0.9935, P<0.001, n = 30). In the total phospholipids 62-63% unsaturated fatty acids were found irrespective of the diet. A negative allometric alteration was found for the total saturated acyl chains (B = -0.012), while a positive value for the calculated unsaturation index (B = 0.026) was obtained. Within the PUFA chains, the n3- n6 balance was markedly changed, on the favour of the n3 fatty acyl chains, namely competitive allometric trends were found for the total n3 (B = 0.087) and n6 (B = 0.032) fatty acid groups. The alterations of the TAG FA chain composition were diet-dependent. The serum creatine kinase activity increased by over one class of magnitude during the trial. The allometric approach was found to be powerful in the characterization of the basic, non diet-dependent ontogenic alterations of the phospholipid fatty acyl chain composition.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    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

  2. 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.

  3. 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

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

    PubMed

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

    2016-05-03

    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.

  5. Density fluctuations in saturated phospholipid bilayers increase as the acyl-chain length decreases.

    PubMed Central

    Ipsen, J H; Jørgensen, K; Mouritsen, O G

    1990-01-01

    A systematic computer simulation study is conducted for a model of the main phase transition of fully hydrated saturated diacyl phosphatidylcholine bilayers (DMPC, DPPC, and DSPC). With particular focus on the fluctuation effects on the thermal properties in the transition region, the study yields data for the specific heat, the lateral compressibility, and the lipid-domain size distribution. Via a simple model assumption the transmembrane passive ion permeability is derived from the lipid-domain interfacial measure. A comparative analysis of the various data shows, in agreement with a number of experiments, that the lateral density fluctuations and hence the response functions increase as the acyl-chain length is decreased. Images FIGURE 2 PMID:2291936

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

    PubMed

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

    2014-03-01

    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 (13)C-labeled diC8PC ((methyl-(13)C)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-(13)C)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.

  7. 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.

  8. 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.

  9. Dual mesomorphic assemblage of chitin normal acylates and rapid enthalpy relaxation of their side chains.

    PubMed

    Teramoto, Yoshikuni; Miyata, Tomoya; Nishio, Yoshiyuki

    2006-01-01

    Chitin derivatives having normalacyl groups (C(n)H(2n-1)O-; n = 4-20) were synthesized with pyridine, p-toluenesulfonyl chloride, and normal alkanoic acid in an N,N-dimethylacetamide-lithium chloride homogeneous system. The products (C(n)-ACs; degree of acyl substitution, DS = 1.7-1.9) showed an n-dependent thermal transition behavior: no evident transition (n = 4-10), a glass transition (n = 12 and 14), and a pseudo-first-order phase transition (n = 16-20), the latter two occurring usually below room temperature when examined by differential scanning calorimetry. Wide-angle X-ray diffractometry (WAXD) at 20 degrees C displayed a sharp diffraction peak (2theta = 2 degrees -7 degrees ) and a diffuse halo (2theta approximately 20 degrees ) for the respective C(n)-ACs. The former d-spacing (1.5-3.6 nm) increased with an increase in n to yield two stages of mutually different increasing rates, which reflects a systematic n-dependence of the period of a layered structure of the main chains. The molecular assembly of C(n)-ACs exhibited "dual mesomorphy"; nematic ordering for the semirigid carbohydrate trunk and smectic one for the flexible side chains. On the other hand, WAXD profiles of C(n)-ACs (n = 14-18) indicated almost no temperature dependence from -150 to +220 degrees C. Therefore, it was reasonably assumed that the pseudo-first-order transition observed in thermograms of C(n)-ACs (n = 16-20) was due to the enthalpy relaxation of the side-chain assemblage. An insight was provided into the kinetics of the characteristic aging behavior as a liquid-crystalline glass, in comparison with the corresponding data for other noncrystalline macromolecules.

  10. Modified acyl-ACP desaturase

    DOEpatents

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

    1998-01-06

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

  11. Modified Acyl-ACP desaturase

    DOEpatents

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

    1999-03-30

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

  12. The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

    PubMed

    Maeda, Isamu; Delessert, Syndie; Hasegawa, Seiko; Seto, Yoshiaki; Zuber, Sophie; Poirier, Yves

    2006-04-28

    The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

    Saraswathi, Viswanathan; Hasty, Alyssa H.

    2009-01-01

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

  15. Altering the sphingolipid acyl chain composition prevents LPS/GLN-mediated hepatic failure in mice by disrupting TNFR1 internalization

    PubMed Central

    Ali, M; Fritsch, J; Zigdon, H; Pewzner-Jung, Y; Schütze, S; Futerman, A H

    2013-01-01

    The involvement of ceramide in death receptor-mediated apoptosis has been widely examined with most studies focusing on the role of ceramide generated from sphingomyelin hydrolysis. We now analyze the effect of the ceramide acyl chain length by studying tumor necrosis factor α receptor-1 (TNFR1)-mediated apoptosis in a ceramide synthase 2 (CerS2) null mouse, which cannot synthesize very-long acyl chain ceramides. CerS2 null mice were resistant to lipopolysaccharide/galactosamine-mediated fulminant hepatic failure even though TNFα secretion from macrophages was unaffected. Cultured hepatocytes were also insensitive to TNFα-mediated apoptosis. In addition, in both liver and in hepatocytes, caspase activities were not elevated, consistent with inhibition of TNFR1 pro-apoptotic signaling. In contrast, Fas receptor activation resulted in the death of CerS2 null mice. Caspase activation was blocked because of the inability of CerS2 null mice to internalize the TNFR1; whereas Fc-TNFα was internalized to a perinuclear region in hepatocytes from wild-type mice, no internalization was detected in CerS2 null mice. Our results indicate that altering the acyl chain composition of sphingolipids inhibits TNFR1 internalization and inhibits selective pro-apoptotic downstream signaling for apoptosis. PMID:24263103

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

    PubMed

    Miklaszewska, Magdalena; Banaś, Antoni

    2016-08-01

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

  17. 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.

  18. 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.

  19. 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. PMID:25788017

  20. Acyl chain-dependent effect of lysophosphatidylcholine on endothelium-dependent vasorelaxation.

    PubMed

    Rao, Shailaja P; Riederer, Monika; Lechleitner, Margarete; Hermansson, Martin; Desoye, Gernot; Hallström, Seth; Graier, Wolfgang F; Frank, Saša

    2013-01-01

    Previously we identified palmitoyl-, oleoyl-, linoleoyl-, and arachidonoyl-lysophosphatidylcholine (LPC 16:0, 18:1, 18:2 and 20:4) as the most prominent LPC species generated by endothelial lipase (EL). In the present study, we examined the impact of those LPC on acetylcholine (ACh)- induced vascular relaxation. All tested LPC attenuated ACh-induced relaxation, measured ex vivo, using mouse aortic rings and wire myography. The rank order of potency was as follows: 18:2>20:4>16:0>18:1. The attenuating effect of LPC 16:0 on relaxation was augmented by indomethacin-mediated cyclooxygenase (COX)-inhibition and CAY10441, a prostacyclin (PGI2)- receptor (IP) antagonist. Relaxation attenuated by LPC 20:4 and 18:2 was improved by indomethacin and SQ29548, a thromboxane A2 (TXA2)- receptor antagonist. The effect of LPC 20:4 could also be improved by TXA2- and PGI2-synthase inhibitors. As determined by EIA assays, the tested LPC promoted secretion of PGI2, TXA2, PGF2α, and PGE2, however, with markedly different potencies. LPC 16:0 was the most potent inducer of superoxide anion production by mouse aortic rings, followed by LPC 18:2, 20:4 and 18:1, respectively. The strong antioxidant tempol recovered relaxation impairment caused by LPC 18:2, 18:1 and 20:4, but not by LPC 16:0. The tested LPC attenuate ACh-induced relaxation through induction of proconstricting prostanoids and superoxide anions. The potency of attenuating relaxation and the relative contribution of underlying mechanisms are strongly related to LPC acyl-chain length and degree of saturation. PMID:23741477

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

    PubMed

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

    2016-09-13

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

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

    PubMed

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

    2016-09-13

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

  3. Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

    PubMed

    Hofbauer, Harald F; Schopf, Florian H; Schleifer, Hannes; Knittelfelder, Oskar L; Pieber, Bartholomäus; Rechberger, Gerald N; Wolinski, Heimo; Gaspar, Maria L; Kappe, C Oliver; Stadlmann, Johannes; Mechtler, Karl; Zenz, Alexandra; Lohner, Karl; Tehlivets, Oksana; Henry, Susan A; Kohlwein, Sepp D

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

  4. 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.

  5. Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation.

    PubMed

    Antharam, Vijay C; Farver, R Suzanne; Kuznetsova, Anna; Sippel, Katherine H; Mills, Frank D; Elliott, Douglas W; Sternin, Edward; Long, Joanna R

    2008-11-01

    Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and (31)P and (2)H solid-state NMR spectroscopy. SP-B(59-80) forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B(59-80) in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B(59-80); in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B(59-80) penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL(4), a peptide mimetic of SP-B which was originally designed using SP-B(59-80) as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment. PMID:18694722

  6. 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.

  7. Identification of Regiospecific Isomers of Diricinoleoyl-acyl-glycerols containing one non-ricinoleoyl chain in Castor Oil by ESI-MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    HPLC fractions of diricinoleoyl-acyl-glycerols containing one non-ricinoleoyl chain from castor oil were used to identify the regiospecific location of this non-ricinoleoyl chain on the glycerol backbone using electrospray ionization-MS3 of lithium adducts. The regiospecific ions used were from...

  8. 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

  9. Physiological Consequences of Compartmentalized Acyl-CoA Metabolism*

    PubMed Central

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

    2015-01-01

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

  10. 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. PMID:27318965

  11. Gating of the Mitochondrial Permeability Transition Pore by Long Chain Fatty Acyl Analogs in Vivo*

    PubMed Central

    Samovski, Dmitri; Kalderon, Bella; Yehuda-Shnaidman, Einav; Bar-Tana, Jacob

    2010-01-01

    The role played by long chain fatty acids (LCFA) in promoting energy expenditure is confounded by their dual function as substrates for oxidation and as putative classic uncouplers of mitochondrial oxidative phosphorylation. LCFA analogs of the MEDICA (MEthyl-substituted DICarboxylic Acids) series are neither esterified into lipids nor β-oxidized and may thus simulate the uncoupling activity of natural LCFA in vivo, independently of their substrate role. Treatment of rats or cell lines with MEDICA analogs results in low conductance gating of the mitochondrial permeability transition pore (PTP), with 10–40% decrease in the inner mitochondrial membrane potential. PTP gating by MEDICA analogs is accounted for by inhibition of Raf1 expression and kinase activity, resulting in suppression of the MAPK/RSK1 and the adenylate cyclase/PKA transduction pathways. Suppression of RSK1 and PKA results in a decrease in phosphorylation of their respective downstream targets, Bad(Ser-112) and Bad(Ser-155). Decrease in Bad(Ser-112, Ser-155) phosphorylation results in increased binding of Bad to mitochondrial Bcl2 with concomitant displacement of Bax, followed by PTP gating induced by free mitochondrial Bax. Low conductance PTP gating by LCFA/MEDICA may account for their thyromimetic calorigenic activity in vivo. PMID:20037159

  12. 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

    2016-01-01

    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

  13. 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.

  14. 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

  15. Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells

    PubMed Central

    Golej, Deidre L.; Askari, Bardia; Kramer, Farah; Barnhart, Shelley; Vivekanandan-Giri, Anuradha; Pennathur, Subramaniam; Bornfeldt, Karin E.

    2011-01-01

    Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E2 (PGE2) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE2. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE2 secretion. Thus, ACSL4 modulates PGE2 release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall. PMID:21242590

  16. Contribution of the Distal Pocket Residue to the Acyl-Chain-Length Specificity of (R)-Specific Enoyl-Coenzyme A Hydratases from Pseudomonas spp.

    PubMed Central

    Sato, Shun; Hiroe, Ayaka; Ishizuka, Koya; Kanazawa, Hiromi; Shiro, Yoshitsugu

    2015-01-01

    (R)-Specific enoyl-coenzyme A (enoyl-CoA) hydratases (PhaJs) are capable of supplying monomers from fatty acid β-oxidation to polyhydroxyalkanoate (PHA) biosynthesis. PhaJ1Pp from Pseudomonas putida showed broader substrate specificity than did PhaJ1Pa from Pseudomonas aeruginosa, despite sharing 67% amino acid sequence identity. In this study, the substrate specificity characteristics of two Pseudomonas PhaJ1 enzymes were investigated by site-directed mutagenesis, chimeragenesis, X-ray crystallographic analysis, and homology modeling. In PhaJ1Pp, the replacement of valine with isoleucine at position 72 resulted in an increased preference for enoyl-coenzyme A (CoA) elements with shorter chain lengths. Conversely, at the same position in PhaJ1Pa, the replacement of isoleucine with valine resulted in an increased preference for enoyl-CoAs with longer chain lengths. These changes suggest a narrowing and broadening in the substrate specificity range of the PhaJ1Pp and PhaJ1Pa mutants, respectively. However, the substrate specificity remains broader in PhaJ1Pp than in PhaJ1Pa. Additionally, three chimeric PhaJ1 enzymes, composed from PhaJ1Pp and PhaJ1Pa, all showed significant hydratase activity, and their substrate preferences were within the range exhibited by the parental PhaJ1 enzymes. The crystal structure of PhaJ1Pa was determined at a resolution of 1.7 Å, and subsequent homology modeling of PhaJ1Pp revealed that in the acyl-chain binding pocket, the amino acid at position 72 was the only difference between the two structures. These results indicate that the chain-length specificity of PhaJ1 is determined mainly by the bulkiness of the amino acid residue at position 72, but that other factors, such as structural fluctuations, also affect specificity. PMID:26386053

  17. Crystal structures of SIRT3 reveal that the α2-α3 loop and α3-helix affect the interaction with long-chain acyl lysine.

    PubMed

    Gai, Wei; Li, He; Jiang, Hualiang; Long, Yaqiu; Liu, Dongxiang

    2016-09-01

    SIRT1-7 play important roles in many biological processes and age-related diseases. In addition to a NAD(+) -dependent deacetylase activity, they can catalyze several other reactions, including the hydrolysis of long-chain fatty acyl lysine. To study the binding modes of sirtuins to long-chain acyl lysines, we solved the crystal structures of SIRT3 bound to either a H3K9-myristoylated- or a H3K9-palmitoylated peptide. Interaction of SIRT3 with the palmitoyl group led to unfolding of the α3-helix. The myristoyl and palmitoyl groups bind to the C-pocket and an allosteric site near the α3-helix, respectively. We found that the residues preceding the α3-helix determine the size of the C-pocket. The flexibility of the α2-α3 loop and the plasticity of the α3-helix affect the interaction with long-chain acyl lysine. PMID:27501476

  18. 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

  19. 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

  20. 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

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

    1990-02-15

    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

  1. Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana*

    PubMed Central

    Chacón, Micaëla G.; Fournier, Ashley E.; Tran, Frances; Dittrich-Domergue, Franziska; Pulsifer, Ian P.; Domergue, Frédéric; Rowland, Owen

    2013-01-01

    Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value. PMID:24005667

  2. The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

    PubMed

    Wang, Yue-Yue; Luo, Hong-Dou; Zhang, Xiao-Sheng; Lin, Tao; Jiang, Hui; Li, Yong-Quan

    2016-03-01

    Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs.

  3. Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

    PubMed Central

    Olety, Balaji; Veatch, Sarah L.

    2015-01-01

    ABSTRACT HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P2-specific pleckstrin homology domain of phospholipase Cδ1 (PHPLCδ1), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P2 with unsaturated or saturated acyl chains affect membrane binding of PHPLCδ1 and Gag. Both unsaturated dioleoyl-PI(4,5)P2 [DO-PI(4,5)P2] and saturated dipalmitoyl-PI(4,5)P2 [DP-PI(4,5)P2] successfully recruited PHPLCδ1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P2 but not DP-PI(4,5)P2 recruited Gag to GUVs, indicating that PI(4,5)P2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P2, cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PHPLCδ1 regardless of PI(4,5)P2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P2 had DO-acyl chains. DP-PI(4,5)P2-containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P2 still promoted recruitment of Gag, but not PHPLCδ1, to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P2 acyl chains in membrane binding of two PI(4,5)P2-binding proteins, Gag and PHPLCδ1. Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P2 containing an unsaturated acyl chain over DP-PI(4,5)P2, suggesting that Gag sensitivity to PI(4,5)P2 acyl chain saturation is determined directly by the matrix-PI(4,5)P2 interaction, rather

  4. Modified Acyl-ACP desaturase

    DOEpatents

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

    1999-03-30

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

  5. 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.

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-08-01

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

  8. Effects of experimental hypo- and hyperthyroidism on hepatic long-chain fatty acyl-CoA synthetase and hydrolase.

    PubMed

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

    1989-07-01

    The effects of T3 treatment and thyroidectomy on rat liver microsomal long-chain fatty acyl-CoA (LCFA-CoA) synthetase and LCFA-CoA hydrolase activities were determined. Hyperthyroid rats had a 36-42% decrease in LCFA-CoA synthetase with no change in hydrolase activity. This may contribute to the redirection of fatty acids from esterification to oxidation reactions in hyperthyroidism. Thyroidectomized rats had a 40-44% decrease in synthetase and a 27-42% decrease in LCFA-CoA hydrolase activity. The decrease in both LCFA-CoA synthetase and hydrolase activities in hypothyroidism may indicate that the LCFA-CoA turnover in this futile cycle is decreased in the liver.

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

    PubMed

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

    2013-01-01

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

  10. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25970403

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

    PubMed

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

    2014-05-01

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

  13. 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

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

    PubMed

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

    2015-04-01

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

  15. 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

  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. 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 ...

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

    PubMed

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

    2012-06-01

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

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

    PubMed

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

    1989-08-15

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

  20. A Peroxisomal Long-Chain Acyl-CoA Synthetase from Glycine max Involved in Lipid Degradation

    PubMed Central

    Jiang, Bingjun; Sun, Xuegang; Gu, Shoulai; Han, Tianfu; Hou, Wensheng

    2014-01-01

    Seed storage oil, in the form of triacylglycerol (TAG), is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL) is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525). Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination. PMID:24992019

  1. Intestinal acyl-CoA synthetase 5: activation of long chain fatty acids and behind.

    PubMed

    Klaus, Christina; Jeon, Min Kyung; Kaemmerer, Elke; Gassler, Nikolaus

    2013-11-14

    The intestinal mucosa is characterized by a high complexity in terms of structure and functions and allows for a controlled demarcation towards the gut lumen. On the one hand it is responsible for pulping and selective absorption of alimentary substances ensuring the immunological tolerance, on the other hand it prevents the penetration of micro-organisms as well as bacterial outgrowth. The continuous regeneration of surface epithelia along the crypt-villus-axis in the small intestine is crucial to assuring these various functions. The core phenomena of intestinal epithelia regeneration comprise cell proliferation, migration, differentiation, and apoptosis. These partly contrarily oriented processes are molecularly balanced through numerous interacting signaling pathways like Wnt/β-catenin, Notch and Hedgehog, and regulated by various modifying factors. One of these modifiers is acyl-CoA synthetase 5 (ACSL5). It plays a key role in de novo lipid synthesis, fatty acid degradation and membrane modifications, and regulates several intestinal processes, primarily through different variants of protein lipidation, e.g., palmitoylation. ACSL5 was shown to interact with proapoptotic molecules, and besides seems to inhibit proliferation along the crypt-villus-axis. Because of its proapoptotic and antiproliferative characteristics it could be of significant relevance for intestinal homeostasis, cellular disorder and tumor development. PMID:24259967

  2. Allometric scaling of fatty acyl chains in fowl liver, lung and kidney, but not in brain phospholipids.

    PubMed

    Szabó, András; Mézes, Miklós; Romvári, Róbert; Fébel, Hedvig

    2010-03-01

    The phospholipid (PL) fatty acyl chain (FA) composition (mol%) was determined in the kidney, liver, lung and brain of 8 avian species ranging in body mass from 150g (Japanese quail, Coturnix coturnix japonica) to 19kg (turkey, Meleagris gallopavo). In all organs except the brain, docosahexaenoic acid (C22:6 n3, DHA) was found to show a negative allometric scaling (allometric exponent: B=-0.18; -0.20 and -0.24, for kidney, liver and lung, respectively). With minor inter-organ differences, smaller birds had more n3 FAs and longer FA chains in the renal, hepatic and pulmonary PLs. Comparing our results with literature data on avian skeletal muscle, liver mitochondria and kidney microsomes and divergent mammalian tissues, the present findings in the kidney, liver and lung PLs seem to be a part of a general relationship termed "membranes as metabolic pacemakers". Marked negative allometric scaling was found furthermore for the tissue malondialdehyde concentrations in all organs except the brain (B=-0.17; -0.13 and -0.05, respectively). In the liver and kidney a strong correlation was found between the tissue MDA and DHA levels, expressing the role of DHA in shaping the allometric properties of membrane lipids.

  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. PMID:25069536

  4. 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.

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

    PubMed Central

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

    2008-01-01

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

  6. Effects of cholesterol and saturated sphingolipids on acyl chain order in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers--a comparative study with phase-selective fluorophores.

    PubMed

    Engberg, Oskar; Nurmi, Henrik; Nyholm, Thomas K M; Slotte, J Peter

    2015-04-14

    Saturated sphingolipids have high acyl chain order. Our aim was to study how palmitoylated sphingomyelin (PSM), ceramide (PCer), glucosyl (GlcPCer)-, and galactosylceramide (GalPCer) were able to order the bulk acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in comparison with cholesterol. For this reason, we used lipid probes which had preferred phases that were either the disordered phase (1-oleoyl-2-propionyl[DPH-sn-glycero-3-phosphcholine (18:1-DPH-PC) or the ordered phase (trans parinaric acid (tPA). DPH was also used, although it has no clear phase preference. We measured steady-state anisotropy (all probes) and performed fluorescence lifetime analysis (tPA) as a function of composition and temperature. At concentrations where the saturated sphingolipids were not aggregated into ordered domains (and 23 °C), they did not increase POPC acyl chain order as determined from 18:1-DPH-PC anisotropy. As expected, cholesterol increased the POPC acyl chain order linearly as a function of concentration (0-28 mol %). Since PCer already forms ordered domains below 5 mol % (at 23 °C), we measured the acyl chain ordering effect of PCer at 50 °C (0-13 mol %) and observed that PCer ordered POPC acyl chains as efficiently as cholesterol. We conclude that the bulk acyl chain order of POPC was not markedly affected in bilayers where disordered and ordered domains coexist.

  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

    SciTech Connect

    Vandevoorde, Severine; Saha, Bijali; Mahadevan, Anu; Razdan, Raj K.; Pertwee, Roger G.; Martin, Billy R.; Fowler, Christopher J. . E-mail: cf@pharm.umu.se

    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 (IC{sub 50} values in the range 5.1-8.2 {mu}M), whereas the two compounds with a single unsaturated bond were less potent (IC{sub 50} values 19 and 21 {mu}M). 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 IC{sub 50} values of 12 and 32 {mu}M, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC{sub 50} value 4.5 {mu}M). 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. Development and pathomechanisms of cardiomyopathy in very long-chain acyl-CoA dehydrogenase deficient (VLCAD(-/-)) mice.

    PubMed

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

    2014-05-01

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

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

    PubMed

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

    2016-01-01

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

  11. 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

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

    PubMed

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

    2015-09-01

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

  13. Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities.

    PubMed

    Han, Gongshe; Gupta, Sita D; Gable, Kenneth; Niranjanakumari, Somashekarappa; Moitra, Prasun; Eichler, Florian; Brown, Robert H; Harmon, Jeffrey M; Dunn, Teresa M

    2009-05-19

    Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells. PMID:19416851

  14. 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.

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

    PubMed

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

    2014-05-01

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

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

    PubMed

    Tucci, Sara; Flögel, Ulrich; Spiekerkoetter, Ute

    2015-07-01

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

  17. 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.

  18. Oxidative acylation using thioacids

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.

    1997-01-01

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

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed Central

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

    2010-01-01

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

  1. 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.

  2. 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/.

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

    PubMed

    Varela, A; Savino, E A

    1987-06-01

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

  4. 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.

  5. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

    PubMed

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-07-19

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

  6. 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

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

    SciTech Connect

    Chen, H.H.

    1989-01-01

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

  8. Reversible competitive α-ketoheterocycle inhibitors of fatty acid amide hydrolase containing additional conformational constraints in the acyl side chain: orally active, long-acting analgesics.

    PubMed

    Ezzili, Cyrine; Mileni, Mauro; McGlinchey, Nicholas; Long, Jonathan Z; Kinsey, Steven G; Hochstatter, Dustin G; Stevens, Raymond C; Lichtman, Aron H; Cravatt, Benjamin F; Bilsky, Edward J; Boger, Dale L

    2011-04-28

    A series of α-ketooxazoles containing conformational constraints in the C2 acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase (FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs R enantiomer), and their potency is comparable or improved relative to 2, indicating that the conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure of the α-ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding details, confirmed that the (S)-enantiomer is the bound active inhibitor, shed light on the origin of the enantiomeric selectivity, and confirmed that the catalytic Ser241 is covalently bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain anandamide levels following either intraperitoneal (ip) or oral (po) administration indicative of effective in vivo FAAH inhibition. Significantly, the oral administration of 12 caused dramatic accumulation of anandamide in the brain, with peak levels achieved between 1.5 and 3 h, and these elevations were maintained over 9 h. Additional studies of these two representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the demonstration that 12 administered orally significantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained periods consistent with its long-acting effects in raising the endogenous concentration of anandamide.

  9. 2H nuclear magnetic resonance order parameter profiles suggest a change of molecular shape for phosphatidylcholines containing a polyunsaturated acyl chain.

    PubMed Central

    Holte, L. L.; Peter, S. A.; Sinnwell, T. M.; Gawrisch, K.

    1995-01-01

    change to a more wedge-shaped space available for the acyl chain. PMID:7647244

  10. 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.

  11. 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. PMID:27091636

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-01-01

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

  14. Kinetic and structural basis for acyl-group selectivity and NAD+-dependence in Sirtuin-catalyzed deacylation

    PubMed Central

    Thelen, Julie N.; Ito, Akihiro; Yoshida, Minoru; Denu, John M.

    2015-01-01

    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.1 Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+ and acyl-group length for a diverse series of human Sirtuins, SIRT1, SIRT2, 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. Co-crystallization 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 solved co-complex structures containing either a myristoylated peptide or 2′-O-myristoyl-ADP-ribose indicate 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. PMID:25897714

  15. Antiproliferative activity of long chain acylated esters of quercetin-3-O-glucoside in hepatocellular carcinoma HepG2 cells

    PubMed Central

    Sudan, Sudhanshu

    2015-01-01

    Despite their strong role in human health, poor bioavailability of flavonoids limits their biological effects in vivo. Enzymatically catalyzed acylation of fatty acids to flavonoids is one of the approaches of increasing cellular permeability and hence, biological activities. In this study, six long chain fatty acid esters of quercetin-3-O-glucoside (Q3G) acylated enzymatically and were used for determining their antiproliferative action in hepatocellular carcinoma cells (HepG2) in comparison to precursor compounds and two chemotherapy drugs (Sorafenib and Cisplatin). Fatty acid esters of Q3G showed significant inhibition of HepG2 cell proliferation by 85 to 90% after 6 h and 24 h of treatment, respectively. The cell death due to these novel compounds was associated with cell-cycle arrest in S-phase and apoptosis observed by DNA fragmentation, fluorescent microscopy and elevated caspase-3 activity and strong DNA topoisomerase II inhibition. Interestingly, Q3G esters showed significantly low toxicity to normal liver cells than Sorafenib (P < 0.05), a chemotherapy drug for hepatocellular carcinoma. Among all, oleic acid ester of Q3G displayed the greatest antiproliferation action and a high potential as an anti-cancer therapeutic. Overall, the results of the study suggest strong antiproliferative action of these novel food-derived compounds in treatment of cancer. PMID:25681471

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Resh, Marilyn D

    2016-07-01

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

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

    PubMed

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

    2015-11-01

    Because hearts with a temporally induced knockout of acyl-CoA synthetase 1 (Acsl1(T-/-)) 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, Acsl1(T-/-) 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 Acsl1(T-/-) and littermate control mice with rapamycin or vehicle alone for 2 wk. Rapamycin treatment normalized mitochondrial structure, number, and the maximal respiration rate in Acsl1(T-/-) 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 Acsl1(T-/-) hearts. The turnover of microtubule associated protein light chain 3b in Acsl1(T-/-) 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. PMID:26220174

  19. 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

  20. Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

    SciTech Connect

    Safford, R.; de Silva, J.; Lucas, C.; Windust, J.H.C.; Shedden, J.; James, C.M.; Sidebottom, C.M.; Slabas, A.R.; Tombs, M.P.; Hughes, S.G.

    1987-03-10

    Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approx. 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.

  1. Substrate Effects on Electronic Properties of Adatom Chains

    NASA Technical Reports Server (NTRS)

    Toshishige, Yamada; Saini, Subhash (Technical Monitor)

    1998-01-01

    About VI author/originator verification in form 1676: (1) There is no export controlled, confidential commercial information. (2) Regarding the patent, the technical field covered here is related to ARC-14246, 'Doping Method of Semiconducting Atomic Chains." Most results are covered by ARC-14246.

  2. 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.

  3. 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

  4. 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

  5. Regioselective self-acylating cyclodextrins in organic solvent

    PubMed Central

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

    2016-01-01

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

  6. 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.

  7. Regioselective self-acylating cyclodextrins in organic solvent.

    PubMed

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

    2016-01-01

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

  8. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2002-11-01

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

  11. Product Diversity Linked to Substrate Usage in Chain Elongation by Mixed-Culture Fermentation.

    PubMed

    Coma, Marta; Vilchez-Vargas, Ramiro; Roume, Hugo; Jauregui, Ruy; Pieper, Dietmar H; Rabaey, Korneel

    2016-06-21

    Acetate and ethanol can be converted to caproic acid by microorganisms through reverse β-oxidation. There is limited insight into the versatility of chain elongation in view of different starting substrates, including even- and odd-carbon carboxylates and alcohols other than ethanol. Thermodynamic analyses show that most elongation pathways are energetically feasible. Through incubations of microbial communities with different substrate-pair combinations, we established that ethanol and propanol were both highly suitable for chain elongation. As an electron acceptor, acetate, propionate, and butyrate readily elongated with ethanol, whereas an adaptation period was necessary for formate. Isobutyrate and longer-chained fatty acids above butyrate were not elongated. The microbial communities converged, and consistent enrichment of Clostridium spp. was observed, independent of the supplied alcohol or carboxylate, with a strain related to Clostridium kluyveri dominating the enrichments. Community analysis also showed phylotypes related to Bacteroidaceae and Microbacteriaceae families in all tests that are capable of converting the base substrates to useful intermediates. These organisms were mainly enriched with methanol or formate. Our overall conclusion is thus that multiple substrates can be used for chain elongation and that this process is carried out by highly similar organisms for direct chain elongation irrespective of the substrate. PMID:27162101

  12. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaene variabilis

    SciTech Connect

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

    1987-04-01

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

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

    PubMed

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

    2012-10-01

    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 Ca(2+) 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 Ca(2+) 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.

  14. 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.

  15. Crystal structure and substrate specificity of the β-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    PubMed Central

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

    2005-01-01

    β-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 Å 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. PMID:15987898

  16. 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.

  17. 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

  18. 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

  19. Acyl-lipid metabolism.

    PubMed

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

    2013-01-01

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

  20. Mechanisms of chain adsorption on porous substrates and critical conditions of polymer chromatography.

    PubMed

    Cimino, Richard T; Rasmussen, Christopher J; Brun, Yefim; Neimark, Alexander V

    2016-11-01

    Polymer adsorption is a ubiquitous phenomenon with numerous technological and healthcare applications. The mechanisms of polymer adsorption on surfaces and in pores are complex owing to a competition between various entropic and enthalpic factors. Due to adsorption of monomers to the surface, the chain gains in enthalpy yet loses in entropy because of confining effects. This competition leads to the existence of critical conditions of adsorption when enthalpy gain and entropy loss are in balance. The critical conditions are controlled by the confining geometry and effective adsorption energy, which depends on the solvent composition and temperature. This phenomenon has important implications in polymer chromatography, since the retention at the critical point of adsorption (CPA) is chain length independent. However, the mechanisms of polymer adsorption in pores are poorly understood and there is an ongoing discussion in the theoretical literature about the very existence of CPA for polymer adsorption on porous substrates. In this work, we examine the mechanisms of chain adsorption on a model porous substrate using Monte Carlo (MC) simulations. We distinguish three adsorption mechanisms depending on the chain location: on external surface, completely confined in pores, and also partially confined in pores in so-called "flower" conformations. The free energies of different conformations of adsorbed chains are calculated by the incremental gauge cell MC method that allows one to determine the partition coefficient as a function of the adsorption potential, pore size, and chain length. We confirm the existence of the CPA for chain length independent separation on porous substrates, which is explained by the dominant contributions of the chain adsorption at the external surface, in particular in flower conformations. Moreover, we show that the critical conditions for porous and nonporous substrates are identical and depend only on the surface chemistry. The theoretical

  1. Mechanisms of chain adsorption on porous substrates and critical conditions of polymer chromatography.

    PubMed

    Cimino, Richard T; Rasmussen, Christopher J; Brun, Yefim; Neimark, Alexander V

    2016-11-01

    Polymer adsorption is a ubiquitous phenomenon with numerous technological and healthcare applications. The mechanisms of polymer adsorption on surfaces and in pores are complex owing to a competition between various entropic and enthalpic factors. Due to adsorption of monomers to the surface, the chain gains in enthalpy yet loses in entropy because of confining effects. This competition leads to the existence of critical conditions of adsorption when enthalpy gain and entropy loss are in balance. The critical conditions are controlled by the confining geometry and effective adsorption energy, which depends on the solvent composition and temperature. This phenomenon has important implications in polymer chromatography, since the retention at the critical point of adsorption (CPA) is chain length independent. However, the mechanisms of polymer adsorption in pores are poorly understood and there is an ongoing discussion in the theoretical literature about the very existence of CPA for polymer adsorption on porous substrates. In this work, we examine the mechanisms of chain adsorption on a model porous substrate using Monte Carlo (MC) simulations. We distinguish three adsorption mechanisms depending on the chain location: on external surface, completely confined in pores, and also partially confined in pores in so-called "flower" conformations. The free energies of different conformations of adsorbed chains are calculated by the incremental gauge cell MC method that allows one to determine the partition coefficient as a function of the adsorption potential, pore size, and chain length. We confirm the existence of the CPA for chain length independent separation on porous substrates, which is explained by the dominant contributions of the chain adsorption at the external surface, in particular in flower conformations. Moreover, we show that the critical conditions for porous and nonporous substrates are identical and depend only on the surface chemistry. The theoretical

  2. 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.

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

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

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

  4. 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

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

    PubMed Central

    Soupene, Eric; Kuypers, Frans A.

    2015-01-01

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

  6. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction.

    PubMed

    Stroka, Kimberly M; Aranda-Espinoza, Helim

    2011-08-11

    A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte transmigration. However, no studies have explored the effects of substrate stiffness on transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness. PMID:21652678

  7. Two Activities of Long-Chain Acyl-Coenzyme A Synthetase Are Involved in Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis1

    PubMed Central

    Jessen, Dirk; Roth, Charlotte; Wiermer, Marcel

    2015-01-01

    In plants, fatty acids are synthesized within the plastid and need to be distributed to the different sites of lipid biosynthesis within the cell. Free fatty acids released from the plastid need to be converted to their corresponding coenzyme A thioesters to become metabolically available. This activation is mediated by long-chain acyl-coenzyme A synthetases (LACSs), which are encoded by a family of nine genes in Arabidopsis (Arabidopsis thaliana). So far, it has remained unclear which of the individual LACS activities are involved in making plastid-derived fatty acids available to cytoplasmic glycerolipid biosynthesis. Because of its unique localization at the outer envelope of plastids, LACS9 was regarded as a candidate for linking plastidial fatty export and cytoplasmic use. However, data presented in this study show that LACS9 is involved in fatty acid import into the plastid. The analyses of mutant lines revealed strongly overlapping functions of LACS4 and LACS9 in lipid trafficking from the endoplasmic reticulum to the plastid. In vivo labeling experiments with lacs4 lacs9 double mutants suggest strongly reduced synthesis of endoplasmic reticulum-derived lipid precursors, which are required for the biosynthesis of glycolipids in the plastids. In conjunction with this defect, double-mutant plants accumulate significant amounts of linoleic acid in leaf tissue. PMID:25540329

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

    PubMed Central

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

    1994-01-01

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

  9. 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.

  10. Long-chain polyunsaturated fatty acid biosynthesis in the euryhaline herbivorous teleost Scatophagus argus: Functional characterization, tissue expression and nutritional regulation of two fatty acyl elongases.

    PubMed

    Xie, Dizhi; Chen, Fang; Lin, Siyuan; You, Cuihong; Wang, Shuqi; Zhang, Qinghao; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

    2016-08-01

    Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n-3/18:2n-6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish. PMID:27050407

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

    PubMed

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

    2016-06-01

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

  12. Long-chain polyunsaturated fatty acid biosynthesis in the euryhaline herbivorous teleost Scatophagus argus: Functional characterization, tissue expression and nutritional regulation of two fatty acyl elongases.

    PubMed

    Xie, Dizhi; Chen, Fang; Lin, Siyuan; You, Cuihong; Wang, Shuqi; Zhang, Qinghao; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

    2016-08-01

    Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n-3/18:2n-6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish.

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

    PubMed

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

    2000-10-01

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

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

    PubMed

    Heath, R J; Rock, C O

    1996-01-26

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

  15. 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

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

    SciTech Connect

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

    1993-06-01

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

  17. ABHD4 regulates multiple classes of N-acyl phospholipids in the mammalian central nervous system

    PubMed Central

    Lee, Hyeon-Cheol; Simon, Gabriel M.; Cravatt, Benjamin F.

    2016-01-01

    N-acyl phospholipids are atypical components of cell membranes that bear three acyl chains and serve as potential biosynthetic precursors for lipid mediators such as endocannabinoids. Biochemical studies have implicated ABHD4 as a brain N-acyl phosphatidylethanolamine (NAPE) lipase, but in vivo evidence for this functional assignment is lacking. Here, we describe ABHD4−/− mice and their characterization using untargeted lipidomics to discover that ABHD4 regulates multiple classes of brain N-acyl phospholipids. In addition to showing reductions in brain glycerophospho-NAEs (GP-NAEs) and plasmalogen-based lyso-NAPEs (lyso-pNAPEs), ABHD4−/− mice exhibited decreases in a distinct set of brain lipids that were structurally characterized as N-acyl lysophosphatidylserines (lyso-NAPSs). Biochemical assays confirmed that NAPS lipids are direct substrates of ABHD4. These findings, taken together, designate ABHD4 as a principal regulator of N-acyl phospholipid metabolism in the mammalian nervous system. PMID:25853435

  18. Essential Role of the Donor Acyl Carrier Protein in Stereoselective Chain Translocation to a Fully Reducing Module of the Nanchangmycin Polyketide Synthase†

    PubMed Central

    Guo, Xun; Liu, Tiangang; Deng, Zixin; Cane, David E.

    2012-01-01

    Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl- NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2- methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a kcat of 4.4±0.9 min−1 and a kcat/Km 67 min−1 mM−1, corresponding to a ~2300-fold increase compared to the kcat/Km for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2. PMID:22229794

  19. Essential role of the donor acyl carrier protein in stereoselective chain translocation to a fully reducing module of the nanchangmycin polyketide synthase.

    PubMed

    Guo, Xun; Liu, Tiangang; Deng, Zixin; Cane, David E

    2012-01-31

    Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a k(cat) of 4.4 ± 0.9 min⁻¹ and a k(cat)/K(m) of 67 min⁻¹ mM⁻¹, corresponding to a ∼2300-fold increase compared to the k(cat)/K(m) for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2. PMID:22229794

  20. Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro.

    PubMed Central

    Mitropoulos, K A; Venkatesan, S

    1984-01-01

    The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the

  1. Miscibility of phospholipids with identical headgroups and acyl chain lengths differing by two methylene units: effects of headgroup structure and headgroup charge.

    PubMed

    Garidel, P; Blume, A

    1998-04-22

    We have investigated the influence of the chemical structure and charge of the hydrophillic headgroup on the miscibility of saturated phospholipids with acyl chain lengths differing by two methylene units, namely DMPA/DPPA, DMPC/DPPC, DMPE/DPPE and DMPG/DPPG (0.1 M NaCl). All four mixtures were analysed by DSC at pH 7. To study the influence of a change in headgroup charge, we additionally investigated DMPA/DPPA mixtures at pH 4 and 12, and DMPG/DPPG mixtures at pH 2. The experimental DSC thermograms were fitted using methods described before [Johann et al., Biophys. J. 71 (1996), 3215-3228] to obtain the temperatures of onset and end of melting and first approximations for the non-ideality parameters as a function of composition. The resulting phase diagrams were then fitted using a four non-ideality parameter model for non-ideal, non-symmetric mixing in both phases. The phase diagram of the system DMPG/DPPG has a lens-like shape, the non-ideality parameters rhog and rhol for the gel and the liquid-crystalline phase, respectively, are zero, indicating ideal mixing in both phases. For the other mixtures, differences in miscibility are observed depending on the structure of the headgroup. At pH 7, rhog > rhol, i.e., the miscibility in the liquid-crystalline phase is more ideal than in the gel state. All rhog values are positive and the sequence for rhog observed is PA>PE>PC>PG. Partial protonation of PA at pH 4 or complete deprotonation at pH 12 leads to negative non-ideality parameters for both phases, indicating a preference for mixed pair formation. Protonation of PG in DMPG/DPPG mixtures at pH 2 leads to positive non-ideality parameters for both phases, indicating a tendency for demixing. The results show, that the miscibility of phospholipids with identical headgroups but chain lengths differing by two methylene groups is dependent on headgroup structure and on headgroup charge.

  2. Effect of Uniformly Applied Force and Molecular Characteristics of a Polymer Chain on Its Adhesion to Graphene Substrates.

    PubMed

    Kumar, Sunil; Pattanayek, Sudip K; Pereira, Gerald G; Mohanty, Sanat

    2016-03-22

    The force-induced desorption of a polymer chain from a graphene substrate is studied with molecular dynamics (MD). A critical force needs to be exceeded before detachment of the polymer from the substrate. It is found that for a chain to exhibit good adhesive properties the chain configuration should consist of fibrils-elongated, aligned sections of polymers and cavities which dissipate the applied energy. A fibrillation index is defined to quantify the quality of fibrils. We focus on the molecular properties of the polymer chain, which can lead to large amounts of fibrillation, and find that both strong attraction between the polymer and substrate and good solvency conditions are important conditions for this. We also vary the stiffness of the chain and find that for less stiff chains a plateau in the stress-strain curve gives rise to good adhesion however for very stiff chains there is limited elongation of the chain but the chain can still exhibit good fibrillation by a lamella-like rearrangement. Finally, it is found that the detachment time, t, of a polymer from the adsorbed substrate is inversely proportional to force, F (i.e., t ∝ F(-γ)), where exponent γ depends on the solvent quality, polymer-substrate attraction, and chain stiffness.

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

    PubMed Central

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

    2000-01-01

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

  4. Minor modifications to the phosphate groups and the C3' acyl chain length of lipid A in two Bordetella pertussis strains, BP338 and 18-323, independently affect Toll-like receptor 4 protein activation.

    PubMed

    Shah, Nita R; Albitar-Nehme, Sami; Kim, Emma; Marr, Nico; Novikov, Alexey; Caroff, Martine; Fernandez, Rachel C

    2013-04-26

    Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

  5. Structural effects of substrate utilization on the adenosinetriphosphatase chains of sarcoplasmic reticulum.

    PubMed

    Watanabe, T; Inesi, G

    1982-07-01

    Addition of ATP to suspensions of fragmented sarcoplasmic reticulum (SR) containing low concentrations of a detergent that does not by itself produce major vesicular disruption is followed by a transient reduction in turbidity accompanied by solubilization of the vesicles. The effect of ATP is Ca2+ dependent and proceeds in parallel with utilization of the nucleotide as a substrate for the SR ATPase. Analogous effects are observed with other substrates producing enzyme phosphorylation at the catalytic site. The effect of ATP can also be detected in studies of fluorescence energy transfer between enzyme chains, by using the technique of Vanderkooi et al. [Vanderkooi, J., Ierokomas, A., Nakamura, H., & Martonosi, A. (1977) Biochemistry 16, 1262]. For this purpose, ATPase chains are labeled separately with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) and 6-(iodoacetamido) fluorescein (IAF). Samples of vesicles uniformly labeled with either IAEDANS or IAF, mixtures of two populations of vesicles uniformly labeled with either fluorophore, and vesicles containing randomized chains labeled with either fluorophore are used as experimental systems. In the last system, significant energy transfer from IAEDANS (donor) to IAF (acceptor) is revealed by fluorescence spectra and measurements of donor fluorescence intensity and lifetime. This is attributed to close interactions between ATPase chains within the membrane bilayer. It is then found that in the presence of low detergent concentrations, ATP changes the extent of energy transfer between labeled ATPase chains, consistent with destabilization of the interaction of chains. The observed effects are attributed to a reversible structural transition concomitant with enzyme phosphorylation and related to catalytic and transport function.

  6. 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

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

    PubMed

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

    2008-04-25

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    1988-12-16

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

  12. 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

  13. 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.

  14. 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.

  15. 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. PMID:27052113

  16. 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

  17. 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; 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 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

  18. 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.

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. 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.

  3. Identification and molecular characterization of acyl-CoA synthetase in human erythrocytes and erythroid precursors.

    PubMed

    Malhotra, K T; Malhotra, K; Lubin, B H; Kuypers, F A

    1999-11-15

    Full-length cDNA species encoding two forms of acyl-CoA synthetase from a K-562 human erythroleukaemic cell line were cloned, sequenced and expressed. The first form, named long-chain acyl-CoA synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase with high similarity to rat brain ACS2 (91% identical). The second form (66% identical with LACS5) was 97% identical with human liver LACS1. The LACS5 gene encodes a highly expressed 2.9 kb mRNA transcript in human haemopoietic stem cells from cord blood, bone marrow, reticulocytes and fetal blood cells derived from fetal liver. An additional 6.3 kb transcript is also found in these erythrocyte precursors; 2.9 and 9.6 kb transcripts of LACS5 are found in human brain, but transcripts are virtually absent from human heart, kidney, liver, lung, pancreas, spleen and skeletal muscle. The 78 kDa expressed LACS5 protein used the long-chain fatty acids palmitic acid, oleic acid and arachidonic acid as substrates. Antibodies directed against LACS5 cross-reacted with erythrocyte membranes. We conclude that early erythrocyte precursors express at least two different forms of acyl-CoA synthetase and that LACS5 is present in mature erythrocyte plasma membranes.

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

    PubMed

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

    2014-09-01

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

  5. In Vitro Ubiquitination: Self-Ubiquitination, Chain Formation, and Substrate Ubiquitination Assays.

    PubMed

    Maspero, Elena; Polo, Simona

    2016-01-01

    Ubiquitination of proteins in vitro has evolved as an indispensable tool for the functional analysis of this posttranslational modification. In vitro ubiquitination is particularly helpful to study conjugation mechanisms. The efficiency of the ubiquitination reaction depends in part on the quality of the enzymes utilized. Here we introduce the assay developed in our lab to study HECT E3 ligases. It involves bacterially expressed E1, His-tagged Ube2D3 (also called UbcH5c, the best E2 for Nedd4), untagged Nedd4, and untagged ubiquitin (Ub). As tags may impair specific activity of the enzymes or even interfere with the enzymatic reaction, they should be avoided, removed, or kept to a minimal size whenever possible, unless proven to be without consequence. The protocol described here is suitable for other E3 ligases capable of forming Ub chains as pseudo-product of the enzyme reaction. It is also adapted to include substrates. In this case, substrates should be tagged and purified after the reaction is completed to allow the detection of the ubiquitinated products. PMID:27613033

  6. Alcohol-to-acid ratio and substrate concentration affect product structure in chain elongation reactions initiated by unacclimatized inoculum.

    PubMed

    Liu, Yuhao; Lü, Fan; Shao, Liming; He, Pinjing

    2016-10-01

    The objective of the study was to investigate whether the ratio of ethanol to acetate affects yield and product structure in chain elongation initiated by unacclimatized mixed cultures. The effect of varying the substrate concentration, while maintaining the same ratio of alcohol to acid, was also investigated. With a high substrate concentration, an alcohol to acid ratio >2:1 provided sufficient electron donor capacity for the chain elongation reaction. With an ethanol to acetate ratio of 3:1 (300mM total carbon), the highest n-caproate concentration (3033±98mg/L) was achieved during the stable phase of the reaction. A lower substrate concentration (150mM total carbon) gave a lower yield of products and led to reduced carbon transformation efficiency compared with other reaction conditions. The use of unacclimatized inoculum in chain elongation can produce significant amounts of odd-carbon-number carboxylates as a result of protein hydrolysis. PMID:27469095

  7. Alcohol-to-acid ratio and substrate concentration affect product structure in chain elongation reactions initiated by unacclimatized inoculum.

    PubMed

    Liu, Yuhao; Lü, Fan; Shao, Liming; He, Pinjing

    2016-10-01

    The objective of the study was to investigate whether the ratio of ethanol to acetate affects yield and product structure in chain elongation initiated by unacclimatized mixed cultures. The effect of varying the substrate concentration, while maintaining the same ratio of alcohol to acid, was also investigated. With a high substrate concentration, an alcohol to acid ratio >2:1 provided sufficient electron donor capacity for the chain elongation reaction. With an ethanol to acetate ratio of 3:1 (300mM total carbon), the highest n-caproate concentration (3033±98mg/L) was achieved during the stable phase of the reaction. A lower substrate concentration (150mM total carbon) gave a lower yield of products and led to reduced carbon transformation efficiency compared with other reaction conditions. The use of unacclimatized inoculum in chain elongation can produce significant amounts of odd-carbon-number carboxylates as a result of protein hydrolysis.

  8. 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

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

    PubMed

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

    2013-11-01

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

  10. 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

    2016-09-01

    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.

  11. 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

    2016-09-01

    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

  12. 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

  13. Dissecting the role of critical residues and substrate preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis.

    PubMed

    Khare, Garima; Gupta, Vibha; Gupta, Rakesh K; Gupta, Radhika; Bhat, Rajiv; Tyagi, Anil K

    2009-01-01

    Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487) resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172) and Ala(302) to be involved in the binding of ATP and Ser(404) in the binding of CoenzymeA. The influence of mutations of the residues Val(209) and Trp(377) emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26)/C(24) fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172), Ala(302) and Lys(487) and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the

  14. A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

    PubMed

    Loch, Christian M; Strickler, James E

    2012-11-01

    Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

  15. LasR receptor for detection of long-chain quorum-sensing signals: identification of N-acyl-homoserine lactones encoded by the avsI locus of Agrobacterium vitis.

    PubMed

    Savka, Michael A; Le, Phuong T; Burr, Thomas J

    2011-01-01

    Bacterial biosensor strains have greatly facilitated the rapid discovery, isolation, and study of quorum-sensing systems. In this study, we determined the relative sensitivity of a LasR-based E. coli bacterial bioluminescence biosensor JM109 (pSB1075) for 13 diverse long-chain N-acyl-homoserine lactones (AHLs) including oxygen-substituted and -unsubstituted AHLs containing 14, 16, and 18 carbons and with and without double bonds. Furthermore, we show by bioassay, HPLC, and GC/MS that four long-chain AHLs of the C16-HSL family are encoded by the avsI gene of Agrobacterium vitis strain F2/5, a non-tumorigenic strain that inhibits pathogenic strains of A. vitis from causing crown gall on grape. The four C16-HSLs include: C16-HSL, N-hexadecanoyl homoserine lactone; 3-oxo-C16-HSL, N-(3-oxohexadecanoyl)homoserine lactone; C16:1-HSL, N-(cis-9-octadecenoyl)homoserine lactone; and 3-oxo-C16:1-HSL, N-(3-oxo-cis-11-hexadecenoyl)homoserine lactone. Thus, the LasR-based bioluminescent biosensor tested in this study should serve as a useful tool for the detection of various long-chain AHLs with and without double bonds as well as those oxylated at the third carbon from uninvestigated species. PMID:20514483

  16. Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

    PubMed Central

    Chankhamhaengdecha, Surang; Hongvijit, Suphatra; Srichaisupakit, Akkaraphol; Charnchai, Pattra; Panbangred, Watanalai

    2013-01-01

    Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces. PMID:23484156

  17. Endophytic actinomycetes: a novel source of potential acyl homoserine lactone degrading enzymes.

    PubMed

    Chankhamhaengdecha, Surang; Hongvijit, Suphatra; Srichaisupakit, Akkaraphol; Charnchai, Pattra; Panbangred, Watanalai

    2013-01-01

    Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

  18. Topology and acylation of spiralin.

    PubMed Central

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

    1989-01-01

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

  19. 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

  20. 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

  1. Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum.

    PubMed

    Lindemann, Andrea; Pessi, Gabriella; Schaefer, Amy L; Mattmann, Margrith E; Christensen, Quin H; Kessler, Aline; Hennecke, Hauke; Blackwell, Helen E; Greenberg, E Peter; Harwood, Caroline S

    2011-10-01

    Many species of Proteobacteria communicate by using LuxI-LuxR-type quorum-sensing systems that produce and detect acyl-homoserine lactone (acyl-HSL) signals. Most of the known signals are straight-chain fatty acyl-HSLs, and evidence indicates that LuxI homologs prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis. Two related LuxI homologs, RpaI and BtaI from Rhodopseudomonas palustris and photosynthetic stem-nodulating bradyrhizobia, direct production of the aryl-HSLs p-coumaroyl-HSL and cinnamoyl-HSL, respectively. Here we report that BjaI from the soybean symbiont Bradyrhizobium japonicum USDA110 is closely related to RpaI and BtaI and catalyzes the synthesis of isovaleryl-HSL (IV-HSL), a branched-chain fatty acyl-HSL. We show that IV-HSL induces expression of bjaI, and in this way IV-HSL functions like many other acyl-HSL quorum-sensing signals. Purified histidine-tagged BjaI was an IV-HSL synthase, which was active with isovaleryl-CoA but not detectably so with isovaleryl-ACP. This suggests that the RpaI-BtaI-BjaI subfamily of acyl-HSL synthases may use CoA- rather than ACP-linked substrates for acyl-HSL synthesis. The bjaI-linked bjaR(1) gene is involved in the response to IV-HSL, and BjaR(1) is sensitive to IV-HSL at concentrations as low as 10 pM. Low but sufficient levels of IV-HSL (about 5 nM) accumulate in B. japonicum culture fluid. The low levels of IV-HSL synthesis have likely contributed to the fact that the quorum-sensing signal from this bacterium has not been described elsewhere.

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

    SciTech Connect

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

    2008-04-02

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

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

    PubMed

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

    2016-05-01

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

  4. Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

    PubMed Central

    Alcala, Diego B.; Haldeman, Brian D.; Brizendine, Richard K.; Krenc, Agata K.; Baker, Josh E.; Rock, Ronald S.; Cremo, Christine R.

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. PMID:27528075

  5. Lessons from the gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) null mouse model: a role in steroidogenesis, but not result in X-ALD phenotype.

    PubMed

    Sheng, Yi; Tsai-Morris, Chon-Hwa; Li, Jie; Dufau, Maria L

    2009-03-01

    Gonadotropin-regulated long chain fatty acid Acyl-CoA synthetase (GR-LACS), is a member of the LACS family that is regulated by gonadotropin in the rat Leydig cell (LC). Its mouse/human homologs, lipidosin/bubblegum, have been suggested to participate in X-linked adrenoleukodystrophy (X-ALD), an adreno/neurodegenerative disorder with accumulation of very long chain fatty acids (VLCFA) in tissues and plasma. To further gain insights into its regulatory function, a GR-LACS/lipidosin null mouse was generated. No apparent phenotypic abnormalities were observed in the X-ALD target tissues (brain, testis, adrenal). Nuclear inclusions seen in mice >15 month-old, were present in LC of 9 month-old GR-LACS(-/-) mice. LC of the null mice showed refractoriness to the gonadotropin-induced desensitization of testosterone production that is observed in adult animals. LCFAs were moderately increased in the testis, ovary and brain, but not in the adrenal gland of GR-LACS(-/-) mice, with no major changes in VLCFA. No change in LACS activity was observed in these tissues, suggesting a compensatory mechanism exhibited by other LACS members. The GR-LACS(-/-) model did not support its association with X-ALD. These studies revealed a role of GR-LACS in reducing the aging process of the LC, and its participation in gonadotropin-induced testicular desensitization of testosterone production.

  6. 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.

  7. 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

  8. Acyl Coenzyme A Synthetase Long-Chain 1 (ACSL1) Gene Polymorphism (rs6552828) and Elite Endurance Athletic Status: A Replication Study

    PubMed Central

    Santiago, Catalina; Hu, Yang; Li, Yan-Chun; Gómez-Gallego, Félix; Fiuza-Luces, Carmen; Verde, Zoraida; Muniesa, Carlos A.; Oliván, Jesús; Santalla, Alfredo; Ruiz, Jonatan R.; Lucia, Alejandro

    2012-01-01

    The aim of this study was to determine the association between the rs6552828 polymorphism in acyl coenzyme A synthetase (ACSL1) and elite endurance athletic status. We studied 82 Caucasian (Spanish) World/Olympic-class endurance male athletes, and a group of sex and ethnically matched healthy young adults (controls, n = 197). The analyses were replicated in a cohort of a different ethnic origin (Chinese of the Han ethnic group), composed of elite endurance athletes (runners) [cases, n = 241 (128 male)] and healthy sedentary adults [controls, n = 504 (267 male)]. In the Spanish cohort, genotype (P = 0.591) and minor allele (A) frequencies were similar in cases and controls (P = 0.978). In the Chinese cohort, genotype (P = 0.973) and minor allele (G) frequencies were comparable in female endurance athletes and sedentary controls (P = 0.881), whereas in males the frequency of the G allele was higher in endurance athletes (0.40) compared with their controls (0.32, P = 0.040). The odds ratio (95%CI) for an elite endurance Chinese athlete to carry the G allele compared with ethnically matched controls was 1.381 (1.015–1.880) (P-value = 0.04). Our findings suggest that the ACSL1 gene polymorphism rs6552828 is not associated with elite endurance athletic status in Caucasians, yet a marginal association seems to exist for the Chinese (Han) male population. PMID:22829935

  9. 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

  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. Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthases

    SciTech Connect

    Blacklock, Brenda J. . E-mail: blacklock@chem.iupui.edu; Jaworski, Jan G.

    2006-07-28

    The very long chain fatty acids (VLCFA) incorporated into plant lipids are derived from the iterative addition of C2 units provided by malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoA synthase (KCS) component of a fatty acid elongase (FAE) complex. Mining of the Arabidopsis genome sequence database revealed 20 genes with homology to seed-specific FAE1 KCS. Eight of the 20 putative KCSs were cloned, expressed in yeast, and isolated as (His){sub 6} fusion proteins. Five of the eight (At1g71160, At1g19440, At1g07720, At5g04530, and At4g34250) had little or no activity with C16 to C20 substrates while three demonstrated activity with C16, C18, and C20 saturated acyl-CoA substrates. At1g01120 KCS (KCS1) and At2g26640 KCS had broad substrate specificities when assayed with saturated and mono-unsaturated C16 to C24 acyl-CoAs while At4g34510 KCS was specific for saturated fatty acyl-CoA substrates.

  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. Characterization of the promoter region of the bovine long-chain acyl-CoA synthetase 1 gene: Roles of E2F1, Sp1, KLF15, and E2F4

    PubMed Central

    Zhao, Zhi-Dong; Zan, Lin-Sen; Li, An-Ning; Cheng, Gong; Li, Shi-Jun; Zhang, Ya-Ran; Wang, Xiao-Yu; Zhang, Ying-Ying

    2016-01-01

    The nutritional value and eating qualities of beef are enhanced when the unsaturated fatty acid content of fat is increased. Long-chain acyl-CoA synthetase 1 (ACSL1) plays key roles in fatty acid transport and degradation, as well as lipid synthesis. It has been identified as a plausible functional and positional candidate gene for manipulations of fatty acid composition in bovine skeletal muscle. In the present study, we determined that bovine ACSL1was highly expressed in subcutaneous adipose tissue and longissimus thoracis. To elucidate the molecular mechanisms involved in bovine ACSL1 regulation, we cloned and characterized the promoter region of ACSL1. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified multiple transcriptional start sites (TSSs) in its promoter region. Using a series of 5′ deletion promoter plasmids in luciferase reporter assays, we found that the proximal minimal promoter of ACSL1 was located within the region −325/−141 relative to the TSS and it was also located in the predicted CpG island. Mutational analysis and electrophoretic mobility shift assays demonstrated that E2F1, Sp1, KLF15 and E2F4 binding to the promoter region drives ACSL1 transcription. Together these interactions integrate and frame a key functional role for ACSL1 in mediating the lipid composition of beef. PMID:26782942

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

    PubMed

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

    2012-05-17

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

  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. Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain.

    PubMed

    Jia, Kaimin; Cao, Ruikai; Hua, Duy H; Li, Ping

    2016-04-11

    Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility, and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogues. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analogue (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analogue (HABCoA) was determined to be 2.86 × 10(5) M(-1) s(-1), which was 6.2% of the value of HBCoA (4.62 × 10(6) M(-1) s(-1)) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically. PMID:26974339

  17. Versatility of acyl-acyl carrier protein synthetases.

    PubMed

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

    2014-10-23

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

  18. Detailed characterization of the substrate specificity of mouse wax synthase.

    PubMed

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  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. Amine, Alcohol and Phosphine Catalysts for Acyl Transfer Reactions

    NASA Astrophysics Data System (ADS)

    Spivey, Alan C.; Arseniyadis, Stellios

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

  1. Effect of the non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activity toward medium-chain, long-chain and polyunsaturated fatty acids in mitochondria of mouse liver and brain.

    PubMed

    Kasuya, Fumiyo; Kazuhiro, Misumi; Tatsuya, Hasegawa; Nakamoto, Kazuo; Tokuyama, Shogo; Masuyama, Teiichi

    2013-02-01

    Effect of eleven non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activities toward octanoic, palmitic, arachidonic and docosahexaenoic acids was evaluated in mouse liver and brain mitochondria. The drugs tested were aspirin, salicylic acid, diflunisal, mefenamic acid, indomethacin, etodolac, ibuprofen, ketoprofen, naproxen, loxoprofen, flurbiprofen. In mouse liver mitochondria, diflunisal and mefenamic acid exhibited the inhibitory activities not only for octanoic acid (IC(50) = 78.7 and 64.7 µM) and but also for palmitic acid (IC(50) = 236.5 and 284.4 µM), respectively. Aspirin was an inhibitor for the activation of octanoic acid only (IC(50) = 411.0 µM). In the brain, mefenamic acid and diflunisal inhibited strongly palmitoyl-CoA formation (IC(50) = 57.3 and 114.0 µM), respectively. The activation of docosahexaenoic acid in brain was sensitive to inhibition by diflunisal and mefenamic acid compared with liver.

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

    PubMed Central

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

    2008-01-01

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

  3. 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

  4. The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata).

    PubMed

    Kuah, Meng-Kiat; Jaya-Ram, Annette; Shu-Chien, Alexander Chong

    2015-03-01

    The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.

  5. The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata).

    PubMed

    Kuah, Meng-Kiat; Jaya-Ram, Annette; Shu-Chien, Alexander Chong

    2015-03-01

    The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores. PMID:25542509

  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. 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

  8. Z-Selective olefin metathesis on peptides: investigation of side-chain influence, preorganization, and guidelines in substrate selection.

    PubMed

    Mangold, Shane L; O'Leary, Daniel J; Grubbs, Robert H

    2014-09-01

    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

  9. Z-Selective olefin metathesis on peptides: investigation of side-chain influence, preorganization, and guidelines in substrate selection.

    PubMed

    Mangold, Shane L; O'Leary, Daniel J; Grubbs, Robert H

    2014-09-01

    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.

  10. 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

  11. The halo-substituent effect on Pseudomonas cepacia lipase-mediated regioselective acylation of nucleosides: A comparative investigation.

    PubMed

    Wang, Zhao-Yu; Bi, Yan-Hong; Yang, Rong-Ling; Duan, Zhang-Qun; Nie, Ling-Hong; Li, Xiang-Qian; Zong, Min-Hua; Wu, Jie

    2015-10-20

    In this work, comparative experiments were explored to investigate the substrate specificity of Pseudomonas cepacia lipase in regioselective acylation of nucleosides carrying various substituents (such as the H, F, Cl, Br, I) at 2'- and 5-positions. Experimental data indicated that the catalytic performance of the enzyme depended very much on the halo-substituents in nucleosides. The increased bulk of 2'-substituents in ribose moiety of the nucleoside might contribute to the improved 3'-regioselectivity (90-98%, nucleosides a-d) in enzymatic decanoylation, while the enhancement of regioselectivity (93-99%) in 3'-O-acylated nucleosides e-h could be attributable to the increasing hydrophobicity of the halogen atoms at 5-positions. With regard to the chain-length selectivity, P. cepacia lipase displayed the highest 3'-regioselectivity toward the longer chain (C14) as compared to shorter (C6 and C10) ones. The position, orientation and property of the substituent, specific structure of the lipase's active site, and acyl structure could account for the diverse results. PMID:26325198

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Olson, M A; Scovill, J P; Hack, D 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.

  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. PMID:23186160

  15. Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB.

    PubMed

    Schaeffer, M L; Agnihotri, G; Volker, C; Kallender, H; Brennan, P J; Lonsdale, J T

    2001-12-14

    Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery. PMID:11600501

  16. Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

    PubMed

    Foulon, Veerle; Asselberghs, Stanny; Geens, Wendy; Mannaerts, Guy P; Casteels, Minne; Van Veldhoven, Paul P

    2003-12-01

    Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.

  17. Endogenous N-acyl taurines regulate skin wound healing.

    PubMed

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

    2016-07-26

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

  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. PMID:22705534

  19. Modification of supramolecular binding motifs induced by substrate registry: formation of self-assembled macrocycles and chain-like patterns.

    PubMed

    Fendt, Leslie-Anne; Stöhr, Meike; Wintjes, Nikolai; Enache, Mihaela; Jung, Thomas A; Diederich, François

    2009-10-26

    The self-assembly properties of two Zn(II) porphyrin isomers on Cu(111) are studied at different coverage by means of scanning tunneling microscopy (STM). Both isomers are substituted in their meso-positions by two voluminous 3,5-di(tert-butyl)phenyl and two rod-like 4'-cyanobiphenyl groups, respectively. In the trans-isomer, the two 4'-cyanobiphenyl groups are opposite to each other, whereas they are located at right angle in the cis-isomer. For coverage up to one monolayer, the cis-substituted porphyrins self-assemble to form oligomeric macrocycles held together by antiparallel CNCN dipolar interactions and CNH-C(sp(2)) hydrogen bonding. Cyclic trimers and tetramers occur most frequently but everything from cyclic dimers to hexamers can be observed. Upon annealing of the samples at temperatures >150 degrees C, dimeric macrocyclic structures are observed, in which the two porphyrins are bridged by Cu atoms, originating from the surface, under formation of two CNCuNC coordination bonds. The trans-isomer builds up linear chains on Cu(111) at low coverage, whereas for higher coverage the molecules assemble in a periodic, densely packed structure. Both cis- and trans-bis(4'-cyanobiphenyl)-substituted Zn(II) porphyrins behave very differently on Cu(111) compared to similar porphyrins in literature on less reactive surfaces such as Au(111) and Ag(111). On the latter surfaces, there is no signal visible between molecular orientation and the crystal directions of the substrate, whereas on Cu(111), very strong adsorbate-substrate interactions have a dominating influence on all observed structures. This strong porphyrin-substrate interaction enables a much broader variety of structures, including also less favorable intermolecular bonding motifs and geometries.

  20. 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.

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

    PubMed Central

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

    2015-01-01

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

  2. Acyl-CoA synthetase 1 deficiency alters cardiolipin species and impairs mitochondrial function

    PubMed Central

    Grevengoed, Trisha J.; Martin, Sarah A.; Katunga, Lalage; Cooper, Daniel E.; Anderson, Ethan J.; Murphy, Robert C.; Coleman, Rosalind A.

    2015-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (Acsl1T−/−) have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. Acsl1T−/− hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in Acsl1T−/− hearts, control and Acsl1T−/− mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function. PMID:26136511

  3. The Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase from Acinetobacter sp. Strain ADP1: Characterization of a Novel Type of Acyltransferase

    PubMed Central

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

    2005-01-01

    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. PMID:15687201

  4. Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

    PubMed Central

    Lu, J Y; Verkruyse, L A; Hofmann, S L

    1996-01-01

    Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8816748

  5. 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

  6. Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates.

    PubMed

    Tagami, Takayoshi; Okuyama, Masayuki; Nakai, Hiroyuki; Kim, Young-Min; Mori, Haruhide; Taguchi, Kazunori; Svensson, Birte; Kimura, Atsuo

    2013-01-01

    Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K(m) for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K(m) values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K(m) values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K(m) value of maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs. PMID:23457711

  7. 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. PMID:25030454

  8. 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

  9. 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.

  10. The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.

    PubMed

    Simic, Zeljko; Weiwad, Matthias; Schierhorn, Angelika; Steegborn, Clemens; Schutkowski, Mike

    2015-11-01

    Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl-CoA compounds. PMID:26382620

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

    PubMed

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

    2016-08-18

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

  12. 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…

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

    PubMed

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

    1998-08-14

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

  14. Glucose/lipid mixed substrates as a means of controlling the properties of medium chain length poly(hydroxyalkanoates).

    PubMed

    Ashby, R D; Solaiman, D K; Foglia, T A; Liu, C K

    2001-01-01

    Glucose-triacylglycerol (TAG) mixed substrates were used to modulate the physical and mechanical properties of medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs). Pseudomonas resinovorans NRRL B-2649 grew and produced mcl-PHAs on glucose and TAGs (coconut oil, C; soybean oil, S) after 24 h in a shake flask culture. However, with the exception of coconut oil, maximum cell productivity was not reached in any of the cultures until 72 h post-inoculation. Here, 50:50 mixtures of glucose and coconut oil (glc/C) or glucose and soybean oil (glc/S) resulted in intermediate cell productivities with a maximum of 57% and 48% of the CDW at 72 h, respectively. In addition, mixed substrates resulted in mcl-PHAs with compositions that varied slightly over time. PHA-glc/C and PHA-glc/S were composed of 7 mol % and 8 mol % 3-hydroxydodecenoic acid (C(12:1)), respectively at 72 h. These concentrations were intermediate to the C(12:1) concentration of PHA-glc and respective PHA-TAG. Also, significant amounts of 3-hydroxytetradecanoic acid (C(14:0)), 3-hydroxytetradecenoic acid (C(14:1)), and 3-hydroxytetradecadienoic acid (C(14:2)) were present in PHA-glc/C and PHA-glc/S, which were derived from the respective TAG, as glucose resulted in almost no C(14:)(X) monomers. The molar masses of each of the polymers remained relatively constant between 24 and 96 h. At 72 h, the number-average molar masses (M(n)) of PHA-glc/C and PHA-glc/S were 178,000 and 163,000 g/mol, respectively, which were also intermediate to the M(n) of PHA-glc (225,000 g/mol) and the respective PHA-TAG (PHA-C = 153,000 g/mol; PHA-S = 75,000 g/mol). These physical differences caused variations in the mechanical properties of mcl-PHA films, thus providing a new and effective method of modifying their properties.

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  17. A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis†

    PubMed Central

    Liu, Ce Feng; Liu, Dali; Momb, Jessica; Thomas, Pei W.; Lajoie, Ashley; Petsko, Gregory A.; Fast, Walter; Ringe, Dagmar

    2013-01-01

    AiiA is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity, but shows preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center, but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and F107W results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate’s N-acyl chain. Two aromatic residues, F64 and F68 form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure’s decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics. PMID:23387521

  18. 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.

  19. 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

  20. N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF.

    PubMed

    Imker, Heidi J; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T

    2010-10-29

    Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on coexpression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr(1) amino group and generate the fatty acyl-Thr(1)-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis.

  1. 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

  2. Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.

    PubMed

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

    2008-01-01

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

  3. An Open-label Phase 2 Study of UX007 (Triheptanoin) in Subjects With Long-Chain Fatty Acid Oxidation Disorders (LC-FAOD)

    ClinicalTrials.gov

    2015-12-15

    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

  4. Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

    PubMed

    Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

    2013-01-17

    Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis.

  5. Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

    PubMed

    Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

    2013-01-01

    Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis. PMID:23344208

  6. 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.

  7. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation.

    PubMed

    Abbadi, Amine; Domergue, Fréderic; Bauer, Jörg; Napier, Johnathan A; Welti, Ruth; Zähringer, Ulrich; Cirpus, Petra; Heinz, Ernst

    2004-10-01

    Omega6- and omega3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Delta6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Delta6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Delta6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.

  8. Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation.

    PubMed

    Sinha, Dipali; Marcinkiewicz, Mariola; Navaneetham, Duraiswamy; Walsh, Peter N

    2007-08-28

    Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). To gain further insight into the mechanisms of activation of FIX by FXIa, we have investigated the kinetic properties of FXIa-light chain (FXIa-LC) with its active site occupied by either a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB) or a small peptidyl substrate (S-2366) and have examined FIX cleavage products resulting from activation by FXIa or FXIa-LC. PAB inhibited the hydrolysis of S-2366 by FXIa-LC in a classically competitive fashion. In contrast, PAB was found to be a noncompetitive inhibitor of the activation of the macromolecular substrate FIX. Occupancy of the active site of the FXIa-LC by S-2366 also resulted in noncompetitive inhibition of FIX activation. These results demonstrate the presence of an exosite for FIX binding on the FXIa-LC remote from its active site. Furthermore, examination of the cleavage products of FIX indicated that in the absence of either Ca2+ or the heavy chain of FXIa there was substantial accumulation of the inactive intermediate FIXalpha, indicating a slower rate of cleavage of the scissile bond Arg180-Val181. We conclude that binding to two substrate-binding exosites one on the heavy chain and the other on the light chain of FXIa is required to mediate the formation of the Michaelis complex and efficient cleavages of the two spatially separated scissile bonds of FIX. PMID:17676929

  9. Atomic-Level Characterization of the Chain-Flipping Mechanism in Fatty-Acids Biosynthesis.

    PubMed

    Colizzi, Francesco; Masetti, Matteo; Recanatini, Maurizio; Cavalli, Andrea

    2016-08-01

    During fatty acids biosynthesis the elongating acyl chain is sequestered within the core of the highly conserved acyl carrier protein (ACP). At each catalytic step, the acyl intermediates are transiently delivered from ACP to the active site of the enzymatic counterparts and, at the same time, are protected from the solvent to prevent nonselective reactivity. Yet, the molecular determinants of such a universal transition-termed chain flipping-remain poorly understood. Here we capture the atomic-level details of the chain-flipping mechanism by using metadynamics simulations. We observe the fatty-acid chain gliding through the protein-protein interface with barely 30% of its surface exposed to water molecules. The small ACP's helix III acts as gatekeeper of the process, and we find its conformational plasticity critical for a successful substrate transfer. The results are in agreement with a wide range of experimental observations and provide unprecedented insight on the molecular determinants and driving forces of the chain-flipping process. PMID:27409360

  10. Atomic-Level Characterization of the Chain-Flipping Mechanism in Fatty-Acids Biosynthesis.

    PubMed

    Colizzi, Francesco; Masetti, Matteo; Recanatini, Maurizio; Cavalli, Andrea

    2016-08-01

    During fatty acids biosynthesis the elongating acyl chain is sequestered within the core of the highly conserved acyl carrier protein (ACP). At each catalytic step, the acyl intermediates are transiently delivered from ACP to the active site of the enzymatic counterparts and, at the same time, are protected from the solvent to prevent nonselective reactivity. Yet, the molecular determinants of such a universal transition-termed chain flipping-remain poorly understood. Here we capture the atomic-level details of the chain-flipping mechanism by using metadynamics simulations. We observe the fatty-acid chain gliding through the protein-protein interface with barely 30% of its surface exposed to water molecules. The small ACP's helix III acts as gatekeeper of the process, and we find its conformational plasticity critical for a successful substrate transfer. The results are in agreement with a wide range of experimental observations and provide unprecedented insight on the molecular determinants and driving forces of the chain-flipping process.

  11. 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

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

    PubMed

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

    1995-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-09-01

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

  15. 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

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

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

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

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

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

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

  18. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    SciTech Connect

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H. )

    1991-04-15

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC.

  19. Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX.

    PubMed

    Ogawa, Taketoshi; Verhamme, Ingrid M; Sun, Mao-Fu; Bock, Paul E; Gailani, David

    2005-06-24

    Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions. PMID:15829482

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

    PubMed

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

    2016-01-01

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

  1. Measurement of β-oxidation capacity of biological samples by respirometry: a review of principles and substrates.

    PubMed

    Ojuka, Edward; Andrew, Brittany; Bezuidenhout, Nicole; George, Siddiqah; Maarman, Gerald; Madlala, Hlengiwe P; Mendham, Amy; Osiki, Prisca Ofure

    2016-05-01

    Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The β-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.

  2. Experimental and computational studies on the unusual substrate specificity of branched-chain amino acid aminotransferase from Thermoproteus uzoniensis.

    PubMed

    Bezsudnova, Ekaterina Yu; Stekhanova, Tatiana N; Suplatov, Dmitry A; Mardanov, Andrey V; Ravin, Nikolai V; Popov, Vladimir O

    2016-10-01

    PLP-Dependent fold-type IV branched-chain amino acid aminotransferases (BCATs) from archaea have so far been poorly characterized. A new BCAT from the hyperthermophilic archaeon Thermoproteus uzoniensis (TUZN1299) has been studied. TUZN1299 was found to be highly active toward branched-chain amino acids (BCAAs), positively charged amino acids, l-methionine, l-threonine, l-homoserine, l-glutamine, as well as toward 2-oxobutyrate and keto analogs of BCAAs, whereas l-glutamate and α-ketoglutarate were not converted in the overall reaction. According to stopped-flow experiments, the enzyme showed the highest specificity to BCAAs and their keto analogs. In order to explain the molecular mechanism of the unusual specificity of TUZN1299, bioinformatic analysis was implemented to identify the subfamily-specific positions in the aminotransferase class IV superfamily of enzymes. The role of the selected residues in binding of various ligands in the active site was further studied using molecular modeling. The results indicate that Glu188 forms a novel binding site for positively charged and polar side-chains of amino acids. Lack of accommodation for α-ketoglutarate and l-glutamate is due to the unique orientation and chemical properties of residues 102-106 in the loop forming the A-pocket. The likely functional roles of TUZN1299 in cellular metabolism - in the synthesis and degradation of BCAAs - are discussed. PMID:27523731

  3. Experimental and computational studies on the unusual substrate specificity of branched-chain amino acid aminotransferase from Thermoproteus uzoniensis.

    PubMed

    Bezsudnova, Ekaterina Yu; Stekhanova, Tatiana N; Suplatov, Dmitry A; Mardanov, Andrey V; Ravin, Nikolai V; Popov, Vladimir O

    2016-10-01

    PLP-Dependent fold-type IV branched-chain amino acid aminotransferases (BCATs) from archaea have so far been poorly characterized. A new BCAT from the hyperthermophilic archaeon Thermoproteus uzoniensis (TUZN1299) has been studied. TUZN1299 was found to be highly active toward branched-chain amino acids (BCAAs), positively charged amino acids, l-methionine, l-threonine, l-homoserine, l-glutamine, as well as toward 2-oxobutyrate and keto analogs of BCAAs, whereas l-glutamate and α-ketoglutarate were not converted in the overall reaction. According to stopped-flow experiments, the enzyme showed the highest specificity to BCAAs and their keto analogs. In order to explain the molecular mechanism of the unusual specificity of TUZN1299, bioinformatic analysis was implemented to identify the subfamily-specific positions in the aminotransferase class IV superfamily of enzymes. The role of the selected residues in binding of various ligands in the active site was further studied using molecular modeling. The results indicate that Glu188 forms a novel binding site for positively charged and polar side-chains of amino acids. Lack of accommodation for α-ketoglutarate and l-glutamate is due to the unique orientation and chemical properties of residues 102-106 in the loop forming the A-pocket. The likely functional roles of TUZN1299 in cellular metabolism - in the synthesis and degradation of BCAAs - are discussed.

  4. Fat Metabolism in Higher Plants: LXII. Stearl-acyl Carrier Protein Desaturase from Spinach Chloroplasts.

    PubMed

    Jacobson, B S; Jaworski, J G; Stumpf, P K

    1974-10-01

    Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds.

  5. Clarification on the decarboxylation mechanism in KasA based on the protonation state of key residues in the acyl-enzyme state.

    PubMed

    Lee, Wook; Engels, Bernd

    2013-07-11

    The β-ketoacyl ACP synthase I (KasA) is a promising drug target because it is essential for the survival of Mycobacterium tuberculosis , a causative agent of tuberculosis. It catalyzes a condensation reaction that comprises three steps. The resulting elongated acyl chains are subsequently needed for the cell wall construction. While the mechanism of the first step (acylation of Cys171 in the active site) is straightforward already, the second step (decarboxylation of malonyl substrate) has been controversial due to the difficulty in determining the correct protonation states of the involved residues (His311, His345, Lys340, Glu354). Available experimental data suggest three possible mechanisms which differ considerably. They are not consistent with each other because these studies could not be performed for KasA at the beginning of decarboxylation step (acyl-enzyme state of KasA). Instead, different mutants had to be used which are expected to resemble this situation. In this first computational study about this topic, we use the free energy perturbation (FEP) method to compute the relevant pKa values in the acyl-enzyme state of KasA and use molecular dynamics (MD) simulations to rationalize the results. Subsequent density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) MD simulations and umbrella samplings have been used to disentangle the close relationships between the protonation states of the involved residues. By these simulations, we can address the preferred protonation states and roles of the residues involved in decarboxylation reaction, thereby suggesting the possible mechanism for the decarboxylation step. PMID:23768199

  6. Characterization of the Mycobacterial Acyl-CoA Carboxylase Holo Complexes Reveals Their Functional Expansion into Amino Acid Catabolism

    PubMed Central

    Ehebauer, Matthias T.; Zimmermann, Michael; Jakobi, Arjen J.; Noens, Elke E.; Laubitz, Daniel; Cichocki, Bogdan; Marrakchi, Hedia; Lanéelle, Marie-Antoinette; Daffé, Mamadou; Sachse, Carsten; Dziembowski, Andrzej; Sauer, Uwe; Wilmanns, Matthias

    2015-01-01

    Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these - the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α−β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications. PMID:25695631

  7. The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila.

    PubMed

    Jaspers, Martin H J; Pflanz, Ralf; Riedel, Dietmar; Kawelke, Steffen; Feussner, Ivo; Schuh, Reinhard

    2014-01-01

    The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.

  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. ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.

    PubMed

    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

    2015-03-01

    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

  10. Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.

    PubMed

    Arndt, Joseph W; Chai, Qing; Christian, Todd; Stevens, Raymond C

    2006-03-14

    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.

  11. 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 ...

  12. 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.

  13. Determination of residues responsible for substrate and product specificity of Solanum habrochaites short-chain cis-prenyltransferases.

    PubMed

    Kang, Jin-Ho; Gonzales-Vigil, Eliana; Matsuba, Yuki; Pichersky, Eran; Barry, Cornelius S

    2014-01-01

    Isoprenoids are diverse compounds that have their biosynthetic origin in the initial condensation of isopentenyl diphosphate and dimethylallyl diphosphate to form C10 prenyl diphosphates that can be elongated by the addition of subsequent isopentenyl diphosphate units. These reactions are catalyzed by either cis-prenyltransferases (CPTs) or trans-prenyltransferases. The synthesis of volatile terpenes in plants typically proceeds through either geranyl diphosphate (C10) or trans-farnesyl diphosphate (C15), to yield monoterpenes and sesquiterpenes, respectively. However, terpene biosynthesis in glandular trichomes of tomato (Solanum lycopersicum) and related wild relatives also occurs via the cis-substrates neryl diphosphate (NPP) and 2Z,6Z-farnesyl diphosphate (Z,Z-FPP). NPP and Z,Z-FPP are synthesized by neryl diphosphate synthase1 (NDPS1) and Z,Z-farnesyl diphosphate synthase (zFPS), which are encoded by the orthologous CPT1 locus in tomato and Solanum habrochaites, respectively. In this study, comparative sequence analysis of NDPS1 and zFPS enzymes from S. habrochaites accessions that synthesize either monoterpenes or sesquiterpenes was performed to identify amino acid residues that correlate with the ability to synthesize NPP or Z,Z-FPP. Subsequent structural modeling, coupled with site-directed mutagenesis, highlighted the importance of four amino acids located within conserved domain II of CPT enzymes that form part of the second α-helix, for determining substrate and product specificity of these enzymes. In particular, the relative positioning of aromatic amino acid residues at positions 100 and 107 determines the ability of these enzymes to synthesize NPP or Z,Z-FPP. This study provides insight into the biochemical evolution of terpene biosynthesis in the glandular trichomes of Solanum species.

  14. 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. PMID:26795690

  15. 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. PMID:26635357

  16. 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.

  17. 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. PMID:21071236

  18. 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

  19. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

    PubMed

    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. PMID:27540631

  20. Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs.

    PubMed

    Romano, Andrea; van der Plas, Linus H W; Witholt, Bernard; Eggink, Gerrit; Mooibroek, Hans

    2005-01-01

    Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants. PMID:15351883

  1. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed Central

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase. PMID:12228351

  2. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

  3. Biochemical Competition Makes Fatty-Acid β-Oxidation Vulnerable to Substrate Overload

    PubMed Central

    van Eunen, Karen; Simons, Sereh M. J.; Gerding, Albert; Bleeker, Aycha; den Besten, Gijs; Touw, Catharina M. L.; Houten, Sander M.; Groen, Bert K.; Krab, Klaas; Reijngoud, Dirk-Jan; Bakker, Barbara M.

    2013-01-01

    Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD+]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration. PMID:23966849

  4. Biochemical competition makes fatty-acid β-oxidation vulnerable to substrate overload.

    PubMed

    van Eunen, Karen; Simons, Sereh M J; Gerding, Albert; Bleeker, Aycha; den Besten, Gijs; Touw, Catharina M L; Houten, Sander M; Groen, Bert K; Krab, Klaas; Reijngoud, Dirk-Jan; Bakker, Barbara M

    2013-01-01

    Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD⁺]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration.

  5. 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.

  6. 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. PMID:25958146

  7. 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.

  8. A Novel Auxin Conjugate Hydrolase from Wheat with Substrate Specificity for Longer Side-Chain Auxin Amide Conjugates1

    PubMed Central

    Campanella, James J.; Olajide, Adebanke F.; Magnus, Volker; Ludwig-Müller, Jutta

    2004-01-01

    This study investigates how the ILR1-like indole acetic acid (IAA) amidohydrolase family of genes has functionally evolved in the monocotyledonous species wheat (Triticum aestivum). An ortholog for the Arabidopsis IAR3 auxin amidohydrolase gene has been isolated from wheat (TaIAR3). The TaIAR3 protein hydrolyzes negligible levels of IAA-Ala and no other IAA amino acid conjugates tested, unlike its ortholog IAR3. Instead, TaIAR3 has low specificity for the ester conjugates IAA-Glc and IAA-myoinositol and high specificity for the conjugates of indole-3-butyric acid (IBA-Ala and IBA-Gly) and indole-3-propionic-acid (IPA-Ala) so far tested. TaIAR3 did not convert the methyl esters of the IBA conjugates with Ala and Gly. IBA and IBA conjugates were detected in wheat seedlings by gas chromatography-mass spectrometry, where the conjugate of IBA with Ala may serve as a natural substrate for this enzyme. Endogenous IPA and IPA conjugates were not detected in the seedlings. Additionally, crude protein extracts of wheat seedlings possess auxin amidohydrolase activity. Temporal expression studies of TaIAR3 indicate that the transcript is initially expressed at day 1 after germination. Expression decreases through days 2, 5, 10, 15, and 20. Spatial expression studies found similar levels of expression throughout all wheat tissues examined. PMID:15299127

  9. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    PubMed

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A; Meredith, Timothy C

    2012-10-01

    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.

  10. Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates.

    PubMed

    Magomedova, Zalina; Grecu, Andreea; Sensen, Christoph W; Schwab, Helmut; Heidinger, Petra

    2016-03-10

    Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.

  11. Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol.

    PubMed

    Fisher, Michael A; Boyarskiy, Sergey; Yamada, Masaki R; Kong, Niwen; Bauer, Stefan; Tullman-Ercek, Danielle

    2014-01-17

    The microbial conversion of sugars to fuels is a promising technology, but the byproducts of biomass pretreatment processes and the fuels themselves are often toxic at industrially relevant levels. One promising solution to these problems is to engineer efflux pumps to secrete fuels and inhibitory chemicals from the cell, increasing microbial tolerance and enabling higher fuel titer. Toward that end, we used a directed evolution strategy to generate variants of the Escherichia coli AcrB efflux pump that act on the non-native substrate n-butanol, enhancing growth rates of E. coli in the presence of this biofuel by up to 25%. Furthermore, these variants confer improved tolerance to isobutanol and straight-chain alcohols up to n-heptanol. Single amino acid changes in AcrB responsible for this phenotype were identified. We have also shown that both the chemical and genetic inactivation of pump activity eliminate the tolerance conferred by AcrB pump variants, supporting our assertion that the variants secrete the non-native substrates. This strategy can be applied to create an array of efflux pumps that modulate the intracellular concentrations of small molecules of interest to microbial fuel and chemical production. PMID:23991711

  12. 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. PMID:27262877

  13. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-01

    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. PMID:7669809

  14. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-01

    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.

  15. Characterization of a Bifunctional Archaeal Acyl Coenzyme A Carboxylase

    PubMed Central

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

    2003-01-01

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

  16. 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

    2014-03-01

    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.

  17. 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

  18. Stability-increasing effects of anthocyanin glycosyl acylation.

    PubMed

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

    2017-01-01

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

  19. Echium oil increased the expression of a Δ4 Fads2 fatty acyl desaturase and the deposition of n-3 long-chain polyunsaturated fatty acid in comparison with linseed oil in striped snakehead (Channa striata) muscle.

    PubMed

    Jaya-Ram, Annette; Shu-Chien, Alexander Chong; Kuah, Meng-Kiat

    2016-08-01

    Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species.

  20. Echium oil increased the expression of a Δ4 Fads2 fatty acyl desaturase and the deposition of n-3 long-chain polyunsaturated fatty acid in comparison with linseed oil in striped snakehead (Channa striata) muscle.

    PubMed

    Jaya-Ram, Annette; Shu-Chien, Alexander Chong; Kuah, Meng-Kiat

    2016-08-01

    Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species. PMID:26842427

  1. Acyl lipidation of a peptide: effects on activity and epidermal permeability in vitro

    PubMed Central

    Rocco, Daniel; Ross, James; Murray, Paul E; Caccetta, Rima

    2016-01-01

    Short-chain lipid conjugates can increase permeability of a small peptide across human epidermis; however, the emerging lipoaminoacid (LAA) conjugation technique is costly and can deliver mixed synthetic products of varied biological potential. LAA conjugation using a racemic mixture produces a mixture of D- and L-stereoisomers. Individual enantiomers can be produced at an extra cost. We investigated an affordable technique that produces only one synthetic product: short-chain (C7–C8) acyl lipidation. Acyl lipidation of Ala-Ala-Pro-Val, an inhibitor of human neutrophil elastase (HNE; believed to lead to abnormal tissue destruction and disease development), was investigated as an alternative to LAA conjugation. The current study aimed to assess the effects of acyl lipidation (either at the N-terminal or at the C-terminal) on neutrophil elastase activity in vitro and on transdermal delivery ex vivo. The inhibitory capacity of the acyl conjugates was compared to LAA conjugates (conjugated at the N-terminal) of the same peptide. The L-stereoisomer appears to rapidly degrade, but it represents a significantly (P<0.05) better inhibitor of HNE than the parent peptide (Ala-Ala-Pro-Val). Although the D-stereoisomer appears to permeate human epidermal skin sections in a better fashion than the L-stereoisomer, it is not a significantly better inhibitor of HNE than the parent peptide. Acyl lipidation (with a C7 lipid chain) at either end of the peptide substantially enhances the permeability of the peptide across human skin epidermis as well as significantly (P<0.005) increases its elastase inhibitory potential. Therefore, our current study indicates that acyl lipidation of a peptide is a more economical and effective alternative to LAA conjugation. PMID:27468224

  2. Acyl lipidation of a peptide: effects on activity and epidermal permeability in vitro.

    PubMed

    Rocco, Daniel; Ross, James; Murray, Paul E; Caccetta, Rima

    2016-01-01

    Short-chain lipid conjugates can increase permeability of a small peptide across human epidermis; however, the emerging lipoaminoacid (LAA) conjugation technique is costly and can deliver mixed synthetic products of varied biological potential. LAA conjugation using a racemic mixture produces a mixture of D- and L-stereoisomers. Individual enantiomers can be produced at an extra cost. We investigated an affordable technique that produces only one synthetic product: short-chain (C7-C8) acyl lipidation. Acyl lipidation of Ala-Ala-Pro-Val, an inhibitor of human neutrophil elastase (HNE; believed to lead to abnormal tissue destruction and disease development), was investigated as an alternative to LAA conjugation. The current study aimed to assess the effects of acyl lipidation (either at the N-terminal or at the C-terminal) on neutrophil elastase activity in vitro and on transdermal delivery ex vivo. The inhibitory capacity of the acyl conjugates was compared to LAA conjugates (conjugated at the N-terminal) of the same peptide. The L-stereoisomer appears to rapidly degrade, but it represents a significantly (P<0.05) better inhibitor of HNE than the parent peptide (Ala-Ala-Pro-Val). Although the D-stereoisomer appears to permeate human epidermal skin sections in a better fashion than the L-stereoisomer, it is not a significantly better inhibitor of HNE than the parent peptide. Acyl lipidation (with a C7 lipid chain) at either end of the peptide substantially enhances the permeability of the peptide across human skin epidermis as well as significantly (P<0.005) increases its elastase inhibitory potential. Therefore, our current study indicates that acyl lipidation of a peptide is a more economical and effective alternative to LAA conjugation. PMID:27468224

  3. Physical characterisation of high amylose maize starch and acylated high amylose maize starches.

    PubMed

    Lim, Ya-Mei; Hoobin, Pamela; Ying, DanYang; Burgar, Iko; Gooley, Paul R; Augustin, Mary Ann

    2015-03-01

    The particle size, water sorption properties and molecular mobility of high amylose maize starch (HAMS) and high amylose maize starch acylated with acetate (HAMSA), propionate (HAMSP) and butyrate (HAMSB) were investigated. Acylation increased the mean particle size (D(4,3)) and lowered the specific gravity (G) of the starch granules with an inverse relationship between the length of the fatty acid chain and particle size. Acylation of HAMS with fatty acids lowered the monolayer moisture content with the trend being HAMSBchain. Measurement of molecular mobility of the starch granules by NMR spectroscopy with Carr-Purcell-Meiboom-Gill (CMPG) experiments showed that T2 long was reduced in acylated starches and that drying and storage of the starch granules further reduced T2 long. Analysis of the Free Induction Decay (FID) focussing on the short components of T2 (correlated to the solid matrix), indicated that drying and subsequent storage resulted in alterations of starch at 0.33a(w) and that these changes were reduced with acylation. In vitro enzymatic digestibility of heated starch dispersions by bacterial α-amylase was increased by acylation (HAMSchain. Digestibility was enhanced with an increase in particle size, or decrease in G, and inversely proportional to the total T2 signal. It is suggested that both external surface area and an internal network of pores and channels collectively influence the digestibility of starch. PMID:25498636

  4. Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins.

    PubMed

    Feldman, Jessica L; Baeza, Josue; Denu, John M

    2013-10-25

    Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD(+)-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ε-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.

  5. Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

    PubMed

    Carpenter, K; Pollitt, R J; Middleton, B

    1992-03-16

    We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.

  6. Novel cinchona carbamate selectors with complementary enantioseparation characteristics for N-acylated amino acids.

    PubMed

    Krawinkler, Karl Heinz; Maier, Norbert M; Ungaro, Rocco; Sansone, Francesco; Casnati, Alessandro; Lindner, Wolfgang

    2003-01-01

    The synthesis and chromatographic evaluation of the enantiomer separation capabilities of covalently immobilized calix[4]arene-cinchona carbamate hybrid type receptors derived from quinine (QN) and its corresponding C9-epimer (eQN) in different solvents are reported. The receptors display complementary enantiomer separation profiles in terms of elution order, chiral substrate specificity, and mobile phase characteristics, indicating the existence of two distinct chiral recognition mechanisms. The QN-derived receptor binds the (S)-enantiomers of N-acylated amino acids more strongly, shows preferential recognition of open-chained amino acids, and superior enantioselectivity in polar media such as methanol/acetic acid. In contrast, the eQN congener preferentially recognizes the corresponding (R)-enantiomers, displays good enantioselectivity (alpha up to 1.74) for cyclic amino acids, and enhanced stereodiscriminating properties in apolar mobile phases, e.g., chloroform/acetic acid. A comparison of the enantiomer separation profiles with those of the corresponding QN and eQN tert-butyl carbamate congeners indicates no significant level of cooperativity between the calix[4]arene module and the cinchona units in terms of overall chiral recognition, most probably as a consequence of residual conformational flexibility of the calixarene module and the carbamate linkage.

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

    SciTech Connect

    Hayashi, H.; Miwa, A. )

    1989-11-01

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

  8. Efficient mono-acylation of fructose by lipase-catalyzed esterification in ionic liquid co-solvents.

    PubMed

    Li, Lu; Ji, Fangling; Wang, Jingyun; Jiang, Bo; Li, Yachen; Bao, Yongming

    2015-10-30

    Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi-Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents.

  9. Efficient mono-acylation of fructose by lipase-catalyzed esterification in ionic liquid co-solvents.

    PubMed

    Li, Lu; Ji, Fangling; Wang, Jingyun; Jiang, Bo; Li, Yachen; Bao, Yongming

    2015-10-30

    Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi-Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents. PMID:26343327

  10. Site‐Selective Acylations with Tailor‐Made Catalysts

    PubMed Central

    Huber, Florian

    2016-01-01

    Abstract The acylation of alcohols catalyzed by N,N‐dimethylamino pyridine (DMAP) is, despite its widespread use, sometimes confronted with substrate‐specific problems: For example, target compounds with multiple hydroxy groups may show insufficient selectivity for one hydroxyl, and the resulting product mixtures are hardly separable. Here we describe a concept that aims at tailor‐made catalysts for the site‐specific acylation. To this end, we introduce a catalyst library where each entry is constructed by connecting a variable and readily tuned peptide scaffold with a catalytically active unit based on DMAP. For selected examples, we demonstrate how library screening leads to the identification of optimized catalysts, and the substrates of interest can be converted with a markedly enhanced site‐selectivity compared with only DMAP. Furthermore, substrate‐optimized catalysts of this type can be used to selectively convert “their” substrate in the presence of structurally similar compounds, an important requisite for reactions with mixtures of substances. PMID:26970553

  11. 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.

  12. 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...

  13. 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.

  14. Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

    PubMed

    Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

    2008-12-25

    Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

  15. Study of Triheptanoin for Treatment of Long-Chain Fatty Acid Oxidation Disorder

    ClinicalTrials.gov

    2015-04-20

    Very Long-chain acylCoA Dehydrogenase (VLCAD) Deficiency; Carnitine Palmitoyltransferase 2 (CPT2) Deficiency; Mitochondrial Trifunctional Protein (TFP) Deficiency; Long-chain 3 hydroxyacylCoA Dehydrogenase (LCHAD) Deficiency

  16. Antifibrotic Activity of Acylated and Unacylated Ghrelin

    PubMed Central

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

    2015-01-01

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

  17. Topo-optical reactions for the identification of O-acyl sugars in amyloid deposits.

    PubMed

    Richter, Susann; Makovitzky, Josef

    2009-01-01

    The aldehyde bisulfite toluidine blue (ABT) reaction with former saponification (KOH-ABT) and periodic acid-borohydride reduction-saponification (PB-KOH-ABT) were applied to sections of human amyloid deposits in the respiratory tract. The saponification-induced increase in ABT-reactivity was confined to the presence of O-acyl sugars associated with the amyloid fibrils. The anisotropic and metachromatic effect in the ABT and KOH-ABT reaction was reduced in the corresponding PB-KOH-ABT reaction, a difference attributed to the removal of staining due to neutral carbohydrate residues. Since the periodic acid-borohydride reduction abolishes all pre-existing ABT-reactivity of neutral sugar vicinal diols, the isolated KOH-effect could be shown using the PB-KOH-ABT reaction. By application of this sequence, the problem identifying small quantities of O-acyl sugars was solved. It is suggested that the KOH-effect depends upon the removal of O-acyl substituents located on the polyhydroxy side chain (C7, C8, C9) of sialic acid residues. An advantage of such topo-optical reactions over biochemical techniques is the exact localization of O-acyl sugars in tissue sites. By means of the KOH-ABT and PB-KOH-ABT reactions we have demonstrated, for the first time, that O-acyl sugars occur within amyloid deposits.

  18. Novel deletion in a patient with an isolated peroxisoml acyl-CoA oxidase deficiency

    SciTech Connect

    Poll-The, B.T.; Fournier, B.; Clevers, H.; Wanders, R.J.A.

    1994-09-01

    Disorders with defective peroxisome assembly are associated with multiple peroxisomal enzymatic abnormalities. Besides these diseases patients have been described suspected of having a single enzyme defect in the peroxisomal {beta}-oxidation pathway. Laboratory findings for these patients include elevated plasma very long chain fatty acids (VLCFA) and impaired VLCFA oxidation in fibroblasts. Complementation analysis between these patients and those with a proven single enzyme deficiency, using peroxisomal {beta}-oxidation of VLCFA as the criterion for complementation, has been used to show whether the patients are deficient in acyl-CoA oxidase, peroxisomal trifunctional protein or thiolase activity. Fibroblasts from a patient showing the clinical and biochemical abnormalities of isolated acyl-CoA oxidase deficiency (using cell complementation) were analyzed at the molecular level. Isolation of RNA from patient`s fibroblasts was followed by random reverse transcription of RNA and PCR amplification. PCR products were blotted and hybridized with the human acyl-CoA oxidase cDNA. A fragment 150 bp shorter than normal was found. Upon sequencing, exon 7 was found to be deleted leading to a frameshift in the acyl-CoA oxidase mRNA. Southern blot analysis of the patient`s DNA did not reveal any deletion in contrast to two siblings previously reported as having a deletion of at least 17 kb in the acyl-CoA oxidase gene.

  19. Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

    SciTech Connect

    de Vetten, M.P.; Agre, P.

    1988-12-05

    The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with (3H)palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.

  20. 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.

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

    PubMed Central

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

    2013-01-01

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

  2. 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.

  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. 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.

  5. 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.

  6. 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

  7. Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.

    PubMed

    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 mitochondrial

  8. Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.

    PubMed

    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 mitochondrial

  9. 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.

  10. Microbial Tailoring of Acyl Peptidic Siderophores

    PubMed Central

    2015-01-01

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

  11. Laser desorption/ionization mass spectrometry of lipids using etched silver substrates.

    PubMed

    Schnapp, Andreas; Niehoff, Ann-Christin; Koch, Annika; Dreisewerd, Klaus

    2016-07-15

    Silver-assisted laser desorption/ionization mass spectrometry can be used for the analysis of small molecules. For example, adduct formation with silver cations enables the molecular analysis of long-chain hydrocarbons, which are difficult to ionize via conventional matrix-assisted laser desorption ionization (MALDI). Here we used highly porous silver foils, produced by etching with nitric acid, as sample substrates for LDI mass spectrometry. As model system for the analysis of complex lipid mixtures, cuticular extracts of fruit flies (Drosophila melanogaster) and worker bees (Apis mellifera) were investigated. The mass spectra obtained by spotting extract onto the etched silver substrates demonstrate the sensitive detection of numerous lipid classes such as long-chain saturated and unsaturated hydrocarbons, fatty acyl alcohols, wax esters, and triacylglycerols. MS imaging of cuticular surfaces with a lateral resolution of a few tens of micrometers became possible after blotting, i.e., after transferring lipids by physical contact with the substrate. The examples of pheromone-producing male hindwings of the squinting bush brown butterfly (Bicyclus anynana) and a fingermark are shown. Because the substrates are also easy to produce, they provide a viable alternative to colloidal silver nanoparticles and other so far described silver substrates.

  12. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

    PubMed Central

    Nguyen, Chi; Haushalter, Robert W.; Lee, D. John; Markwick, Phineus R. L.; Bruegger, Joel; Caldara-Festin, Grace; Finzel, Kara; Jackson, David R.; Ishikawa, Fumihiro; O’Dowd, Bing; McCammon, J. Andrew; Opella, Stanley J.; Tsai, Shiou-Chuan; Burkart, Michael D.

    2015-01-01

    Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious

  13. 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.

  14. Membrane Topology and Transient Acylation of Toxoplasma gondii Glycosylphosphatidylinositols

    PubMed Central

    Kimmel, Jürgen; Smith, Terry K.; Azzouz, Nahid; Gerold, Peter; Seeber, Frank; Lingelbach, Klaus; Dubremetz, Jean-François; Schwarz, Ralph T.

    2006-01-01

    Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-3H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-3H]mannose or UDP-[6-3H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER. PMID:16896225

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

    PubMed

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

    2014-02-01

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

  16. 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.

  17. 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

  18. Merging Photoredox with Palladium Catalysis: Decarboxylative ortho-Acylation of Acetanilides with α-Oxocarboxylic Acids under Mild Reaction Conditions.

    PubMed

    Zhou, Chao; Li, Pinhua; Zhu, Xianjin; Wang, Lei

    2015-12-18

    A room temperature decarboxylative ortho-acylation of acetanilides with α-oxocarboxylic acids has been developed via a novel Eosin Y with Pd dual catalytic system. This dual catalytic reaction shows a broad substrate scope and good functional group tolerance, and an array of ortho-acylacetanilides can be afforded in high yields under mild conditions.

  19. 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.

  20. 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.

  1. 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.

  2. Pleiotropic Effect of AccD5 and AccE5 Depletion in Acyl-Coenzyme A Carboxylase Activity and in Lipid Biosynthesis in Mycobacteria

    PubMed Central

    Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Cabruja, Matías; Bardou, Fabienne; Quémard, Annaïk; Gago, Gabriela; Gramajo, Hugo

    2014-01-01

    Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs. PMID:24950047

  3. The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

    PubMed

    van Nocker, S; Sadis, S; Rubin, D M; Glickman, M; Fu, H; Coux, O; Wefes, I; Finley, D; Vierstra, R D

    1996-11-01

    The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

  4. Kinetic mechanism and order of substrate binding for sn-glycerol-3-phosphate acyltransferase from squash (Cucurbita moschata).

    PubMed

    Hayman, Matthew W; Fawcett, Tony; Slabas, Antoni R

    2002-03-13

    sn-Glycerol-3-phosphate acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three-dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451-453; Turnbull et al. (2001) Structure 9, 347-353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl-acyl carrier protein (acyl-ACP) substrates. The reaction proceeds via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate. We have also determined that the reaction will proceed with C(4:0)-CoA, C(6:0)-CoA and C(12:0)-ACP substrates, allowing a wider choice of acyl groups for future co-crystallisation studies.

  5. An alternative pathway for the effective production of the omega-3 long-chain polyunsaturates EPA and ETA in transgenic oilseeds.

    PubMed

    Ruiz-Lopez, Noemi; Haslam, Richard P; Usher, Sarah; Napier, Johnathan A; Sayanova, Olga

    2015-12-01

    The synthesis and accumulation of omega-3 long-chain polyunsaturated fatty acids in transgenic Camelina sativa is demonstrated using the so-called alternative pathway. This aerobic pathway is found in a small number of taxonomically unrelated unicellular organisms and utilizes a C18 Δ9-elongase to generate C20 PUFAs. Here, we evaluated four different combinations of seed-specific transgene-derived activities to systematically determine the potential of this pathway to direct the synthesis of eicosapentaenoic acid (EPA) in transgenic plants. The accumulation of EPA and the related omega-3 LC-PUFA eicosatetraenoic acid (ETA) was observed up to 26.4% of total seed fatty acids, of which ETA was 9.5%. Seed oils such as these not only represent an additional source of EPA, but also an entirely new source of the bona fide fish oil ETA. Detailed lipidomic analysis of the alternative pathway in Camelina revealed that the acyl-substrate preferences of the different activities in the pathway can still generate a substrate-dichotomy bottleneck, largely due to inefficient acyl-exchange from phospholipids into the acyl-CoA pool. However, significant levels of EPA and ETA were detected in the triacylglycerols of transgenic seeds, confirming the channelling of these fatty acids into this storage lipid.

  6. GOAT induced ghrelin acylation regulates hedonic feeding.

    PubMed

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

    2012-11-01

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

  7. Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

    PubMed Central

    Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

    2015-01-01

    Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

  8. Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia).

    PubMed

    Ghosh, Santosh K; Bhattacharjee, Ashish; Jha, Jyoti K; Mondal, Ashis K; Maiti, Mrinal K; Basu, Asitava; Ghosh, Dolly; Ghosh, Sudhamoy; Sen, Soumitra K

    2007-12-01

    Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a

  9. Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation.

    PubMed

    Weinhold, Alexander; Baldwin, Ian Thomas

    2011-05-10

    Plant glandular trichomes exude secondary metabolites with defensive functions, but these epidermal protuberances are surprisingly the first meal of Lepidopteran herbivores on Nicotiana attenuata. O-acyl sugars, the most abundant metabolite of glandular trichomes, impart a distinct volatile profile to the body and frass of larvae that feed on them. The headspace composition of Manduca sexta larvae is dominated by the branched chain aliphatic acids hydrolyzed from ingested O-acyl sugars, which waxes and wanes rapidly with trichome ingestion. In native habitats a ground-hunting predator, the omnivorous ant Pogonomyrmex rugosus, but not the big-eyed bug Geocoris spp., use these volatile aliphatic acids to locate their prey.

  10. Kinetic resolution of racemic mandelic acid esters by N,N'-dioxide-scandium-complex-catalyzed enantiomer-selective acylation.

    PubMed

    Zhang, Yuheng; Liu, Xiaohua; Zhou, Lin; Wu, Wangbin; Huang, Tianyu; Liao, Yuting; Lin, Lili; Feng, Xiaoming

    2014-11-24

    A simple and efficient acylative kinetic resolution of racemic mandelic acid esters was accomplished with a chiral N,N'-dioxide-scandium(III) complex under mild and base-free reaction conditions. A variety of mandelic acid esters performed well in the reaction, obtaining both acylated products (up to 49% yield, 97% ee) and recovered substrates (up to 49% yield, 95% ee) in high enantioselectivities with perfect selectivity factors (up to 247). The enantioselective recognition and catalytic models were also proposed for the catalytic KR reaction.

  11. Visible-light-promoted iminyl-radical formation from acyl oximes: a unified approach to pyridines, quinolines, and phenanthridines.

    PubMed

    Jiang, Heng; An, Xiaode; Tong, Kun; Zheng, Tianyi; Zhang, Yan; Yu, Shouyun

    2015-03-23

    A unified strategy involving visible-light-induced iminyl-radical formation has been established for the construction of pyridines, quinolines, and phenanthridines from acyl oximes. With fac-[Ir(ppy)3 ] as a photoredox catalyst, the acyl oximes were converted by 1 e(-) reduction into iminyl radical intermediates, which then underwent intramolecular homolytic aromatic substitution (HAS) to give the N-containing arenes. These reactions proceeded with a broad range of substrates at room temperature in high yield. This strategy of visible-light-induced iminyl-radical formation was successfully applied to a five-step concise synthesis of benzo[c]phenanthridine alkaloids.

  12. Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination.

    PubMed

    Karkabounas, Athanassios; Georgiadou, Dimitra G; Argitis, Panagiotis; Psycharis, Vassilios; Nakos, George; Kosmas, Agni M; Lekka, Marilena E

    2015-12-01

    The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A(2) (PLA(2)) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C(12)-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA(2). Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA(2). Mass spectrometry showed that only C(12)-NBD-FA, the PLA(2 )hydrolysis product, was detected in the reaction mixture, indicating that PLA(2) recognizes PVA-SbQ/C(12)-NBD-PtdCho as a surface to perform catalysis. PMID:26449236

  13. The Two Functional Enoyl-Acyl Carrier Protein Reductases of Enterococcus faecalis Do Not Mediate Triclosan Resistance

    PubMed Central

    Zhu, Lei; Bi, Hongkai; Ma, Jincheng; Hu, Zhe; Zhang, Wenbin; Cronan, John E.; Wang, Haihong

    2013-01-01

    ABSTRACT Enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. The Enterococcus faecalis genome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of an Escherichia coli fabI temperature-sensitive mutant strain under nonpermissive conditions. In vitro assays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression of E. faecalis fabK confers to E. coli high levels of resistance to the antimicrobial triclosan, deletion of fabK from the E. faecalis genome showed that FabK does not play a detectable role in the inherent triclosan resistance of E. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion of fabK grow normally without fatty acid supplementation, whereas fabI deletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs. PMID:24085780

  14. Effect of a mutagenized acyl-ACP thioesterase FATA allele from sunflower with improved activity in tobacco leaves and Arabidopsis seeds.

    PubMed

    Moreno-Pérez, Antonio Javier; Venegas-Calerón, Mónica; Vaistij, Fabián E; Salas, Joaquin J; Larson, Tony R; Garcés, Rafael; Graham, Ian A; Martínez-Force, Enrique

    2014-03-01

    The substrate specificity of the acyl-acyl carrier protein (ACP) thioesterases significantly determines the type of fatty acids that are exported from plastids. Thus, designing acyl-ACP thioesterases with different substrate specificities or kinetic properties would be of interest for plant lipid biotechnology to produce oils enriched in specialty fatty acids. In the present work, the FatA thioesterase from Helianthus annuus was used to test the impact of changes in the amino acids present in the binding pocket on substrate specificity and catalytic efficiency. Amongst all the mutated enzymes studied, Q215W was especially interesting as it had higher specificity towards saturated acyl-ACP substrates and higher catalytic efficiency compared to wild-type H. annuus FatA. Null, wild type and high-efficiency alleles were transiently expressed in tobacco leaves to check their effect on lipid biosynthesis. Expression of active FatA thioesterases altered the composition of leaf triacylglycerols but did not alter total lipid content. However, the expression of the wild type and the high-efficiency alleles in Arabidopsis thaliana transgenic seeds resulted in a strong reduction in oil content and an increase in total saturated fatty acid content. The role and influence of acyl-ACP thioesterases in plant metabolism and their possible applications in lipid biotechnology are discussed.

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

    PubMed

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

    2014-04-01

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

  16. On the mechanism of N-heterocyclic carbene-catalyzed reactions involving acyl azoliums.

    PubMed

    Mahatthananchai, Jessada; Bode, Jeffrey W

    2014-02-18

    , however, date back to the intense investigation of thiamine-dependent enzymatic processes in the 1960s. Acyl azoliums are remarkably reactive in acylation chemistry and are unusually chemoselective. These two properties have led to a new wave of reactions such as redox esterification reaction (1) and the catalytic kinetic resolution of challenging substrates (i.e., 3). Our group and others have also developed methods to generate and exploit α,β-unsaturated acyl azoliums, which have facilitated new C-C bond-forming annulations, including a catalytic, enantioselective variant of the Claisen rearrangement (2). From essentially one class of catalysts, the N-mesityl derived triazolium salts, researchers can easily prepare highly enantioenriched dihydropyranones and dihydropyridinones. Although this field is now one of the most explored areas of enantioselective C-C bond forming reactions, many mechanistic details remained unsolved and in dispute. In this Account, we address the mechanistic inquiries about the characterization of the unsaturated acyl triazolium species and its kinetic profile under catalytically relevant conditions. We also provide explanations for the requirement and effect of the N-mesityl group in NHC catalysis based on detailed experimental data within given specific reactions or conditions. We hope that our studies provide a roadmap for catalyst design/selection and new reaction discovery based on a fundamental understanding of the mechanistic course of NHC reactions.

  17. 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.

  18. 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

  19. Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function

    PubMed Central

    Stanley, Peter; Koronakis, Vassilis; Hughes, Colin

    1998-01-01

    The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a “double-anchor” motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation. PMID:9618444

  20. 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.

  1. 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.

  2. 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.

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

    PubMed

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

    2016-05-01

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

  4. Acyl Coenzyme A Thioesterase Them5/Acot15 Is Involved in Cardiolipin Remodeling and Fatty Liver Development

    PubMed Central

    Gut, Heinz; Hynx, Debby; Marcellin, David; Bleck, Christopher K. E.; Genoud, Christel; Cron, Peter; Keusch, Jeremy J.; Dummler, Bettina; Esposti, Mauro Degli

    2012-01-01

    Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5−/− mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions. PMID:22586271

  5. Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development.

    PubMed

    Zhuravleva, Elena; Gut, Heinz; Hynx, Debby; Marcellin, David; Bleck, Christopher K E; Genoud, Christel; Cron, Peter; Keusch, Jeremy J; Dummler, Bettina; Esposti, Mauro Degli; Hemmings, Brian A

    2012-07-01

    Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5(-/-) mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions. PMID:22586271

  6. 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

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

    PubMed

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

    2010-10-01

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

  8. Microbial production of short-chain alkanes.

    PubMed

    Choi, Yong Jun; Lee, Sang Yup

    2013-10-24

    Increasing concerns about limited fossil fuels and global environmental problems have focused attention on the need to develop sustainable biofuels from renewable resources. Although microbial production of diesel has been reported, production of another much in demand transport fuel, petrol (gasoline), has not yet been demonstrated. Here we report the development of platform Escherichia coli strains that are capable of producing short-chain alkanes (SCAs; petrol), free fatty acids (FFAs), fatty esters and fatty alcohols through the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway. First, the β-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo. To increase the formation of short-chain fatty acids suitable for subsequent conversion to SCAs in vivo, the activity of 3-oxoacyl-ACP synthase (FabH), which is inhibited by unsaturated fatty acyl-ACPs, was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene; deletion of the fadR gene prevents upregulation of the fabA and fabB genes responsible for unsaturated fatty acids biosynthesis. A modified thioesterase was used to convert short-chain fatty acyl-ACPs to the corresponding FFAs, which were then converted to SCAs by the sequential reactions of E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase and Arabidopsis thaliana fatty aldehyde decarbonylase. The final engineered strain produced up to 580.8 mg l(-1) of SCAs consisting of nonane (327.8 mg l(-1)), dodecane (136.5 mg l(-1)), tridecane (64.8 mg l(-1)), 2-methyl-dodecane (42.8 mg l(-1)) and tetradecane (8.9 mg l(-1)), together with small amounts of other hydrocarbons. Furthermore, this platform strain could produce short-chain FFAs using a fadD-deleted strain, and short-chain fatty esters by introducing the Acinetobacter sp. ADP1 wax ester synthase (atfA) and the E. coli mutant

  9. Protease-catalyzed peptide synthesis using inverse substrates: the synthesis of Pro-Xaa-bonds by trypsin.

    PubMed

    Schellenberger, V; Schellenberger, U; Jakubke, H D; Zapevalova, N P; Mitin, Y V

    1991-07-01

    Benzyloxycarbonyl-L-proline p-guanidinophenyl ester is an "inverse substrate" for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin-catalyzed acyl-transfer reactions leading to the synthesis of Pro-Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine-containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P(1)'-position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl-enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl-enzyme. PMID:18600766

  10. [Antibacterial Activity of Alkylated and Acylated Derivatives of Low-Molecular Weight Chitosan].

    PubMed

    Shagdarova, B Ts; Il'ina, A V; Varlamov, V P

    2016-01-01

    A number of alkylated (quaternized) and acylated derivatives of low-molecular weight chitosan were obtained. The structure and composition of the compounds were confirmed by the results of IR and PMR spectroscopy, as well as conductometric titration. The effect of the acyl substituent and the degree of substitution of N-(2-hydroxy-3-trimethylammonium) with the propyl fragment appended to amino groups of the C2 atom of polymer chains on antibacterial activity against typical representatives of gram-positive and gram-negative microorganisms (Staphylococcus epidermidis and Escherichia coli) was studied. The highest activity was in the case of N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride with the maximal substitution (98%). The minimal inhibitory concentration of the derivative was 0.48 µg/mL and 3.90 µg/mL for S. epidermis and E. coli, respectively. PMID:27266254

  11. New Acylated Flavonol Glycosides and a Phenolic Profile of Pritzelago alpina, a Forgotten Edible Alpine Plant.

    PubMed

    Corradi, Elisabetta; De Mieri, Maria; Cadisch, Larissa; Abbet, Christian; Hamburger, Matthias; Potterat, Olivier

    2016-02-01

    Thirteen acylated flavonoid glycosides, 1-13, including eleven new congeners, 3-13, were isolated from the aerial parts of Pritzelago alpina (Brassicaceae) by a combination of column chromatography on Sephadex LH-20, and preparative and semi-preparative HPLC. The structures were established by extensive NMR and MS experiments in combination with acid hydrolysis and sugar analysis by GC/MS. The new compounds were shown to be kaempferol and quercetin glycosides acylated for most of them by a branched short chain fatty acid or a hydroxycinnamic acid residue on the sugar portion. As shown by a HPLC-DAD analysis of a MeOH extract, these compounds are the main phenolic constituents in the aerial parts of the plant. PMID:26880431

  12. Inhibition of Lux quorum-sensing system by synthetic N-acyl-L-homoserine lactone analogous.

    PubMed

    Wang, Wenzhao; Morohoshi, Tomohiro; Ikeda, Tsukasa; Chen, Liang

    2008-12-01

    In the present study, we investigated the inhibition of the Lux quorum-sensing system by N-acyl cyclopentylamine (Cn-CPA). The Lux quorum-sensing system regulates luminescence gene expression in Vibrio fischeri. We have already reported on the synthesis of Cn-CPA and their abilities as inhibitors of the quorum-sensing systems in Pseudomonas aeruginosa and Serratia marcescens. In the case of Pseudomonas aeruginosa (Las and Rhl quorum-sensing system) and Serratia marcescens (Spn quorum-sensing system), specific Cn-CPA with a particular acyl chain length showed the strongest inhibitory effect. In the case of the Lux quorum-sensing system, it was found that several kinds of Cn-CPA with a range from C5 to C10 showed similar strong inhibitory effects. Moreover, the inhibitory effect of Cn-CPA on the Lux quorum-sensing system was stronger than that of halogenated furanone, a natural quorum-sensing inhibitor.

  13. 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

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

    PubMed Central

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

    2014-01-01

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

  15. Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase.

    PubMed

    Joshi, Anil K; Witkowski, Andrzej; Berman, Harvey A; Zhang, Lei; Smith, Stuart

    2005-03-15

    A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long-chain

  16. Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

    PubMed Central

    Schäfer, Franziska; Schuster, Judith; Würz, Birgit; Härtig, Claus; Harms, Hauke; Müller, Roland H.

    2012-01-01

    The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed. PMID:22752178

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

    PubMed

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

    2014-11-14

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

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

    PubMed

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

    1986-01-01

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

  19. Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo.

    PubMed

    Parkes, Heidi A; Preston, Elaine; Wilks, Donna; Ballesteros, Mercedes; Carpenter, Lee; Wood, Leonie; Kraegen, Edward W; Furler, Stuart M; Cooney, Gregory J

    2006-10-01

    Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 +/- 3 vs. 67 +/- 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 +/- 6 vs. 100 +/- 6 nmol/g protein in controls, P < 0.05) and increased [(14)C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 +/- 4 vs. 20 +/- 2 mumol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-(14)C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver. PMID:16705061

  20. Site-specific saturation mutagenesis on residues 132 and 450 of Candida rugosa LIP2 enhances catalytic efficiency and alters substrate specificity in various chain lengths of triglycerides and esters.

    PubMed

    Yen, Chih-Chung; Malmis, Conmar C; Lee, Guan-Chiun; Lee, Li-Chiun; Shaw, Jei-Fu

    2010-10-27

    The catalytic versatility of recombinant Candida rugosa LIP2 has been known to have potential applications in industry. In this study, site-specific saturation mutagenesis on residues L132 and G450 of recombinant LIP2 has been employed to investigate the impact of both residues on substrate specificity of LIP2. Point mutations on L132 and G450 were done separately using mutagenic degenerate primer sets containing 32 codons to generate two libraries of mutants in Pichia pastoris . Replacements of amino acid on these mutants were identified as L132A, L132I, G450S, and G450A. In lipase activity assay, L132A and L132I mutants showed a shift of preference from short- to medium-chain triglyceride, whereas G450S and G450A mutants retained preferences as compared to wild-type LIP2. Among mutants, G450A has the highest activity on tributyrin. However, hydrolysis of p-nitrophenyl (p-NP) esters with L132A, L132I, and G450S did not show differences of preferences over medium- to long-chain esters except in G450A, which prefers only medium-chain ester as compared to wild-type LIP2. All mutants showed an enhanced catalytic activity and higher optimal temperature and pH stability as compared to wild-type LIP2.

  1. Substrate specificity modification of the stromal glycerol-3-phosphate acyltransferase.

    PubMed

    Ferri, S R; Toguri, T

    1997-01-15

    The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared. The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA. In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA. By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras. All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered. The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs. Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.

  2. Genetic replacement of tesB with PTE1 affects chain-length proportions of 3-hydroxyalkanoic acids produced through β-oxidation of oleic acid in Escherichia coli.

    PubMed

    Seto, Yoshiaki; Kang, Junkyu; Ming, Li; Habu, Naoto; Nihei, Ken-ichi; Ueda, Shunsaku; Maeda, Isamu

    2010-10-01

    Acyl-CoA thioesterase II (TesB), which catalyzes hydrolysis of acyl-CoAs to free fatty acids and CoA, is involved in 3-hydroxyalkanoic acid production in Escherichia coli. Effects of genetic replacement of tesB with Saccharomyces cerevisiae acyl-CoA thioesterase gene PTE1 on 3-hydroxyalkanoic acid production from oleic acid through β-oxidation were examined. Kinetic analyses using β-oxidation intermediates showed that hydrolyses of C4-acyl substrates are more efficient by PTE1 than by TesB. Deletion of tesB in E. coli decreased 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and hexanoic acid in medium after cultivation with oleic acid as a sole carbon source. Hexanoic acid concentration was much lower than those of 3-hydroxyacids. In genetic complementation of tesB deletion, use of PTE1, instead of tesB, affected proportions of the 3-hydroxyalkanoic acids. Proportion of 3-hydroxybutyric acid was higher in a PTE1-complemented strain than in a tesB-complemented strain, while proportions of 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid markedly increased in the tesB-complemented strain. Proportion of 3-hydroxyoctanoic acid did not significantly increase in the PTE1-complemented strain. These data indicate possibilities of 3-hydroxyalkanoic acid production from oleic acid through β-oxidation and customization of their chain-length proportions by genetic replacement of tesB with a gene encoding acyl-CoA thioesterase with a different kinetic property.

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

    SciTech Connect

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

    2015-09-18

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

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

    DOE PAGES

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

    2015-09-18

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

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

    PubMed

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

    2015-10-01

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

  6. Acylated iridoids with cytotoxicity from Valeriana jatamansi.

    PubMed

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

    2009-04-01

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

  7. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.

    PubMed

    Peitzsch, R M; McLaughlin, S

    1993-10-01

    We studied the binding of fatty acids and acylated peptides to phospholipid vesicles by making electrophoretic mobility and equilibrium dialysis measurements. The binding energies of the anionic form of the fatty acids and the corresponding acylated glycines were identical; the energies increased by 0.8 kcal/mol per number of carbons in the acyl chain (Ncarbon = 10, 12, 14, 16), a value identical to that for the classical entropy-driven hydrophobic effect discussed by Tanford [The Hydrophobic Effect (1980) Wiley, New York]. The unitary Gibbs free binding energy, delta Gou, of myristoylated glycine, 8 kcal/mol, is independent of the nature of the electrically neutral lipids used to form the vesicles. Similar binding energies were obtained with other myristoylated peptides (e.g., Gly-Ala, Gly-Ala-Ala). The 8 kcal/mol, which corresponds to an effective dissociation constant of 10(-4) M for myristoylated peptides with lipids, provides barely enough energy to attach a myristoylated protein in the cytoplasm to the plasma membrane. Thus, other factors that reduce (e.g., hydrophobic interaction of myristate with the covalently attached protein) or enhance (e.g., electrostatic interactions of basic residues with acidic lipids; protein-protein interactions with intrinsic receptor proteins) the interaction of myristoylated proteins with membranes are likely to be important and may cause reversible translocation of these proteins to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. 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. PMID:26521810

  9. Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

    PubMed Central

    Vamecq, J; Schepers, L; Parmentier, G; Mannaerts, G P

    1987-01-01

    Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation. PMID:3435468

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

    SciTech Connect

    Towler, D.; Glaser, L.

    1986-05-01

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

  11. Effects of branched-chain amino acid supplementation on plasma concentrations of free amino acids, insulin, and energy substrates in young men.

    PubMed

    Zhang, Yi; Kobayashi, Hisamine; Mawatari, Kazunori; Sato, Juichi; Bajotto, Gustavo; Kitaura, Yasuyuki; Shimomura, Yoshiharu

    2011-01-01

    The present study was conducted to examine alterations in the concentrations of plasma free amino acids, glucose, insulin, free fatty acids (FFAs), and urea nitrogen induced by branched-chain amino acid (BCAA) supplementation in young men. Overnight-fasted subjects ingested drinks containing 1 or 5 g of a BCAA mixture (weight ratio of 1 : 2.3 : 1.2 for isoleucine : leucine : valine), and blood was intermittently collected for 3 h after ingestion. Ingestion of the BCAA mixture resulted in significant increases in the plasma concentrations of individual BCAAs, corresponding to the amounts of amino acids ingested. On the other hand, plasma concentrations of methionine and aromatic amino acids tended to decrease in the trial with 5 g BCAAs, suggesting that BCAA ingestion affects the metabolism of these amino acids. The ingestion of BCAAs temporarily increased plasma insulin levels and affected plasma concentrations of FFAs, but had almost no effect on glucose or urea nitrogen. PMID:21512300

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

    PubMed Central

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

    2014-01-01

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

  13. Prediction and experimental validation of enzyme substrate specificity in protein structures

    PubMed Central

    Amin, Shivas R.; Erdin, Serkan; Ward, R. Matthew; Lua, Rhonald C.; Lichtarge, Olivier

    2013-01-01

    Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase–like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. PMID:24145433

  14. Unique plasma metabolomic signatures of individuals with inherited disorders of long-chain fatty acid oxidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Blood and urine acylcarnitine profiles are commonly used to diagnose long-chain fatty acid oxidation disorders (FAOD: i.e., long-chain hydroxy-acyl-CoA dehydrogenase [LCHAD] and carnitine palmitoyltransferase 2 [CPT2] deficiency), but the global metabolic impact of long-chain FAOD has not been repor...

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

    PubMed

    Kandra, L; Wagner, G J

    1990-11-01

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

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

    PubMed Central

    Kandra, Lili; Wagner, George J.

    1990-01-01

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