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Sample records for acyl-coa-binding protein acyl-coa-binding

  1. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    International Nuclear Information System (INIS)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-01-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. 14 C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell

  2. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    Energy Technology Data Exchange (ETDEWEB)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-05-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

  3. Disruption of the acyl-coa binding protein gene delays hepatic adaptation to metabolic changes at weaning

    DEFF Research Database (Denmark)

    Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek

    2011-01-01

    The acyl-CoA binding protein/diazepam binding inhibitor (ACBP/DBI) is an intracellular protein that binds C14-C22 acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however....... The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads...

  4. Acyl-CoA binding proteins; structural and functional conservation over 2000 MYA

    DEFF Research Database (Denmark)

    Faergeman, Nils J; Wadum, Majken; Feddersen, Søren

    2007-01-01

    -CoA binding protein, ACBP, has been proposed to play a pivotal role in the intracellular trafficking and utilization of long-chain fatty acyl-CoA esters. Depletion of acyl-CoA binding protein in yeast results in aberrant organelle morphology incl. fragmented vacuoles, multi-layered plasma membranes...... and accumulation of vesicles of variable sizes. In contrast to synthesis and turn-over of glycerolipids, the levels of very-long-chain fatty acids, long-chain bases and ceramide are severely affected by Acb1p depletion, suggesting that Acb1p, rather than playing a general role, serves specific roles in cellular...

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

    DEFF Research Database (Denmark)

    Wadum, M.C.; Villadsen, J.K.; Feddersen, S.

    2002-01-01

    methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl...... of ligand (excitation 387nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460nm upon binding of C14-C20 saturated...

  6. Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Seker, Tamay; Møller, Kasper; Nielsen, Jens

    2005-01-01

    The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl Co......A, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we...... used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase...

  7. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

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

    Directory of Open Access Journals (Sweden)

    Yi Yu

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

  9. Determination of hydrophobic coenzyme a esters and other lipids using a biosensor comprising a modified coenzyme a- and acyl-coa binding protein (acbp)

    DEFF Research Database (Denmark)

    2002-01-01

    , food and feed preparations, tissue extracts, acyl-CoA synthetase reaction media and various laboratory conditions using a modified Coenzyme A- and acyl-CoA binding protein (ACBP) is provided. Furthermore the invention relates to a construct comprising a peptide and a signal moiety for performing...

  10. Acyl CoA Binding Domain Containing 3 (ACBD3) Protein in Huntington’s Disease 
Human Skin Fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, H.; Rodinová, M.; Sládková, J.; Klempíř, J.; Lišková, Irena; Motlík, Jan; Zeman, J.; Hansíková, H.; Tesařová, M.

    2015-01-01

    Roč. 78, Suppl. 2 (2015), s. 34-38 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. Liblice, 08.11.2015-10.11.2015] R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : Huntington’s disease * Acyl-CoA binding domain containing 3 protein * human skin fibroblasts Subject RIV: FH - Neurology Impact factor: 0.209, year: 2015

  11. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous mod...... have molecularly cloned and characterized the ACBP/DBI gene family in rat. The rat ACBP/DBI gene family comprises one expressed gene and four processed pseudogenes of which one was shown to exist in two allelic forms. The expressed gene is organized into four exons and three introns...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-02-18

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

  13. Evolution of the acyl-CoA binding protein (ACBP)

    DEFF Research Database (Denmark)

    Burton, Mark; Rose, Timothy M; Faergeman, Nils J

    2005-01-01

    -CoA pool size, donation of acyl-CoA esters for beta-oxidation, vesicular trafficking, complex lipid synthesis and gene regulation. In the present study, we delineate the evolutionary history of ACBP to get a complete picture of its evolution and distribution among species. ACBP homologues were identified...... duplication and/or retrotransposition events. The ACBP protein is highly conserved across phylums, and the majority of ACBP genes are subjected to strong purifying selection. Experimental evidence indicates that the function of ACBP has been conserved from yeast to humans and that the multiple lineage...

  14. Acyl-CoA binding protein and epidermal barrier function

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Neess, Ditte; Færgeman, Nils J

    2014-01-01

    The acyl-CoA binding protein (ACBP) is a 10kDa intracellular protein expressed in all eukaryotic species and mammalian tissues investigated. It binds acyl-CoA esters with high specificity and affinity and is thought to act as an intracellular transporter of acyl-CoA esters between different...... includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced...

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

    Energy Technology Data Exchange (ETDEWEB)

    Halavaty, Andrei S. [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Kim, Youngchang [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Zhou, Min [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Onopriyenko, Olena; Skarina, Tatiana [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N. [Center for Structural Genomics of Infectious Diseases, (United States); J. Craig Venter Institute, Rockville, MD 20850 (United States); Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Savchenko, Alexei [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Anderson, Wayne F., E-mail: wf-anderson@northwestern.edu [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States)

    2012-10-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  17. Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Ferreira, Natalia Santos; Engelsby, Hanne; Neess, Ditte

    2017-01-01

    and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very......-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes...... of ACBP(-/-) mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial...

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

    Science.gov (United States)

    Thomas, Jacob

    2009-01-01

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

  19. Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for beta-oxidation and glycerolipid synthesis

    DEFF Research Database (Denmark)

    Rasmussen, J T; Færgeman, Nils J.; Kristiansen, K

    1994-01-01

    The dissociation constants for octanoyl-CoA, dodecanoyl-CoA and hexadecanoyl-CoA binding to acyl-CoA-binding protein (ACBP) were determined by using titration microcalorimetry. The KD values obtained, (0.24 +/- 0.02) x 10(-6) M, (0.65 +/- 0.2) x 10(-8) M and (0.45 +/- 0.2) x 10(-13) M respectively......, were much lower than expected. ACBP was able to extract hexadecanoyl-CoA from phosphatidylcholine membranes immobilized on a nitrocellulose membrane. The acyl-CoA/ACBP complex formed was able to transport acyl-CoA to mitochondria or microsomes in suspension, or to microsomes immobilized...

  20. Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

    Science.gov (United States)

    Wang, Nan; Rudolf, Jeffrey D; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2018-06-04

    Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

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

    Science.gov (United States)

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

    2016-02-22

    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.

  2. Role of acylCoA binding protein in acylCoA transport, metabolism and cell signaling

    DEFF Research Database (Denmark)

    Knudsen, J; Jensen, M V; Hansen, J K

    1999-01-01

    and pool formation and therefore also for the function of LCAs as metabolites and regulators of cellular functions [1]. The major factors controlling the free concentration of cytosol long chain acylCoA ester (LCA) include ACBP [2], sterol carrier protein 2 (SCP2) [3] and fatty acid binding protein (FABP...

  3. Disruption of the Acyl-CoA binding protein gene delays hepatic adaptation to metabolic changes at weaning

    DEFF Research Database (Denmark)

    Neess, Ditte; Marcher, Ann-Britt; Bloksgaard, Maria

    The acyl-CoA binding protein/diazepam binding inhibitor (ACBP/DBI) is an evolutionary conserved intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity. ACBP is thought to act as an acyl-CoA transporter, and in vitro analyses have indicated that ACBP can transport acyl......-CoA esters between different enzymatic systems. However, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP-/-). These mice are viable and fertile and develop normally. However, around weaning the ACBP-/- mice show decreased growth......) family, around the weaning period. As a result, the hepatic de novo cholesterogenesis is significantly decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP...

  4. Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast

    DEFF Research Database (Denmark)

    Mandrup, S; Jepsen, R; Skøtt, H

    1993-01-01

    We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovi...

  5. Up-regulation of hepatic Acyl CoA: Diacylglycerol acyltransferase-1 (DGAT-1) expression in nephrotic syndrome.

    Science.gov (United States)

    Vaziri, Nosratola D; Kim, Choong H; Phan, Dennis; Kim, Sara; Liang, Kaihui

    2004-07-01

    Nephrotic syndrome is associated with hypercholesterolemia, hypertriglyceridemia, and marked elevations of plasma low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Hypertriglyceridemia in nephrotic syndrome is accompanied by increased hepatic fatty acid synthesis, elevated triglyceride secretion, as well as lipoprotein lipase, VLDL-receptor, and hepatic triglyceride lipase deficiencies, which lead to impaired clearance of triglyceride-rich lipoproteins. Acyl CoA: diacylglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl CoA to 1, 2-diacylglycerol to form triglyceride. Two distinct DGATs (DGAT-1 and DGAT2) have recently been identified in the liver and other tissues. The present study tested the hypothesis that the reported increase in hepatic triglyceride secretion in nephrotic syndrome may be caused by up-regulation of DGAT. Male Sprague-Dawley rats were rendered nephrotic by two sequential injections of puromycin aminonucleoside (130 mg/kg on day 1 and 60 mg/kg on day 14) and studied on day 30. Placebo-treated rats served as controls. Hepatic DGAT-1 and DGAT-2 mRNA abundance and enzymatic activity were measured. The nephrotic group exhibited heavy proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, and marked elevation of VLDL concentration. Hepatic DGAT-1 mRNA, DGAT-1, and total DGAT activity were significantly increased, whereas DGAT-2 mRNA abundance and activity were unchanged in the nephrotic rats compared to the control animals. The functional significance of elevation of DGAT activity was illustrated by the reduction in microsomal free fatty acid concentration in the liver of nephrotic animals. Nephrotic syndrome results in up-regulation of hepatic DGAT-1 expression and activity, which can potentially contribute to the associated hypertriglyceridemia by enhancing triglyceride synthesis. Thus, it appears that both depressed catabolism and increased synthetic capacity contribute to

  6. Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

    Science.gov (United States)

    Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

    2016-03-01

    To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

  7. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show...

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Dominik M. Peter

    2016-04-01

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

  10. Acyl-CoA metabolism and partitioning

    DEFF Research Database (Denmark)

    Grevengoed, Trisha J; Klett, Eric L; Coleman, Rosalind A

    2014-01-01

    Long-chain fatty acyl-coenzyme As (CoAs) are critical regulatory molecules and metabolic intermediates. The initial step in their synthesis is the activation of fatty acids by one of 13 long-chain acyl-CoA synthetase isoforms. These isoforms are regulated independently and have different tissue...

  11. Inhibition of 3T3-L1 adipocyte differentiation by expression of acyl-CoA-binding protein antisense RNA

    DEFF Research Database (Denmark)

    Mandrup, S; Sorensen, R V; Helledie, T

    1998-01-01

    Several lines of evidence have recently underscored the significance of fatty acids or fatty acid-derived metabolites as signaling molecules in adipocyte differentiation. The acyl-CoA-binding protein (ACBP), which functions as an intracellular acyl-CoA pool former and transporter, is induced duri...

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT).

    Science.gov (United States)

    Holmes, Roger S

    2010-03-01

    BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron-exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals. Copyright 2009 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T.

    2006-01-01

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

  15. Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

    DEFF Research Database (Denmark)

    Kragelund, B B; Poulsen, K; Andersen, K V

    1999-01-01

    In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability...

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt 2 cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 μM) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 μm/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of [ 14 C]-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis

  18. The acyl-CoA binding protein affects Monascus pigment production in Monascus ruber CICC41233.

    Science.gov (United States)

    Long, Chuannan; Liu, Mengmeng; Chen, Xia; Wang, Xiaofang; Ai, Mingqiang; Cui, Jingjing; Zeng, Bin

    2018-02-01

    The present study verified whether acyl-coenzyme A (acyl-CoA)-binding protein (ACBP) affected the production of Monascus pigments (MPs) in Monascus ruber CICC41233 (MrACBP). Phylogenetic analysis revealed that the cloned Mracbp gene, which encoded the MrACBP protein, exhibited the closest match (99% confidence level) to the gene from Penicilliopsis zonata . The MrACBP and maltose-binding protein (MBP) were simultaneously expressed in Escherichia coli Rosetta DE3 in the form of a fusion protein. The microscale thermophoresis binding assay revealed that the purified MBP-MrACBP exhibited a higher affinity for myristoyl-CoA (Kd = 88.16 nM) than for palmitoyl-CoA (Kd = 136.07 nM) and octanoyl-CoA (Kd = 270.9 nM). Further, the Mracbp gene was homologously overexpressed in M. ruber CICC41233, and a positive transformant M. ruber ACBP5 was isolated. The fatty acid myristic acid in M. ruber ACBP5 was lower than that in the parent strain M. ruber CICC41233. However, when compared with the parent strain, the production of total MPs, water-soluble pigment, and ethanol-soluble pigment in M. ruber ACBP5 increased by 11.67, 9.80, and 12.70%, respectively, after 6 days. The relative gene expression level, as determined by a quantitative real-time polymerase chain reaction analysis, of the key genes acbp , pks , mppr1 , fasA , and fasB increased by 4.03-, 3.58-, 1.67-, 2.11-, and 2.62-fold after 6 days. These data demonstrate the binding preference of MrACBP for myristoyl-CoA, and its influence on MPs production.

  19. A liver-specific defect of Acyl-CoA degradation produces hyperammonemia, hypoglycemia and a distinct hepatic Acyl-CoA pattern.

    Directory of Open Access Journals (Sweden)

    Nicolas Gauthier

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

  20. 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

    Energy Technology Data Exchange (ETDEWEB)

    Melton, Elaina M. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Center for Cardiovascular Sciences, Albany Medical College, Albany, NY (United States); Cerny, Ronald L. [Department of Chemistry, University of Nebraska, Lincoln, NE (United States); DiRusso, Concetta C. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Black, Paul N., E-mail: pblack2@unl.edu [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States)

    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

  1. 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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  2. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  3. Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding

    International Nuclear Information System (INIS)

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

    1987-01-01

    Conditions are described under which monomeric [ 14 C]methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the [ 14 C]methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria

  4. Depletion of Acyl-Coenzyme A-Binding Protein Affects Sphingolipid Synthesis and Causes Vesicle Accumulation and Membrane Defects in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Gaigg, B; Neergaard, T B; Schneiter, R

    2001-01-01

    Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:10(5). A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under contro...

  5. α-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

    International Nuclear Information System (INIS)

    Lee, Young; Naseem, R. Haris; Park, Byung-Hyun; Garry, Daniel J.; Richardson, James A.; Schaffer, Jean E.; Unger, Roger H.

    2006-01-01

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

  6. Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

    Science.gov (United States)

    Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

    2010-11-15

    Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication. Published by Elsevier Ltd.

  7. Cholesterol esterification by mouse liver homogenate. Contribution to the study of ACYL-CoA: Cholesterol ACYL transferase in mammalian liver

    International Nuclear Information System (INIS)

    Soares, M.G.C.B.

    1976-01-01

    A cholesterol- esterifying enzyme from mouse liver has been partially characterized. The enzyme which showed optimum activity at pH 7,1 and required ATP and CoA, was identified as an acyl CoA: cholesterol acyl transferase (E.C.2.3.1.26). As a fuction of time the percentage of esterified cholesterol increased linearly during the first hour of incubation and continued to increase but not linearly with 4 hours, after which time no further net esterefication was observed. The relative concentration of esterified cholesterol remained constant between the fourth and twelveth hours of incubation but afterwards decreased when the incubation continued until 24 hours. The cholesterol- esterifying activity was 24,0+- 2,9 nmoles cholesterol esterified per gram tissue wet weight per minute. The mean percentages of free cholesterol esterified in and 24 hours respectively were 14,8+- 1,6 e 21,9+- 4,5. The subfractionation of labelled cholesteryl esters after one hour incubation of liver homogenate with 4-C 14 -Cholesterol showed the order of preference for the formation of the different ester classes to be monounsatured > diunsatured ≥ saturated >> polyunsaturated. The properties of the enzyme frommouse liver do not markedly differ from those of the previously recorded ACAT activity of rat liver. (Author) [pt

  8. Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

    Science.gov (United States)

    Schultz, D J; Suh, M C; Ohlrogge, J B

    2000-10-01

    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 Delta9 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 [(14)C]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 x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 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.

  9. The acyl-CoA binding protein is required for normal epidermal barrier function in mice

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt

    2012-01-01

    (+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC......The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling...... with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP...

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

    Science.gov (United States)

    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

  11. How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)

    DEFF Research Database (Denmark)

    Andersen, Kell Kleiner; Otzen, Daniel

    2009-01-01

    maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle......Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl...... constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex...

  12. Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase.

    Science.gov (United States)

    Thornburg, Chelsea K; Walter, Tyler; Walker, Kevin D

    2017-11-07

    In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

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

    Science.gov (United States)

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

    2013-06-01

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

  14. Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.; Barb, Adam W.

    2017-11-01

    The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.

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

    Directory of Open Access Journals (Sweden)

    Lovera Cristina

    2012-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Yuxun Zhang

    Full Text Available 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.

  17. Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis.

    Science.gov (United States)

    Lung, Shiu-Cheung; Liao, Pan; Yeung, Edward C; Hsiao, An-Shan; Xue, Yan; Chye, Mee-Len

    2017-07-01

    Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis ( Arabidopsis thaliana ) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1 Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 ( GL2 ), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1 , smo1-1 , and ACBP1 +/- smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1 +/- smo1-1 Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. © 2017 American Society of Plant Biologists. All Rights Reserved.

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

    Science.gov (United States)

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

    2011-10-04

    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.

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

    Science.gov (United States)

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

    2015-10-27

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

  20. Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

    Science.gov (United States)

    Marcella, Aaron M; Culbertson, Sannie J; Shogren-Knaak, Michael A; Barb, Adam W

    2017-11-24

    The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K D =62±13nM, followed by the binding of two more equivalents of holo-ACPP with K D =1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K D =2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: diacylglycerol transferase (DGAT) 1 inhibitor.

    Science.gov (United States)

    Yokoyama, Hideaki; Kobayashi, Akio; Kondo, Kazuma; Oshida, Shin-Ichi; Takahashi, Tadakazu; Masuyama, Taku; Shoda, Toshiyuki; Sugai, Shoichiro

    2018-01-01

    Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.

  2. Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

    Directory of Open Access Journals (Sweden)

    Clément Chevalier

    2010-03-01

    Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

  3. Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

    2016-08-01

    The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

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

    Science.gov (United States)

    Dodd, David T.; Roberts, Richard L.

    1994-01-01

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

  5. Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

    International Nuclear Information System (INIS)

    Sato, Miho; Nakahara, Keiko; Goto, Shintaro; Kaiya, Hiroyuki; Miyazato, Mikiya; Date, Yukari; Nakazato, Masamitsu; Kangawa, Kenji; Murakami, Noboru

    2006-01-01

    Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [ 125 I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord

  6. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

  7. Insulin signaling regulates fatty acid catabolism at the level of CoA activation.

    Directory of Open Access Journals (Sweden)

    Xiaojun Xu

    2012-01-01

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

  8. Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

    Science.gov (United States)

    Hsiao, An-Shan; Xue, Yan

    2017-01-01

    Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

  9. STRUCTURAL AND FUNCTIONAL ASPECTS OF ACYL-COENZYME A BINDING PROTEINS (ACBPs: A COMPREHENSIVE REVIEW

    Directory of Open Access Journals (Sweden)

    Richa Arya

    2012-06-01

    Full Text Available ACBP was originally identified as a mammalian diazepam binding inhibitor – a neuropeptide that has the ability to inhibit diazepam binding to the �-aminobutyric acid (GABA receptor (Guidotti et al., 1983. Typically, ACBPs are small (~10 kDa cytosolic proteins (Burton et al., 2005. However, a number of hybrid ACBPs are reported that are fused with ankyrin repeats, such as ACBP1 and ACBP2 in Arabidopsis thaliana (Chye et al., 1999; Li and Chye, 2003. Other functional domains, such as the human peroxisomal �3/ �2-enoyl-CoA isomerase (Geisbrecht et al., 1999, or any non-functional/ uncharacterized domain are also cited. ACBP predominantly functions as an intracellular acyl-CoA transporter and pool former, and is critical to lipid metabolism in cells (Gossett et al., 1996; Knudsen et al., 2000; Schroeder et al., 1998. Impaired lipid metabolism and other cellular functions in humans arising out of ACBP defects thus need to be explored. ACBP has only been reported in eukaryotes, not in prokaryotes, except for a few pathogenic eubacteria that might have acquired ACBP from eukaryotic hosts via lateral gene transfer (Burton et al., 2005. Whole genome sequences of several prokaryotes and pathogens being available currently, it is worthwhile to extend search for ACBPs beyond eukaryotes as well, to explore their potential as drug targets, given their essential role in lipid metabolism. As a prelude to such investigations, the current review summarizes available knowledge of ACBPs and outlines the scope of future research.

  10. HIF-1-dependent regulation of lifespan in Caenorhabditis elegans by the acyl-CoA-binding protein MAA-1

    DEFF Research Database (Denmark)

    Shamalnasab, Mehrnaz; Dhaoui, Manel; Thondamal, Manjunatha

    2017-01-01

    In yeast, the broadly conserved acyl-CoA-binding protein (ACBP) is a negative regulator of stress resistance and longevity. Here, we have turned to the nematode C. elegans as a model organism in which to determine whether ACBPs play similar roles in multicellular organisms. We systematically...... inactivated each of the seven C. elegans ACBP paralogs and found that one of them, maa-1 (which encodes membrane-associated ACBP 1), is indeed involved in the regulation of longevity. In fact, loss of maa-1 promotes lifespan extension and resistance to different types of stress. Through genetic and gene...... of the proteome. Our work extends to C. elegans the role of ACBP in aging, implicates HIF-1 in the increase of lifespan of maa-1-deficient worms, and sheds light on the anti-aging function of HIF-1. Given that both ACBP and HIF-1 are highly conserved, our results suggest the possible involvement of these proteins...

  11. The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

    Science.gov (United States)

    van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

    2012-01-01

    SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

  12. Endophilin-A1 BAR domain interaction with arachidonyl CoA.

    Science.gov (United States)

    Petoukhov, Maxim V; Weissenhorn, Winfried; Svergun, Dmitri I

    2014-01-01

    Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.

  13. Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

    Science.gov (United States)

    Kawaguchi, Kouhei; Kikuma, Takashi; Higuchi, Yujiro; Takegawa, Kaoru; Kitamoto, Katsuhiko

    2016-11-04

    In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role

    International Nuclear Information System (INIS)

    Krishna, S. Sri; Aravind, L.; Bakolitsa, Constantina; Caruthers, Jonathan; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Rife, Christopher L.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The crystal structure of SSO2064, the first structural representative of Pfam family PF01796 (DUF35), reveals a two-domain architecture comprising an N-terminal zinc-ribbon domain and a C-terminal OB-fold domain. Analysis of the domain architecture, operon organization and bacterial orthologs combined with the structural features of SSO2064 suggests a role involving acyl-CoA binding for this family of proteins. SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-15

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

  16. Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

    Science.gov (United States)

    Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

    2018-05-01

    In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

  17. Lead-Binding Proteins: A Review

    Directory of Open Access Journals (Sweden)

    Harvey C. Gonick

    2011-01-01

    Full Text Available Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte. Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

  18. Thioesterase activity and acyl-CoA/fatty acid cross-talk of hepatocyte nuclear factor-4{alpha}.

    Science.gov (United States)

    Hertz, Rachel; Kalderon, Bella; Byk, Tamara; Berman, Ina; Za'tara, Ghadeer; Mayer, Raphael; Bar-Tana, Jacob

    2005-07-01

    Hepatocyte nuclear factor-4alpha (HNF-4alpha) activity is modulated by natural and xenobiotic fatty acid and fatty acyl-CoA ligands as a function of their chain length, unsaturation, and substitutions. The acyl-CoA site of HNF-4alpha is reported here to consist of the E-F domain, to bind long-chain acyl-CoAs but not the respective free acids, and to catalyze the hydrolysis of bound fatty acyl-CoAs. The free acid pocket, previously reported in the x-ray structure of HNF-4alpha E-domain, entraps fatty acids but excludes acyl-CoAs. The acyl-CoA and free acid sites are distinctive and noncongruent. Free fatty acid products of HNF-4alpha thioesterase may exchange with free acids entrapped in the fatty acid pocket of HNF-4alpha. Cross-talk between the acyl-CoA and free fatty acid binding sites is abrogated by high affinity, nonhydrolyzable acyl-CoA ligands of HNF-4alpha that inhibit its thioesterase activity. Hence, HNF-4alpha transcriptional activity is controlled by its two interrelated acyl ligands and two binding sites interphased in tandem by the thioesterase activity. The acyl-CoA/free-acid and receptor/enzyme duality of HNF-4alpha extends the paradigm of nuclear receptors.

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

    Science.gov (United States)

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

    2014-04-01

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

  20. Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

    DEFF Research Database (Denmark)

    Hansen, Jesper S; Færgeman, Nils J; Kragelund, Birthe B

    2008-01-01

    showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations....... Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence...... recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved...

  1. Role of Feedback Regulation of Pantothenate Kinase (CoaA) in Control of Coenzyme A Levels in Escherichia coli

    Science.gov (United States)

    Rock, Charles O.; Park, Hee-Won; Jackowski, Suzanne

    2003-01-01

    Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4′-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels. PMID:12754240

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

    Science.gov (United States)

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

    1997-04-01

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

  3. S-naproxen-ss-1-O-acyl glucuronide degradation kinetic studies by stopped-flow high-performance liquid chromatography-H-1 NMR and high-performance liquid chromatography-UV

    DEFF Research Database (Denmark)

    Mortensen, R. W.; Corcoran, O.; Cornett, Claus

    2001-01-01

    Acyl-migrated isomers of drug beta -1-O-acyl glucuronides have been implicated in drug toxicity because they can bind to proteins. The acyl migration and hydrolysis of S-naproxen-beta -1-O-acyl glucuronide (S-nap-g) was followed by dynamic stopped-flow HPLC-H-1 NMR and HPLC methods. Nine first or...

  4. A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi.

    Science.gov (United States)

    Byers, D M; Holmes, C G

    1990-01-01

    An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.

  5. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  6. Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

    DEFF Research Database (Denmark)

    Hu, Liping; Deeney, Jude T; Nolan, Christopher J

    2005-01-01

    -cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both....... The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue...

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Reprogramming Acyl Carrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase

    DEFF Research Database (Denmark)

    Ye, Zhixia; Musiol-Kroll, Ewa Maria; Weber, Tilmann

    2014-01-01

    Protein interactions between acyl carrier proteins (ACPs) and trans-acting acyltransferase domains (trans-ATs) are critical for regioselective extender unit installation by many polyketide synthases, yet little is known regarding the specificity of these interactions, particularly for trans-ATs w...

  9. Fractal binding and dissociation kinetics of lecithin cholesterol acyl transferase (LCAT), a heart-related compound, on biosensor surfaces

    Science.gov (United States)

    Doke, Atul M.; Sadana, Ajit

    2006-05-01

    A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg-white apoA-I rHDL immobilized on a biosensor chip surface.1 Single- and dual- fractal models were employed to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (Corel Corporation Limited).2 The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution, and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.

  10. Association of acylated cationic decapeptides with dipalmitoylphosphatidylserine-dipalmitoyl- phosphatidylcholine lipid membranes

    DEFF Research Database (Denmark)

    Pedersen, T. B.; Sabra, Mads Christian; Frokjaer, Sven

    2001-01-01

    decapeptides that are N-terminally linked with C-2, C-8, and C-14 acyl chains contain four basic histidine residues in their identical amino acid sequence. A binding model, based on changes in the intrinsic fluorescent properties of the peptides upon association with the DPPC-DPPS membranes, is used...... DPPC-DPPS lipid mixture. The extent of peptide association deduced from the heat capacity measurements suggests a strong binding and membrane insertion of the C-14 acylated peptide in accordance with the fluorescence measurements....

  11. Human carbonyl reductase 1 participating in intestinal first-pass drug metabolism is inhibited by fatty acids and acyl-CoAs.

    Science.gov (United States)

    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; El-Kabbani, Ossama; Miura, Takeshi; Nishinaka, Toru; Terada, Tomoyuki

    2017-08-15

    Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, reduces a variety of carbonyl compounds including endogenous isatin, prostaglandin E 2 and 4-oxo-2-nonenal. It is also a major non-cytochrome P450 enzyme in the phase I metabolism of carbonyl-containing drugs, and is highly expressed in the intestine. In this study, we found that long-chain fatty acids and their CoA ester derivatives inhibit CBR1. Among saturated fatty acids, myristic, palmitic and stearic acids were inhibitory, and stearic acid was the most potent (IC 50 9µM). Unsaturated fatty acids (oleic, elaidic, γ-linolenic and docosahexaenoic acids) and acyl-CoAs (palmitoyl-, stearoyl- and oleoyl-CoAs) were more potent inhibitors (IC 50 1.0-2.5µM), and showed high inhibitory selectivity to CBR1 over its isozyme CBR3 and other SDR superfamily enzymes (DCXR and DHRS4) with CBR activity. The inhibition by these fatty acids and acyl-CoAs was competitive with respect to the substrate, showing the K i values of 0.49-1.2µM. Site-directed mutagenesis of the substrate-binding residues of CBR1 suggested that the interactions between the fatty acyl chain and the enzyme's Met141 and Trp229 are important for the inhibitory selectivity. We also examined CBR1 inhibition by oleic acid in cellular levels: The fatty acid effectively inhibited CBR1-mediated 4-oxo-2-nonenal metabolism in colon cancer DLD1 cells and increased sensitivity to doxorubicin in the drug-resistant gastric cancer MKN45 cells that highly express CBR1. The results suggest a possible new food-drug interaction through inhibition of CBR1-mediated intestinal first-pass drug metabolism by dietary fatty acids. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. CoaSim Guile Manual — Using the Guile-based CoaSim Simulator

    DEFF Research Database (Denmark)

    Mailund, T

    2006-01-01

    CoaSim is a tool for simulating the coalescent process with recombination and geneconversion, under either constant population size or exponential population growth. It effectively constructs the ancestral recombination graph for a given number of chromosomes and uses this to simulate samples...

  13. CoAs: The line of 3 d demarcation

    Science.gov (United States)

    Campbell, Daniel J.; Wang, Limin; Eckberg, Chris; Graf, Dave; Hodovanets, Halyna; Paglione, Johnpierre

    2018-05-01

    Transition metal-pnictide compounds have received attention for their tendency to combine magnetism and unconventional superconductivity. Binary CoAs lies on the border of paramagnetism and the more complex behavior seen in isostructural CrAs, MnP, FeAs, and FeP. Here we report the properties of CoAs single crystals grown with two distinct techniques along with density functional theory calculations of its electronic structure and magnetic ground state. While all indications are that CoAs is paramagnetic, both experiment and theory suggest proximity to a ferromagnetic instability. Quantum oscillations are seen in torque measurements up to 31.5 T and support the calculated paramagnetic Fermiology.

  14. Analysis of an acyl-CoA binding protein in Aspergillus oryzae that undergoes unconventional secretion.

    Science.gov (United States)

    Kwon, Hee Su; Kawaguchi, Kouhei; Kikuma, Takashi; Takegawa, Kaoru; Kitamoto, Katsuhiko; Higuchi, Yujiro

    2017-11-04

    Acyl-CoA binding protein (ACBP) plays important roles in the metabolism of lipids in eukaryotic cells. In the industrially important filamentous fungus Aspergillus oryzae, although we have previously demonstrated that the A. oryzae ACBP (AoACBP) localizes to punctate structures and exhibits long-range motility, which is dependent on autophagy-related proteins, the physiological role of AoACBP remains elusive. Here, we describe identification and characterization of another ACBP from A. oryzae; we named this ACBP as AoAcb2 and accordingly renamed AoACBP as AoAcb1. The deduced amino acid sequence of AoAcb2 lacked a signal peptide. Phylogenetic analysis classified AoAcb2 into a clade that was same as the ACBP Acb1 of the model yeast Saccharomyces cerevisiae, but was different from that of AoAcb1. In contrast to punctate localization of AoAcb1, AoAcb2 was found to be dispersedly distributed in the cytoplasm, as was previously observed for the S. cerevisiae Acb1. Since we could not generate an Aoacb2 disruptant, we created an Aoacb2 conditional mutant that exhibited less growth under Aoacb2-repressed condition, suggesting that Aoacb2 is an essential gene for growth. Moreover, we observed that A. oryzae AoAcb2, but not A. oryzae AoAcb1, was secreted under carbon-starved condition, suggesting that AoAcb2 might be secreted via the unconventional protein secretion (UPS) pathway, just like S. cerevisiae Acb1. We also demonstrated that the unconventional secretion of AoAcb2 was dependent on the t-SNARE AoSso1, but was independent of the autophagy-related protein AoAtg1, suggesting that the unconventional secretion of AoAcb2, unlike that of S. cerevisiae Acb1, via the UPS pathway, is not regulated by the autophagy machinery. Thus, the filamentous fungus A. oryzae harbors two types of ACBPs, one of which appears to be essential for growth and undergoes unconventional secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals

    DEFF Research Database (Denmark)

    Black, P N; Færgeman, Nils J.; DiRusso, C C

    2000-01-01

    ). Both repression and activation are dependent upon the function of either of the acyl-CoA synthetases Faa1p or Faa4p. In mammals, purified hepatocyte nuclear transcription factor 4alpha (HNF-4alpha) like E. coli FadR, binds long chain acyl-CoA directly. Coexpression of HNF-4alpha and acyl-CoA synthetase...

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

    Science.gov (United States)

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

    2016-09-06

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

  17. Getting Started with CoaSim — An Introduction to the Simulator CoaSim

    DEFF Research Database (Denmark)

    Mailund, T

    2005-01-01

    CoaSim is a tool for simulating the coalescent process with recombination and geneconversion, under either constant population size or exponential population growth. It effectively constructs the ancestral recombination graph for a given number of chromosomes and uses this to simulate samples...

  18. A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli.

    Science.gov (United States)

    Manandhar, Miglena; Cronan, John E

    2018-01-01

    BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in vitro the Escherichia coli and Bacillus sphaericus enzymes have been shown to condense pimeloyl-CoA with l-alanine in a pyridoxal 5'-phosphate-dependent reaction with concomitant CoA release and decarboxylation of l-alanine. However, recent in vivo studies of E. coli and Bacillus subtilis suggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in that E. coli BioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast, B. subtilis BioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic and in vitro data demonstrating that B. subtilis BioF specifically utilizes pimeloyl-CoA. IMPORTANCE Biotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of the Bacillus subtilis pathway and shows that it differs from that of Escherichia coli in the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis. Copyright © 2017 American Society for Microbiology.

  19. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD+ and triclosan

    International Nuclear Information System (INIS)

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-01-01

    Structure of the ternary complex of F. tularensis enoyl-acyl carrier protein reductase reveals the structure of the substrate binding loop whose electron density was missing in an earlier structure, and demonstrates a shift in the position of the NAD + cofactor. Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD + has been solved to a resolution of 2.1 Å. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure which is bound to only NAD + reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD + cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

    International Nuclear Information System (INIS)

    Fehling, E.; Murphy, D.J.; Mukherjee, K.D.

    1990-01-01

    Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1- 14 C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2- 14 C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerols and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1- 14 C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2- 14 C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols

  2. Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement

    DEFF Research Database (Denmark)

    Trier, Sofie; Linderoth, Lars; Bjerregaard, Simon

    2015-01-01

    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...... 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 decrease the permeability. In conclusion, acylated sCT acts as its own in vitro intestinal permeation enhancer, with reversible effects...... on Caco-2 cells, indicating that acylation of sCT may represent a promising tool to increase intestinal permeability without adding oral permeation enhancers....

  3. Xanthomonas campestris RpfB is a Fatty Acyl-CoA Ligase Required to Counteract the Thioesterase Activity of the RpfF Diffusible Signal Factor (DSF) Synthase

    Science.gov (United States)

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

    2014-01-01

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

  4. Characterization and functional assay of a fatty acyl-CoA reductase gene in the scale insect, Ericerus pela Chavannes (Hemiptera: Coccoidae).

    Science.gov (United States)

    Hu, Yan-Hong; Chen, Xiao-Ming; Yang, Pu; Ding, Wei-Feng

    2018-04-01

    Ericerus pela Chavannes (Hemiptera: Coccoidae) is an economically important scale insect because the second instar males secrete a harvestable wax-like substance. In this study, we report the molecular cloning of a fatty acyl-CoA reductase gene (EpFAR) of E. pela. We predicted a 520-aa protein with the FAR family features from the deduced amino acid sequence. The EpFAR mRNA was expressed in five tested tissues, testis, alimentary canal, fat body, Malpighian tubules, and mostly in cuticle. The EpFAR protein was localized by immunofluorescence only in the wax glands and testis. EpFAR expression in High Five insect cells documented the recombinant EpFAR reduced 26-0:(S) CoA and to its corresponding alcohol. The data illuminate the molecular mechanism for fatty alcohol biosynthesis in a beneficial insect, E. pela. © 2017 Wiley Periodicals, Inc.

  5. AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

    Science.gov (United States)

    Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

    2015-04-01

    An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

    Science.gov (United States)

    Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

    2004-03-31

    This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

  7. Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency

    DEFF Research Database (Denmark)

    Gregersen, N; Andresen, B S; Bross, P

    1991-01-01

    . All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing......A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct...... size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed...

  8. Biosynthesis of plasmalogens by the microsomal fraction of Fischer R-3259 sarcoma. Influence of specific 2-acyl chains on the desaturation of 1-alkyl-2-acyl-sn-gycero-3-phosphoethanolamine

    Energy Technology Data Exchange (ETDEWEB)

    Wykle, R.L.; Schremmer, J.M.

    1979-08-07

    In the Fischer R-3259 sarcoma, ethanolamine plasmalogens are synthesized from 1-akyl-2-acyl-sn-glycero-3-phosphoethanolamine by a microsomal desaturase that inserts a ..delta../sup 1/ double bond in the alkyl chain. In the present study, a series of 1-(1-/sup 14/C)hexadecyl-2-acyl-GPE substrates containing specific acyl groups ranging from C/sub 2/ /sub 0/ to C/sub 20/ /sub 4/ at the 2 position were prepared and tested as substrates for the microsomal ..delta../sup 1/-alkyl desaturase. The microsomal preparations contained an acyl hydrolase that removed the C/sub 2/ /sub 0/, C/sub 4/ /sub 0/, and C/sub 7/ /sub 0/ acyl groups from the 2 position. By inhibiting the hydrolase with diisopropyl fluorophosphate, it was possible to test conversion of the unaltered substrates to plasmalogens. The alkyl desaturase exhibited little discrimination among the specific acyl derivatives tested. The highest rate of desaturation was obtained with 1-(1-/sup 14/C)-hexadecyl-2-acyl-GPE synthesized in situ in the microsomes via acylation of 1-(1-/sup 14/C)hexadecyl-GPE; this rate was threefold that observed with exogenously acylated substrates. The 1-(1-/sup 14/C)hexadecyl-2-acyl-GPE synthesized in situ contained highly unsaturated acyl groups; no selectivity of the desaturase for specific acyl chains was detected when the different molecular species of 1-(1-/sup 14/C)alkyl-2-acyl-GPE and 1-(1-/sup 14/C)alk-1'-eyl-2-acyl-GPE were compared. The short-chain substrates, being moe hydrophilic, mimicked the chromatographic behavior of 1-alkyl-GPE, yet they did not resemble the lyso compound in its higher conversion to plasmalogens. Thus, despite their similar R/sub f/ values, the packing of the short-chain acyl homologues in the membrane may be quite different from that of the lyso compound. Binding of 1-hexadecyl-2-acyl-GPE and 1-hexadecyl-GPE to microsomal membranes was similar.

  9. Mutations in COA3 cause isolated complex IV deficiency associated with neuropathy, exercise intolerance, obesity, and short stature.

    Science.gov (United States)

    Ostergaard, Elsebet; Weraarpachai, Woranontee; Ravn, Kirstine; Born, Alfred Peter; Jønson, Lars; Duno, Morten; Wibrand, Flemming; Shoubridge, Eric A; Vissing, John

    2015-03-01

    We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors. The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  10. The frequency of a disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase in sudden infant death syndrome

    DEFF Research Database (Denmark)

    Banner, Jytte; Gregersen, N; Kølvraa, S

    1993-01-01

    A number of rare inherited metabolic disorders are known to lead to death in infancy. Deficiency of medium-chain acyl CoA dehydrogenase has, on clinical grounds, been related particularly to sudden infant death syndrome. The contribution of this disorder to the etiology of sudden infant death...... syndrome is still a matter of controversy. The present study investigated 120 well-defined cases of sudden infant death syndrome in order to detect the frequency of the most common disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase (G985) compared with the frequency...... in the general population. A highly specific polymerase chain reaction assay was applied on dried blood spots. No over-representation of homo- or heterozygosity for G985 appears to exist in such a strictly defined population, for which reason it may be more relevant to look at a broader spectrum of clinical...

  11. Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11.

    Science.gov (United States)

    Herrera, María Georgina; Pignataro, María Florencia; Noguera, Martín Ezequiel; Cruz, Karen Magalí; Santos, Javier

    2018-05-16

    Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.

  12. Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

    International Nuclear Information System (INIS)

    Wilcox, C.A.; Olson, E.N.

    1987-01-01

    The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

  13. Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors.

    Science.gov (United States)

    Caldo, Kristian Mark P; Acedo, Jeella Z; Panigrahi, Rashmi; Vederas, John C; Weselake, Randall J; Lemieux, M Joanne

    2017-10-01

    Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT1 1-113 ) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

  14. Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

    Science.gov (United States)

    Parker, Antony R

    2003-10-01

    The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.

  15. Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase.

    Science.gov (United States)

    Liu, Qin; Chai, Jin; Moche, Martin; Guy, Jodie; Lindqvist, Ylva; Shanklin, John

    2015-09-01

    Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity. © 2015 American Society of Plant Biologists. All Rights Reserved.

  16. COA based robust output feedback UPFC controller design

    Energy Technology Data Exchange (ETDEWEB)

    Shayeghi, H., E-mail: hshayeghi@gmail.co [Technical Engineering Department, University of Mohaghegh Ardabili, Ardabil (Iran, Islamic Republic of); Shayanfar, H.A. [Center of Excellence for Power System Automation and Operation, Electrical Engineering Department, Iran University of Science and Technology, Tehran (Iran, Islamic Republic of); Jalilzadeh, S.; Safari, A. [Technical Engineering Department, Zanjan University, Zanjan (Iran, Islamic Republic of)

    2010-12-15

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

  17. Acylation of Therapeutic Peptides

    DEFF Research Database (Denmark)

    Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

    ) , which promotes intestinal growth and is used to treat bowel disorders such as inflammatory bowel diseases and short bowel syndrome, and the 32 amino acid salmon calcitonin (sCT), which lowers blood calcium and is employed in the treatment of post-menopausal osteoporosis and hypercalcemia. The two...... peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... remained optimal overall. The results indicate that rational acylation of GLP-2 can increase its in vitro intestinal absorption, alone or in combination with permeation enhancers, and are consistent with the initial project hypothesis. For sCT, an unpredicted effect of acylation largely superseded...

  18. The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

    Science.gov (United States)

    Marcella, Aaron M; Barb, Adam W

    2017-12-01

    The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

  19. Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

    DEFF Research Database (Denmark)

    Yang, Liang; Liu, Yang; Sternberg, Claus

    2010-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents...... which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea...... epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent...

  20. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    Science.gov (United States)

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

    2015-03-01

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

  1. Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

    International Nuclear Information System (INIS)

    Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas; Goulding, Celia W.

    2014-01-01

    The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

  2. Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

    Energy Technology Data Exchange (ETDEWEB)

    Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas [UC Irvine, 2212 Natural Sciences I, Irvine, CA 92697 (United States); Goulding, Celia W., E-mail: celia.goulding@uci.edu [UC Irvine, 2212 Natural Sciences I, Irvine, CA 92697 (United States); UC Irvine, 2302 Natural Sciences I, Irvine, CA 92697 (United States)

    2014-04-01

    The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

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

    Science.gov (United States)

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

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase1[OPEN

    Science.gov (United States)

    Liu, Qin; Chai, Jin; Moche, Martin; Guy, Jodie; Lindqvist, Ylva; Shanklin, John

    2015-01-01

    Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity. PMID:26224800

  5. Regulation of schistosome egg production by HMG CoA reductase

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  6. Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH.

    Science.gov (United States)

    Mizutani, Makoto; Atsuchi, Kaori; Asakawa, Akihiro; Matsuda, Norifumi; Fujimura, Masaki; Inui, Akio; Kato, Ikuo; Fujimiya, Mineko

    2009-11-01

    Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

  7. Caveolar fatty acids and acylation of caveolin-1.

    Directory of Open Access Journals (Sweden)

    Qian Cai

    Full Text Available Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.Caveolae were isolated from Chinese hamster ovary (CHO cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS. The caveolin-1 bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5 × 10(7 cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.

  8. 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

    International Nuclear Information System (INIS)

    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

  9. 3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis DPN7T: crystal structure and function of a desulfinase with an acyl-CoA dehydrogenase fold

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Karen van Eunen

    Full Text Available 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.

  11. Development of an activity-based probe for acyl-protein thioesterases

    Science.gov (United States)

    Garland, Megan; Schulze, Christopher J.; Foe, Ian T.; van der Linden, Wouter A.; Child, Matthew A.

    2018-01-01

    Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe. PMID:29364904

  12. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5′-phosphate-binding protein YggS

    OpenAIRE

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-01-01

    Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/M...

  13. Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Fujiyama, A.; Tamanoi, F.

    1986-01-01

    The authors demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO 4 /polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [ 3 H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fraction of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane

  14. Regulation of hepatic level of fatty-acid-binding protein by hormones and clofibric acid in the rat.

    Science.gov (United States)

    Nakagawa, S; Kawashima, Y; Hirose, A; Kozuka, H

    1994-01-01

    Regulation of the hepatic level of fatty-acid-binding protein (FABP) by hormones and p-chlorophenoxyisobutyric acid (clofibric acid) was studied. The hepatic level of FABP, measured as the oleic acid-binding capacity of the cytosolic FABP fraction, was decreased in streptozotocin-diabetic rats. The level of FABP was markedly increased in adrenalectomized rats, and the elevation was prevented by the administration of dexamethasone. Hypothyroidism decreased the level of FABP and hyperthyroidism increased it. A high correlation between the incorporation of [14C]oleic acid in vivo into hepatic triacylglycerol and the level of FABP was found for normal, diabetic and adrenalectomized rats. The level of FABP was increased by administration of clofibric acid to rats in any altered hormonal states, as was microsomal 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, a peroxisome-proliferator-responsive parameter. These results suggest that the hepatic level of FABP is under regulation by multiple hormones and that clofibric acid induces FABP and 1-acyl-GPC acyltransferase by a mechanism which may be distinct from that by which hormones regulate the level of FABP. PMID:8110197

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

    Science.gov (United States)

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

    2016-07-29

    It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-02

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

  17. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    Energy Technology Data Exchange (ETDEWEB)

    Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

    2016-05-06

    Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

  18. EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2014. Scientific Opinion on Dietary Reference Values for pantothenic acid

    DEFF Research Database (Denmark)

    Tetens, Inge

    2014-01-01

    Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies (NDA) derived Dietary Reference Values (DRVs) for pantothenic acid. Pantothenic acid is a water-soluble vitamin, which is a component of coenzyme A (CoA) and acyl-carrier proteins. Pantothenic...

  19. Reduced fat mass in rats fed a high oleic acid-rich safflower oil diet is associated with changes in expression of hepatic PPARalpha and adipose SREBP-1c-regulated genes.

    Science.gov (United States)

    Hsu, Shan-Ching; Huang, Ching-Jang

    2006-07-01

    PPARs and sterol regulatory element-binding protein-1c (SREPB-1c) are fatty acid-regulated transcription factors that control lipid metabolism at the level of gene expression. This study compared a high oleic acid-rich safflower oil (ORSO) diet and a high-butter diet for their effect on adipose mass and expressions of genes regulated by PPAR and SREPB-1c in rats. Four groups of Wistar rats were fed 30S (30% ORSO), 5S (5% ORSO), 30B (29% butter + 1% ORSO), or 5B (4% butter plus 1% ORSO) diets for 15 wk. Compared with the 30B group, the 30S group had less retroperitoneal white adipose tissue (RWAT) mass and lower mRNA expressions of lipoprotein lipase, adipocyte fatty acid-binding protein, fatty acid synthase, acetyl CoA carboxylase, and SREBP-1c in the RWAT, higher mRNA expressions of acyl CoA oxidase, carnitine palmitoyl-transferase 1A, fatty acid binding protein, and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in the liver (P 2 fold those of the 30B group (P < 0.05). These results suggested that the smaller RWAT mass in rats fed the high-ORSO diet might be related to the higher tissue 18:2(n-6) and 20:4(n-6). This in turn could upregulate the expressions of fatty acid catabolic genes through the activation of PPARalpha in the liver and downregulate the expressions of lipid storage and lipogenic gene through the suppression of SREBP-1c in the RWAT.

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

    Science.gov (United States)

    Martinelle, M; Hult, K

    1995-09-06

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

  1. Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.

    Directory of Open Access Journals (Sweden)

    Roberta Leonardi

    2010-06-01

    Full Text Available Coenzyme A (CoA biosynthesis is regulated by the pantothenate kinases (PanK, of which there are four active isoforms. The PanK1 isoform is selectively expressed in liver and accounted for 40% of the total PanK activity in this organ. CoA synthesis was limited using a Pank1(-/- knockout mouse model to determine whether the regulation of CoA levels was critical to liver function. The elimination of PanK1 reduced hepatic CoA levels, and fasting triggered a substantial increase in total hepatic CoA in both Pank1(-/- and wild-type mice. The increase in hepatic CoA during fasting was blunted in the Pank1(-/- mouse, and resulted in reduced fatty acid oxidation as evidenced by abnormally high accumulation of long-chain acyl-CoAs, acyl-carnitines, and triglycerides in the form of lipid droplets. The Pank1(-/- mice became hypoglycemic during a fast due to impaired gluconeogenesis, although ketogenesis was normal. These data illustrate the importance of PanK1 and elevated liver CoA levels during fasting to support the metabolic transition from glucose utilization and fatty acid synthesis to gluconeogenesis and fatty acid oxidation. The findings also suggest that PanK1 may be a suitable target for therapeutic intervention in metabolic disorders that feature hyperglycemia and hypertriglyceridemia.

  2. Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA▿

    OpenAIRE

    Uchino, Keiichi; Saito, Terumi; Gebauer, Birgit; Jendrossek, Dieter

    2007-01-01

    Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in...

  3. Gene expression in plant lipid metabolism in Arabidopsis seedlings.

    Directory of Open Access Journals (Sweden)

    An-Shan Hsiao

    Full Text Available Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3, DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1 and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6 in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1 and LATE ELONGATED HYPOCOTYL (LHY from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis.

  4. Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

    Science.gov (United States)

    MacLean, Lorna; Price, Helen; O'Toole, Peter

    2016-01-01

    Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

  5. Effects of carnitine and its congeners on eicosanoid discharge from rat cells: Implications for release of TNFα

    NARCIS (Netherlands)

    I.M. Garrelds (Ingrid); G.R. Elliott (G.); W.M. Pruimboom (Wanda); F.J. Zijlstra (Freek); I.L. Bonta

    1993-01-01

    textabstractTHE acyl carrier coenzyme A (CoA) is involved in fatty acid metabolism. The carnitine/CoA ratio is of particular importance in regulating the transport of long-chain fatty acids into mitochondria for oxidation. Also CoA has a role in the formation and breakdown of products from both the

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

    Science.gov (United States)

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

    2015-07-01

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

  7. Acyl-Lipid Metabolism

    Science.gov (United States)

    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

  8. Friedel-Crafts Acylation with Amides

    Science.gov (United States)

    Raja, Erum K.; DeSchepper, Daniel J.; Nilsson Lill, Sten O.; Klumpp, Douglas A.

    2012-01-01

    Friedel-Crafts acylation has been known since the 1870s and it is an important organic synthetic reaction leading to aromatic ketone products. Friedel-Crafts acylation is usually done with carboxylic acid chlorides or anhydrides while amides are generally not useful substrates in these reactions. Despite being the least reactive carboxylic acid derivative, we have found a series of amides capable of providing aromatic ketones in good yields (55–96%, 17 examples). We propose a mechanism involving diminished C-N resonance through superelectrophilic activation and subsequent cleavage to acyl cations. PMID:22690740

  9. Endogenous ghrelin-O-acyltransferase (GOAT) acylates local ghrelin in the hippocampus.

    Science.gov (United States)

    Murtuza, Mohammad I; Isokawa, Masako

    2018-01-01

    Ghrelin is an appetite-stimulating peptide. Serine 3 on ghrelin must be acylated by octanoate via the enzyme ghrelin-O-acyltransferase (GOAT) for the peptide to bind and activate the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). Interest in GHSR1a increased dramatically when GHSR1a mRNA was demonstrated to be widespread in the brain, including the cortex and hippocampus, indicating that it has multifaceted functions beyond the regulation of metabolism. However, the source of octanoylated ghrelin for GHSR1a in the brain, outside of the hypothalamus, is not well understood. Here, we report the presence of GOAT and its ability to acylate non-octanoylated ghrelin in the hippocampus. GOAT immunoreactivity is aggregated at the base of the dentate granule cell layer in the rat and wild-type mouse. This immunoreactivity was not affected by the pharmacological inhibition of GHSR1a or the metabolic state-dependent fluctuation of systemic ghrelin levels. However, it was absent in the GHSR1a knockout mouse hippocampus, pointing the possibility that the expression of GHSR1a may be a prerequisite for the production of GOAT. Application of fluorescein isothiocyanate (FITC)-conjugated non-octanoylated ghrelin in live hippocampal slice culture (but not in fixed culture or in the presence of GOAT inhibitors) mimicked the binding profile of FITC-conjugated octanoylated ghrelin, suggesting that extracellularly applied non-octanoylated ghrelin was acylated by endogenous GOAT in the live hippocampus while GOAT being mobilized out of neurons. Our results will advance the understanding for the role of endogenous GOAT in the hippocampus and facilitate the search for the source of ghrelin that is intrinsic to the brain. © 2017 International Society for Neurochemistry.

  10. A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum

    International Nuclear Information System (INIS)

    Sharma, Shailendra Kumar; Modak, Rahul; Sharma, Shilpi; Sharma, Alok Kumar; Sarma, Siddhartha P.; Surolia, Avadhesha; Surolia, Namita

    2005-01-01

    Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton-nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods

  11. Impaired rate of microsomal fatty acid elongation in undernourished neonatal rat brain

    International Nuclear Information System (INIS)

    Yeh, Y.Y.

    1986-01-01

    Hypomyelination caused by undernourishment in characterized by low concentrations of myelin lipids and marked reduction in lignocerate (C/sub 24:0/) and nervonate (C/sub 24:1/) moiety of cerebroside and sulfatide. Since microsomal elongation is the major source of long chain (22 to 24 carbons) fatty acids in the brain, the effect of neonatal undernourishment on acyl elongation was investigated. Undernourishment of suckling rats were induced after birth by restricting maternal dietary intake to 40% of that consumed by dams fed ad libitum. Neonates suckled by the normally fed dams served as controls. Microsomal elongation was measured as nmol from [2- 14 C] malonyl CoA incorporated/h per mg of protein. At 19 days of age, rates of behenoyl CoA (C/sub 22:0/) and erucoyl CoA (C/sub 22:1/) elongation in whole brain of undernourished neonates were 30-40% lower than that of the control, whereas the elongation rates of acyl CoA 16, 18 and 20 carbons in length either saturated or monounsaturated were similar in both groups. Undernourishment had no effect on cytoplasmic de novo fatty acid synthesis from acetyl CoA. If there are multiple elongation factors, the results indicate that the depressed activity of elongating enzyme(s) for C/sub 22:0/ and C/sub 22:1/ is an important contributing factor in lowering S/sub 24:0/ and C/sub 24:1/ content in cerebroside and sulfatide. This impairment may be a specific lesion leading to hypomyelination in undernourished rats

  12. Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

    Science.gov (United States)

    Fedarovich, Alena; Cook, Edward; Tomberg, Joshua; Nicholas, Robert A; Davies, Christopher

    2014-12-09

    A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c-β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

  13. The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii*

    Science.gov (United States)

    Serebryakova, Marina V.; Demina, Irina A.; Galyamina, Maria A.; Kondratov, Ilya G.; Ladygina, Valentina G.; Govorun, Vadim M.

    2011-01-01

    Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In Gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in Gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of Gram-negative bacteria are also present in other mollicutes and Gram-positive bacteria. PMID:21540185

  14. Alpha-synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

    DEFF Research Database (Denmark)

    Golovko, Mikhail Y; Rosenberger, Thad A; Feddersen, Søren

    2007-01-01

    Previously, we demonstrated that ablation of alpha-synuclein (Snca) reduces arachidonate (20:4n-6) turnover in brain phospholipids through modulation of an endoplasmic reticulum-localized acyl-CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n-3) metabolism is unknown...... and turnover in ethanolamine glycerophospholipid, phosphatidylserine, and phosphatidylinositol pools. Increased 22:6n-3-CoA mass was not the result of altered Acsl activity, which was unaffected by the absence of Snca. While Snca bound 22:6n-3, Kd = 1.0 +/- 0.5 micromol/L, it did not bind 22:6n-3-Co...

  15. Diacylglycerol Acyltransferase-2 (DGAT2) and Monoacylglycerol Acyltransferase-2 (MGAT2) Interact to Promote Triacylglycerol Synthesis*

    Science.gov (United States)

    Jin, Youzhi; McFie, Pamela J.; Banman, Shanna L.; Brandt, Curtis; Stone, Scot J.

    2014-01-01

    Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of ∼650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis. PMID:25164810

  16. CoCoa: a software tool for estimating the coefficient of coancestry from multilocus genotype data.

    Science.gov (United States)

    Maenhout, Steven; De Baets, Bernard; Haesaert, Geert

    2009-10-15

    Phenotypic data collected in breeding programs and marker-trait association studies are often analyzed by means of linear mixed models. In these models, the covariance between the genetic background effects of all genotypes under study is modeled by means of pairwise coefficients of coancestry. Several marker-based coancestry estimation procedures allow to estimate this covariance matrix, but generally introduce a certain amount of bias when the examined genotypes are part of a breeding program. CoCoa implements the most commonly used marker-based coancestry estimation procedures and as such, allows to select the best fitting covariance structure for the phenotypic data at hand. This better model fit translates into an increased power and improved type I error control in association studies and an improved accuracy in phenotypic prediction studies. The presented software package also provides an implementation of the new Weighted Alikeness in State (WAIS) estimator for use in hybrid breeding programs. Besides several matrix manipulation tools, CoCoa implements two different bending heuristics, in case the inverse of an ill-conditioned coancestry matrix estimate is needed. The software package CoCoa is freely available at http://webs.hogent.be/cocoa. Source code, manual, binaries for 32 and 64-bit Linux systems and an installer for Microsoft Windows are provided. The core components of CoCoa are written in C++, while the graphical user interface is written in Java.

  17. Acylation of proteins with myristic acid occurs cotranslationally

    International Nuclear Information System (INIS)

    Wilcox, C.; Hu, J.S.; Olson, E.N.

    1987-01-01

    Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC 3 H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [ 3 H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [ 3 H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [ 3 H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification

  18. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    Science.gov (United States)

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  19. Effects of acylation on the functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate.

    Science.gov (United States)

    Yin, Shou-Wei; Tang, Chuan-He; Wen, Qi-Biao; Yang, Xiao-Quan

    2009-01-01

    The effects of succinylation and acetylation on some functional properties and the in vitro trypsin digestibility of kidney bean protein isolate (KPI) were investigated. The extent of succinylation or acetylation progressively increased from 0% to 96% to 97%, as the anhydride-to-protein ratio increased from 0 to 1 g/g. Polyacrylamide gel electrophoresis (PAGE) and zeta potential analyses indicated that acylation, especially succinylation, considerably increased the net charge and hydrodynamic radius of the proteins in KPI, especially vicilin. Acylation treatment at various anhydride-to-protein ratios (0.05 to 1 g/g) remarkably improved the protein solubility (PS) and emulsifying activity index (EAI) at neutral pH, but the improvement by succinylation was much better than that by acetylation. Succinylation resulted in a marked decrease in mechanical moduli of heat-induced gels of KPI, while the mechanical moduli were, on the contrary, increased by acetylation. Additionally, in vitro trypsin digestibility was improved by the acylation in an anhydride-type and level-dependent manner. The results suggest that the functional properties of KPI could be modulated by the chemical acylation treatment, using succinic or acetic anhydride at appropriate anhydride-to-protein ratios.

  20. Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus.

    Science.gov (United States)

    Pereira, Jose H; Goh, Ee-Been; Keasling, Jay D; Beller, Harry R; Adams, Paul D

    2012-10-01

    Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or β-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.

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

    Science.gov (United States)

    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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Age-dependent decline in acyl-ghrelin concentrations and reduced association of acyl-ghrelin and growth hormone in healthy older adults.

    Science.gov (United States)

    Nass, Ralf; Farhy, Leon S; Liu, Jianhua; Pezzoli, Suzan S; Johnson, Michael L; Gaylinn, Bruce D; Thorner, Michael O

    2014-02-01

    Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels. The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups. Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst. Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

  3. Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun-Jung; Son, Hyeoncheol Francis [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of); Kim, Sangwoo [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of); School of Nono-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Ahn, Jae-Woo [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of); Kim, Kyung-Jin, E-mail: kkim@knu.ac.kr [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of)

    2014-02-14

    Highlights: • We determined a crystal structure of β-keto thiolase from Ralstonia eutropha H16 (ReBktB). • Distinct substrate binding mode ReBktB was elucidated. • Enzymatic kinetic parameters of ReBktB were revealed. - Abstract: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K{sub m}, V{sub max}, and K{sub cat} values of 11.58 μM, 1.5 μmol/min, and 102.18 s{sup −1}, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.

  4. Metal complexation by tripodal N-Acyl(thio)urea and picolin(thio)amide compounds: synthesis/extraction and potentiometric studies

    NARCIS (Netherlands)

    Reinoso garcia, M.M.; Dijkman, Arjan; Verboom, Willem; Reinhoudt, David; Malinowska, Elzbieta; Wojciechowska, Dorota; Pietrzak, Mariusz; Selucky, Pavel

    2005-01-01

    The synthesis and binding properties towards different cations of a series of tripodal ligands functionalized with N-acyl(thio)urea and picolin(thio)amide moieties are described. For the extraction of Am3+ and Eu3+ the compounds are not efficient. However, N-acylurea derivative 10 exhibit a

  5. Acylated flavone glycosides from Veronica

    DEFF Research Database (Denmark)

    Albach, Dirk C.; Grayer, Renée J.; Jensen, Søren Rosendal

    2003-01-01

    A survey of the flavonoid glycosides of selected taxa in the genus Veronica yielded two new acylated 5,6,7,3',4'-pentahydroxyflavone (6-hydroxyluteolin) glycosides and two rare allose-containing acylated 5,7,8,4'-tetrahydroxyflavone (isoscutellarein) glycosides. The new compounds were isolated from...

  6. Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis.

    Science.gov (United States)

    Mukherjee, T; Basu, D; Mahapatra, S; Goffin, C; van Beeumen, J; Basu, J

    1996-01-01

    The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR2-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor, behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of M(r) 52,000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin. PMID:8947487

  7. An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine.

    Science.gov (United States)

    Asciolla, James J; Miele, Matthew M; Hendrickson, Ronald C; Resh, Marilyn D

    2017-08-18

    Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC 50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O -acyl transferase family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

    Science.gov (United States)

    Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

    2015-03-01

    The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Acyl transfer from membrane lipids to peptides is a generic process.

    Science.gov (United States)

    Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

    2013-11-15

    The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2012-12-01

    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 (K(D 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 K(m) for phenylbutyryl-CoA were 0.2 mM 34(-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. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

    International Nuclear Information System (INIS)

    Carey, L.M.; Rodriguez, A.; Meighen, E.

    1984-01-01

    The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of > 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system

  12. Crystallization of the acyl-CoA thioesterase TesB from Yersinia pestis

    International Nuclear Information System (INIS)

    Swarbrick, Crystall M. D.; Patterson, Edward I.; Forwood, Jade K.

    2013-01-01

    The expression, purification, crystallization and diffraction of the acyl-CoA thioesterase TesB from Y. pestis are reported. X-ray crystallographic diffraction data to 2.0 Å resolution were collected at the Australian Synchrotron. Yersinia pestis is a highly virulent human pathogen and is the causative agent of bubonic plague. Spread through the bite of infected fleas, plague epidemics have marked important events in history, including the Justinian plague (6th century), the Black Death (14th century) which decimated nearly one quarter of the European population, and more recently the Orientalis plague (1894). To date, deaths are still being reported and, without treatment, the disease kills most people within 4 days. One of the thioesterases from Y. pestis, TesB, is a broad-range acyl-CoA thioesterase and is highly conserved within prokaryotes and throughout evolution, sharing sequence similarity with the HIV Nef binding protein ACOT8. Here the expression, purification, crystallization and diffraction of TesB are reported. TesB has been recombinantly expressed and crystallized using the vapour-diffusion hanging-drop technique at pH 7.0 and 290 K. After optimization, crystals diffracted to 2.0 Å resolution at the Australian Synchrotron and belong to the space group P12 1 1 (a = 73.55, b = 170.82, c = 101.98 Å), with eight molecules likely to be present in the asymmetric unit

  13. N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity.

    Science.gov (United States)

    Weiss, Stefan; Keller, Max; Bernhardt, Günther; Buschauer, Armin; König, Burkhard

    2010-09-01

    N(G)-Acylated argininamides, covering a broad range of lipophilicity (calculated logD values: -1.8-12.5), were synthesized and investigated for NPY Y(1) receptor (Y(1)R) antagonism, Y(1)R affinity and stability in buffer (N(G)-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The K(i) (binding) and K(b) values (Y(1)R antagonism) varied from low nM to one-digit muM. Most of the compounds proved to be sufficiently stable at pH 7.4 over 90min to determine reliable pharmacological data in vitro. Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, probably, due to an intramolecular cleavage mechanism. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. 12-Oxo-Phytodienoic Acid Accumulation during Seed Development Represses Seed Germination in Arabidopsis[C][W][OA

    Science.gov (United States)

    Dave, Anuja; Hernández, M. Luisa; He, Zhesi; Andriotis, Vasilios M.E.; Vaistij, Fabián E.; Larson, Tony R.; Graham, Ian A.

    2011-01-01

    Arabidopsis thaliana COMATOSE (CTS) encodes an ABC transporter involved in peroxisomal import of substrates for β-oxidation. Various cts alleles and mutants disrupted in steps of peroxisomal β-oxidation have previously been reported to exhibit a severe block on seed germination. Oxylipin analysis on cts, acyl CoA oxidase1 acyl CoA oxidase2 (acx1 acx2), and keto acyl thiolase2 dry seeds revealed that they contain elevated levels of 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and JA-Ile. Oxylipin and transcriptomic analysis showed that accumulation of these oxylipins occurs during late seed maturation in cts. Analysis of double mutants generated by crossing cts with mutants in the JA biosynthesis pathway indicate that OPDA, rather than JA or JA-Ile, contributes to the block on germination in cts seeds. We found that OPDA was more effective at inhibiting wild-type germination than was JA and that this effect was independent of CORONATINE INSENSITIVE1 but was synergistic with abscisic acid (ABA). Consistent with this, OPDA treatment increased ABA INSENSITIVE5 protein abundance in a manner that parallels the inhibitory effect of OPDA and OPDA+ABA on seed germination. These results demonstrate that OPDA acts along with ABA to regulate seed germination in Arabidopsis. PMID:21335376

  15. 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.

    Science.gov (United States)

    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.

  16. Fatty Acyl Chains of Mycobacterium marinum Lipooligosaccharides

    Science.gov (United States)

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

    2011-01-01

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

  17. Chromium downregulates the expression of Acetyl CoA Carboxylase 1 gene in lipogenic tissues of domestic goats: a potential strategy for meat quality improvement.

    Science.gov (United States)

    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Zali, Abolfazl; Moradi-Shahrebabak, Hossein; Mousapour, Hojatollah

    2014-06-15

    Acetyl CoA Carboxylase 1 (ACC1) is a biotin-dependent enzyme that catalyzes the carboxylation of Acetyl CoA to form Malonyl CoA, the key intermediate metabolite in fatty acid synthesis. In this study, the mRNA expression of the ACC1 gene was evaluated in four different tissues (liver, visceral fat, subcutaneous fat, and longissimus muscle) of the domestic goat (Capra hircus) kids feeding on four different levels of trivalent chromium (0, 0.5, 1, and 1.5mg/day) as food supplementation. RT-qPCR technique was used for expression analyses and heat shock protein 90 gene (HSP-90) was considered as reference gene for data normalization. Our results revealed that 1.5mg/day chromium significantly reduced the expression of the ACC1 gene in liver, visceral fat, and subcutaneous fat tissues, but not in longissimus muscles (Pmeat quality in domestic animals. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Analytische und Effektor-Studien von N-Acyl-Ethanolaminphosphaten

    OpenAIRE

    Ates, Ebru

    2011-01-01

    Bei N-Acyl-Ethanolaminphosphaten handelt es sich um eine bislang wenig untersuchte Klasse polarer Substanzen, deren Erforschung aufgrund ihrer strukturellen Analogie zu apolaren, physiologisch wirksamen N-Acyl-Ethanolaminen von Interesse ist. Zu bear-beiten waren analytische Fragestellungen, die auch synthetische Aufgaben beinhalteten, wie Methodenentwicklung und Versuche zur Erfassung von N-Acyl-Ethanolamin-phosphaten in ausgewählten Lebensmitteln sowie strukturelle Studien zur „Bioaktivität...

  19. Identification of N-acyl-fumonisin B1 as new cytotoxic metabolites of fumonisin mycotoxins.

    Science.gov (United States)

    Harrer, Henning; Laviad, Elad L; Humpf, Hans Ulrich; Futerman, Anthony H

    2013-03-01

    Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new fumonisin metabolites and to evaluate their cytotoxic potential. MS, molecular biology, and in vitro enzyme assays were used to investigate fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl fumonisins depends on the CerS isoform acylating them. The N-acyl fumonisins are more cytotoxic than the parent fumonisin B1. The identification of N-acyl fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of fumonisins. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. An Efficient, Mild and Solvent-Free Synthesis of Benzene Ring Acylated Harmalines

    Directory of Open Access Journals (Sweden)

    Sabira Begum

    2009-12-01

    Full Text Available A facile synthesis of a series of benzene ring acylated analogues of harmaline has been achieved by Friedel-Crafts acylation under solvent-free conditions at room temperature using acyl halides/acid anhydrides and AlCl3. The reaction afforded 10- and 12-acyl analogues of harmaline in good yield, along with minor quantities of N-acyl-tryptamines and 8-acyl analogues of N-acyltryptamines.

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

    International Nuclear Information System (INIS)

    Hayashi, H.; Miwa, A.

    1989-01-01

    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- 14 C]butyric acid and [1- 14 C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [ 14 C]lignoceric acid into primary bile acids was approximately four times higher than that of [ 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 [ 14 C]lignoceric acid and [ 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, H.; Miwa, A. (Josai Univ., Saitama (Japan))

    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.

  3. Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

    Science.gov (United States)

    Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

    2011-06-17

    To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

  4. Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

    Science.gov (United States)

    Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

    2011-01-01

    To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

  5. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

    Science.gov (United States)

    Ericson, Megan E.; Frank, Matthew W.

    2016-01-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

  6. Expression of a fatty acid-binding protein in yeast

    International Nuclear Information System (INIS)

    Scholz, H.

    1991-06-01

    The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP C ) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size and isoelectric point to native protein, was reached after approximately 16 hours of induction. In contrast, transcription of the gene was induced within half an hour. Both, protein and mRNA were unstable and degraded within 1 h after repression of transcription. Analysis of subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind long chain fatty acids in an in vitro assay. Growth of all transformants on galactose as the carbon source showed no phenotype at temperatures up to 37 deg C, but the growth of FABP-expressing cells at 37 deg C was significantly retarded. Among the biochemical effects of FABP expression on lipid metabolism is a marked reduction of chain elongation and desaturation of exogenously added 14 C-palmitic acid. This effect is most pronounced in triacylglycerols and phospholipids when cells grow at 30 deg C and 37 deg C, respectively. In an in vitro assay determining the desaturation of palmitoyl CoA by microsomal membranes cytosol with or without exo- or endogenous FABP showed the same stimulation of the reaction. The desaturation of exogenously added 14 C-stearic acid, the pattern of unlabelled fatty acids (saturated vs. unsaturated) and the distribution of exogenously added radioactive fatty acids (palmitic, stearic or oleic acid) among lipid classes was not significantly affected. Using high concentrations (1 mM) the uptake of fatty acids was first stimulated and then inhibited when FABP was expressed. (author)

  7. Diacylglycerol acyltransferase-2 (DGAT2) and monoacylglycerol acyltransferase-2 (MGAT2) interact to promote triacylglycerol synthesis.

    Science.gov (United States)

    Jin, Youzhi; McFie, Pamela J; Banman, Shanna L; Brandt, Curtis; Stone, Scot J

    2014-10-10

    Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of ∼ 650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Plasma levels of acylated ghrelin in patients with functional dyspepsia

    Science.gov (United States)

    Kim, Yeon Soo; Lee, Joon Seong; Lee, Tae Hee; Cho, Joo Young; Kim, Jin Oh; Kim, Wan Jung; Kim, Hyun Gun; Jeon, Seong Ran; Jeong, Hoe Su

    2012-01-01

    AIM: To investigate the relationship between plasma acylated ghrelin levels and the pathophysiology of functional dyspepsia. METHODS: Twenty-two female patients with functional dyspepsia and twelve healthy volunteers were recruited for the study. The functional dyspepsia patients were each diagnosed based on the Rome III criteria. Eligible patients completed a questionnaire concerning the severity of 10 symptoms. Plasma acylated ghrelin levels before and after a meal were determined in the study participants using a commercial human acylated enzyme immunoassay kit; electrogastrograms were performed for 50 min before and after a standardized 10-min meal containing 265 kcal. RESULTS: There were no significant differences in plasma acylated ghrelin levels between healthy volunteers and patients with functional dyspepsia. However, in patients with functional dyspepsia, there was a negative correlation between fasting plasma acylated ghrelin levels and the sum score of epigastric pain (r = -0.427, P = 0.047) and a positive correlation between the postprandial/fasting plasma acylated ghrelin ratio and the sum score of early satiety (r = 0.428, P =0.047). Additionally, there was a negative correlation between fasting acylated ghrelin plasma levels and fasting normogastria (%) (r = -0.522, P = 0.013). Interestingly, two functional dyspepsia patients showed paradoxically elevated plasma acylated ghrelin levels after the meal. CONCLUSION: Abnormal plasma acylated ghrelin levels before or after a meal may be related to several of the dyspeptic symptoms seen in patients with functional dyspepsia. PMID:22611317

  9. Erbium trifluoromethanesulfonate-catalyzed Friedel–Crafts acylation using aromatic carboxylic acids as acylating agents under monomode-microwave irradiation

    DEFF Research Database (Denmark)

    Tran, Phuong Hoang; Hansen, Poul Erik; Nguyen, Hai Truong

    2015-01-01

    Erbium trifluoromethanesulfonate is found to be a good catalyst for the Friedel–Crafts acylation of arenes containing electron-donating substituents using aromatic carboxylic acids as the acylating agents under microwave irradiation. An effective, rapid and waste-free method allows the preparation...... of a wide range of aryl ketones in good yields and in short reaction times with minimum amounts of waste...

  10. The Effect of Conformational Variability of Phosphotriesterase upon N-acyl-L-homoserine Lactone and Paraoxon Binding: Insights from Molecular Dynamics Studies

    Directory of Open Access Journals (Sweden)

    Dongling Zhan

    2013-12-01

    Full Text Available The organophosphorous hydrolase (PTE from Brevundimonas diminuta is capable of degrading extremely toxic organophosphorous compounds with a high catalytic turnover and broad substrate specificity. Although the natural substrate for PTE is unknown, its loop remodeling (loop 7-2/H254R led to the emergence of a homoserine lactonase (HSL activity that is undetectable in PTE (kcat/km values of up to 2 × 104, with only a minor decrease in PTE paraoxonase activity. In this study, homology modeling and molecular dynamics simulations have been undertaken seeking to explain the reason for the substrate specificity for the wild-type and the loop 7-2/H254R variant. The cavity volume estimated results showed that the active pocket of the variant was almost two fold larger than that of the wild-type (WT enzyme. pKa calculations for the enzyme (the WT and the variant showed a significant pKa shift from WT standard values (ΔpKa = 3.5 units for the His254residue (in the Arg254 variant. Molecular dynamics simulations indicated that the displacement of loops 6 and 7 over the active site in loop 7-2/H254R variant is useful for N-acyl-L-homoserine lactone (C4-HSL with a large aliphatic chain to site in the channels easily. Thence the expanding of the active pocket is beneficial to C4-HSL binding and has a little effect on paraoxon binding. Our results provide a new theoretical contribution of loop remodeling to the rapid divergence of new enzyme functions.

  11. Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2016-02-01

    Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

  12. The ETFDH c.158A>G Variation Disrupts the Balanced Binding of ESE and ESS Proteins Causing Missplicing and Multiple acyl-CoA Dehydrogenation Deficiency

    DEFF Research Database (Denmark)

    Olsen, Rikke K J; Brøner, Sabrina; Sabaratnam, Rugivan

    2013-01-01

    Multiple acyl-CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostica...

  13. In vivo efficacy of acyl CoA: diacylglycerol acyltransferase (DGAT) 1 inhibition in rodent models of postprandial hyperlipidemia.

    Science.gov (United States)

    King, Andrew J; Segreti, Jason A; Larson, Kelly J; Souers, Andrew J; Kym, Philip R; Reilly, Regina M; Collins, Christine A; Voorbach, Martin J; Zhao, Gang; Mittelstadt, Scott W; Cox, Bryan F

    2010-07-10

    Postprandial serum triglyceride concentrations have recently been identified as a major, independent risk factor for future cardiovascular events. As a result, postprandial hyperlipidemia has emerged as a potential therapeutic target. The purpose of this study was two-fold. Firstly, to describe and characterize a standardized model of postprandial hyperlipidemia in multiple rodent species; and secondly, apply these rodent models to the evaluation of a novel class of pharmacologic agent; acyl CoA:diacylglycerol acyltransferase (DGAT) 1 inhibitors. Serum triglycerides were measured before and for 4h after oral administration of a standardized volume of corn oil, to fasted C57BL/6, ob/ob, apoE(-/-) and CD-1 mice; Sprague-Dawley and JCR/LA-cp rats; and normolipidemic and hyperlipidemic hamsters. Intragastric administration of corn oil increased serum triglycerides in all animals evaluated, however the magnitude and time-course of the postprandial triglyceride excursion varied. The potent and selective DGAT-1 inhibitor A-922500 (0.03, 0.3 and 3 mg/kg, p.o.), dose-dependently attenuated the maximal postprandial rise in serum triglyceride concentrations in all species tested. At the highest dose of DGAT-1 inhibitor, the postprandial triglyceride response was abolished. This study provides a comprehensive characterization of the time-course of postprandial hyperlipidemia in rodents. In addition, the ability of DGAT-1 inhibitors to attenuate postprandial hyperlipidemia in multiple rodent models, including those that feature insulin resistance, is documented. Exaggerated postprandial hyperlipidemia is inherent to insulin-resistant states in humans and contributes to the substantially elevated cardiovascular risk observed in these patients. Therefore, by attenuating postprandial hyperlipidemia, DGAT-1 inhibition may represent a novel therapeutic approach to reduce cardiovascular risk. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Turnover and metabolism of phosphatidylglycerol acyl moieties in E. coli

    International Nuclear Information System (INIS)

    Cooper, C.L.; Rock, C.O.

    1987-01-01

    Fatty acids synthesized in mutants (plsB) blocked in de novo phospholipid biosynthesis were preferentially transferred to phosphatidylglycerol (PtdGro). The ratio of phospholipid species labeled with 32 P and [ 3 H]acetate in the absence of glycerol-3-P acyltransferase activity indicated that [ 3 H]acetate incorporation into PtdGro was due to fatty acid turnover. The magnitude of the turnover process was difficult to estimate due to a significant contraction of the acetyl-CoA pool following the inhibition of phospholipid synthesis. A possible connection between PtdGro turnover and protein acylation was investigated in an E. coli strain containing a lipoprotein expression vector. Cells were prelabeled with [ 3 H]acetate and lipoprotein expression was induced concomitant with the addition of exogenous [ 14 C]-palmitate. [ 14 C] Palmitate was assimilated into the l-position of phosphatidylethanolamine and transferred to the amino terminus of the lipoprotein. In contrast, the ester-linked lipoprotein fatty acids and PtdGro were not enriched in carbon-14 implying a metabolic relationship between these two pools. The data suggest that turnover of PtdGro acyl moieties is related to protein acylation, but a direct link between the two processes remains to be established

  15. Metabolic alkene labeling and in vitro detection of histone acylation via the aqueous oxidative Heck reaction

    NARCIS (Netherlands)

    Ourailidou, Maria E; Dockerty, Paul; Witte, Martin; Poelarends, Gerrit J; Dekker, Frank J

    2015-01-01

    The detection of protein lysine acylations remains a challenge due to lack of specific antibodies for acylations with various chain lengths. This problem can be addressed by metabolic labeling techniques using carboxylates with reactive functionalities. Subsequent chemoselective reactions with a

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  17. OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, Matthew R.; Goblirsch, Brandon R.; Christenson, James K.; Esler, Morgan A.; Mohamed, Fatuma A.; Wackett, Lawrence P.; Wilmot, Carrie M. (UMM)

    2017-10-12

    In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon–carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.

  18. Plasma fatty acyl-carnitines during 8 Weeks of overfeeding: relation to diet energy expenditure and body composition: the PROOF study.

    Science.gov (United States)

    Bray, George A; Redman, Leanne M; de Jonge, Lilian; Rood, Jennifer; Sutton, Elizabeth F; Smith, Steven R

    2018-01-24

    Overfeeding is a strategy for evaluating the effects of excess energy intake. In this secondary analysis we tested the possibility that different levels of dietary protein might differentially modify the response of fatty acyl-carnitines to overfeeding. Twenty-three healthy adult men and women were overfed by 40% for 8 weeks while in-patients with diets containing 5% (LPD), 15% (NPD) or 25% (HPD) protein. Plasma fatty acyl-carnitines were measured by gas chromatography/mass spectrometry (GC/MS) at baseline and after 8 weeks of overfeeding. Measurements included: body composition by DXA, energy expenditure by ventilated hood and doubly-labeled water, fat cell size from subcutaneous fat biopsies, and fat distribution by CT scan. Analysis was done on 5 groups of fatty acyl-carnitines identified by principal components analysis and 6 individual short-chain fatty acyl carnitines. Higher protein intake was associated with significantly lower 8 week levels of medium chain fatty acids and C2, C4-OH and C 6:1, but higher values of C3 and C5:1 acyl-carnitines derived from essential amino acids. In contrast energy and fat intake were only weakly related to changes in fatty acyl-carnitines. A decease or smaller rise in 8 week medium chain acyl-carnitines was associated with an increase in sleeping energy expenditure (P = 0.0004), and fat free mass (P < 0.0001) and a decrease in free fatty acid concentrations (FFA) (P = 0.0067). In contrast changes in short-chain fatty acyl-carnitines were related to changes in resting energy expenditure (P = 0.0026), and fat free mass (P = 0.0007), and C4-OH was positively related to FFA (P = 0006). Protein intake was the major factor influencing changes in fatty acyl carnitines during overfeeding with higher values of most acyl-fatty acids on the low protein diet. The association of dietary protein and fat intake may explain the changes in energy expenditure and metabolic variables resulting in the observed

  19. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    Energy Technology Data Exchange (ETDEWEB)

    Ohlrogge, John B.

    2013-11-05

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

  20. Oxymatrine attenuates hepatic steatosis in non-alcoholic fatty liver disease rats fed with high fructose diet through inhibition of sterol regulatory element binding transcription factor 1 (Srebf1) and activation of peroxisome proliferator activated receptor alpha (Pparα).

    Science.gov (United States)

    Shi, Li-juan; Shi, Lei; Song, Guang-yao; Zhang, He-fang; Hu, Zhi-juan; Wang, Chao; Zhang, Dong-hui

    2013-08-15

    The aim of this study was to examine the therapeutic effect of oxymatrine, a monomer isolated from the medicinal plant Sophora flavescens Ait, on the hepatic lipid metabolism in non-alcoholic fatty liver (NAFLD) rats and to explore the potential mechanism. Rats were fed with high fructose diet for 8 weeks to establish the NAFLD model, then were given oxymatrine treatment (40, 80, and 160 mg/kg, respectively) for another 8 weeks. Body weight gain, liver index, serum and liver lipids, and histopathological evaluation were measured. Enzymatic activity and gene expression of the key enzymes involved in the lipogenesis and fatty acid oxidation were assayed. The results showed that oxymatrine treatment reduced body weight gain, liver weight, liver index, dyslipidemia, and liver triglyceride level in a dose dependant manner. Importantly, the histopathological examination of liver confirmed that oxymatrine could decrease the liver lipid accumulation. The treatment also decreased the fatty acid synthase (FAS) enzymatic activity and increased the carnitine palmitoyltransferase 1A (CPT1A) enzymatic activity. Besides, oxymatrine treatment decreased the mRNA expression of sterol regulatory element binding transcription factor 1(Srebf1), fatty acid synthase (Fasn), and acetyl CoA carboxylase (Acc), and increased the mRNA expression of peroxisome proliferator activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), and acyl CoA oxidase (Acox1) in high fructose diet induced NAFLD rats. These results suggested that the therapeutic effect of oxymatrine on the hepatic steatosis in high fructose diet induced fatty liver rats is partly due to down-regulating Srebf1 and up-regulating Pparα mediated metabolic pathways simultaneously. © 2013 Elsevier B.V. All rights reserved.

  1. [Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

    Science.gov (United States)

    Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

    2003-02-01

    Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

  2. Theoretical Study of Molecular Determinants Involved in Signal Binding to the TraR Protein of Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    N. Kumar

    2005-10-01

    Full Text Available N-acylated homoserine lactone (AHL mediated cell-cell communication in bacteria is dependent on the recognition of the cognate signal by its receptor. This interaction allows the receptor-ligand complex to act as a transcriptional activator, controlling the expression of a range of bacterial phenotypes, including virulence factor expression and biofilm formation. One approach to determine the key features of signal- binding is to model the intermolecular interactions between the receptor and ligand using computational-based modeling software (LigandFit. In this communication, we have modeled the crystal structure of the AHL receptor protein TraR and its AHL signal N-(3- oxooctanoyl-homoserine lactone from Agrobacterium tumefaciens and compared it to the previously reported antagonist behaviour of a number of AHL analogues, in an attempt to determine structural constraints for ligand binding. We conclude that (i a common conformation of the AHL in the hydrophobic and hydrophilic region exists for ligand-binding, (ii a tail chain length threshold of 8 carbons is most favourable for ligand-binding affinity, (iii the positive correlation in the docking studies could be used a virtual screening tool.

  3. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    Science.gov (United States)

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Emerging Signaling Pathway in Arcuate Feeding-Related Neurons: Role of the Acbd7

    Directory of Open Access Journals (Sweden)

    Damien Lanfray

    2017-06-01

    Full Text Available The understanding of the mechanisms whereby energy balance is regulated is essential to the unraveling of the pathophysiology of obesity. In the last three decades, focus was put on the metabolic role played by the hypothalamic neurons expressing proopiomelanocortin (POMC and cocaine and amphetamine regulated transcript (CART and the neurons co-localizing agouti-related peptide (AgRP, neuropeptide Y (NPY, and gamma-aminobutyric acid (GABA. These neurons are part of the leptin-melanocortin pathway, whose role is key in energy balance regulation. More recently, the metabolic involvement of further hypothalamic uncharacterized neuron populations has been suggested. In this review, we discuss the potential homeostatic implication of hypothalamic GABAergic neurons that produce Acyl-Coa-binding domain containing protein 7 (ACBD7, precursor of the nonadecaneuropeptide (NDN, which has recently been characterized as a potent anorexigenic neuropeptide capable of relaying the leptin anorectic/thermogenic effect via the melanocortin system.

  5. Rapid Hydrogen Shift Reactions in Acyl Peroxy Radicals

    DEFF Research Database (Denmark)

    Knap, Hasse Christian; Jørgensen, Solvejg

    2017-01-01

    -shift with X = 6, 7, 8, or 9) in the hydroperoxy acyl peroxy radicals, this H-shift is a reversible reaction and it scrambles between two peroxides, hydroperoxy acyl peroxy and peroxy peroxoic acid radicals. The forward reaction rate constants of the 1,X-OOH H-shift reactions are estimated to be above 103 s–1...... with transition state theory corrected with Eckart quantum tunnelling correction. The ratio between the forward and reverse reaction rate constant of the 1,X-OOH H-shift reactions is around ∼105. Therefore, the equilibrium is pushed toward the production of peroxy peroxoic acid radicals. These very fast 1,X-OOH H......We have used quantum mechanical chemical calculations (CCSD(T)-F12a/cc-pVDZ-F12//M06-2X/aug-cc-pVTZ) to investigate the hydrogen shift (H-shift) reactions in acyl peroxy and hydroperoxy acyl peroxy radicals. We have focused on the H-shift reactions from a hydroperoxy group (OOH) (1,X-OOH H...

  6. Striving towards improved Friedel-Crafts acylation catalysts

    International Nuclear Information System (INIS)

    Scott, N.M.; Deacon, G.B.

    1998-01-01

    Full text: Lanthanum, ytterbium and scandium salts of trifluoromethanesulfonic acid have been shown to act as promising Lewis acid catalysts for the Friedel-Craft acylation reactions. In our study catalytic acylation of anisole by acetic anhydride in nitroethane was investigated. Yields were determined after extraction of para-methoxyacetophenone from the reaction mixture by G.L.C using the external standardisation method. Anhydrous lanthanoid tris-triflate salts [Ln(O 3 SCF 3 ) 3 , Ln La, Y, Nd, Eu and Yb] were initially investigated as catalysts. Ytterbium tris-triflate was found to be the most effective giving ∼90% of the acylation product. The hydrated lanthanide tris-nitrate salts [Ln(NO 3 ) 3 .nH 2 O, Ln = La, Nd, Eu and Yb] were also investigated using in situ dehydration with acetic anhydride. These were found to have low solubility in the reaction mixture and gave poor yields of para-methoxyacetophenone. The formation of side products was suggested by the low total recovery of anisole and para-methoxyacetophenone. The blocking of coordination sites of these catalysts by tetraglyme resulted in a 50% reduction in acylation activity compared with the simple salt. Addition of Li(O 3 SCF 3 ) to Ln(O 3 SCF 3 ) 3 catalysts (ratio of 4:1)had only a slight accelerating effect on the Friedel-Crafts acylation reaction and the yield was only marginally greater than that in the absence of the added salt. In contrast Li(ClO 4 ) dramatically decreased reaction times and improved the yield of para-methoxyace-tophenone, as recently reported

  7. Plasma levels of acylated and total ghrelin in pediatric patients with chronic kidney disease.

    Science.gov (United States)

    Naufel, Maria Fernanda Soares; Bordon, Milena; de Aquino, Talita Marques; Ribeiro, Eliane Beraldi; de Abreu Carvalhaes, João Tomás

    2010-12-01

    This cross-sectional study set out to compare total and acyl ghrelin levels in children with mild chronic kidney disease (CKD) undergoing conservative treatment (n = 19) with children with end-stage renal disease (ESRD) undergoing hemodialysis (n = 24), and with healthy controls (n = 20). The relationship between ghrelin levels and parameters of renal function, nutritional status, and selective hormones were investigated. ESRD patients had higher total ghrelin levels than those with mild CKD or control individuals. However, acyl ghrelin did not differ between groups, indicating that the excess circulating ghrelin was desacylated. Since desacyl ghrelin has been shown to inhibit appetite, increased levels might contribute to protein-energy wasting in pediatric renal patients. When all 43 renal patients were combined, multiple regression analysis found age and glomerular filtration rate (GFR) to be significant negative predictors of total ghrelin. Acyl ghrelin was influenced negatively by age and positively by energy intake. Acyl to total ghrelin ratio related positively to GFR and energy intake. The results indicate that total but not acyl ghrelin is influenced by low GFR in children with CKD and suggests that ghrelin activation may be impaired in these patients. Since energy intake is a positive predictor of acyl ghrelin, the physiological control of ghrelin secretion appears to be altered in pediatric renal patients.

  8. Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus

    International Nuclear Information System (INIS)

    Pereira, Jose H.; Goh, Ee-Been; Keasling, Jay D.; Beller, Harry R.; Adams, Paul D.

    2012-01-01

    In an effort to better understand the control of the formation of branched fatty acids in Micrococcus luteus, the structure of β-ketoacyl-ACP synthase III, which catalyzes the initial step of fatty-acid biosynthesis, has been determined. Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or β-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition

  9. New anthrarobin acyl derivatives as butyrylcholinesterase inhibitors: synthesis, in vitro and in silico studies

    Directory of Open Access Journals (Sweden)

    Mehreen Lateef

    2017-07-01

    Full Text Available To treat Alzheimer's disease (AD, the available candidates are effective only against mild AD or have side effects. So, a study was planned to synthesis new candidates that may have good potential to treat AD. A series of new anthrarobin acyl derivatives (2–8 were synthesized by the reaction of anthrarobin (1 and acetic anhydride/acyl chlorides. The product were characterized by 1H NMR and EI-MS, and evaluated for butyrylcholinesterase (BuChE inhibition activity. Compounds 5 and 4 showed notable BuChE inhibitory potential with IC50 5.3 ± 1.23 and 17.2 ± 0.47 μM, respectively when compared with the standard eserine (IC50 7.8 ± 0.27 μM, compound 5 showed potent BuChE inhibition potential than the standard eserine. The active compounds 5 and 4 have acyl groups at 2-OH and 10-OH positions which may be responsible for inhibitory potential as this orientation is absent in other products. In silico studies of 5 and 4 products revealed the high inhibitory potential due to stable binding of ligand with the BuChE active sites with docking energy score −18.8779 kcal/mol and −23.1159 kcal/mol, respectively. Subsequently, compound 5 that have potent BuChE inhibitory activity could be the potential candidate for drug development for Alzheimer’s disease.

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

    Science.gov (United States)

    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. © 2012 National Research Council of Canada. The Plant Journal © 2012 Blackwell Publishing Ltd.

  11. The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket

    NARCIS (Netherlands)

    Bokhove, Marcel; Jimenez, Pol Nadal; Quax, Wim J.; Dijkstra, Bauke W.

    2010-01-01

    In many Gram-negative pathogens, their virulent behavior is regulated by quorum sensing, in which diffusible signals such as N-acyl homoserine lactones (AHLs) act as chemical messaging compounds. Enzymatic degradation of these diffusible signals by, e. g., lactonases or amidohydrolases abolishes AHL

  12. Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro.

    Science.gov (United States)

    Nancolas, Bethany; Bull, Ian D; Stenner, Richard; Dufour, Virginie; Curnow, Paul

    2017-06-01

    The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium-chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2016-05-11

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

  14. Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study.

    Directory of Open Access Journals (Sweden)

    Carlos Navarro-Retamal

    Full Text Available Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT (EC 2.3.1.84 catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This

  15. N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

    DEFF Research Database (Denmark)

    Térová, B.; Slotte, J.P.; Petersen, G.

    2005-01-01

    -acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel ¿ liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains...... in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak...... interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also...

  16. Evaluation of protein acylation agents for the radioiodination of peptides: Application to labelling octreotide

    International Nuclear Information System (INIS)

    Zalutsky, M.; Vaidyanathan, G.

    2002-01-01

    The purpose of this study was to investigate the utility of two acylation agents originally developed for protein labelling - N-succinimidyl 3-[ 131 I]iodobenzoate and N-succinimidyl 5-[ 131 I]iodopyridine-3- carboxylate - for the radioiodination of peptides. Because of the widespread interest in imaging and treating malignancies that overexpress somatostatin receptors, octreotide was selected as the model peptide. Using these reagents, octreotide was coupled to 3-iodobenzoyl and 3-iodonicotinoyl templates, yielding [N-(3-iodobenzoyl)- D-Phe 1 ]octreotide (IBO) and [N-(3-iodonicotinoyl)-D-Phe 1 ]octreotide (INO), respectively. The IC 50 values for the binding of IBO and INO to somatostatin receptor expressing CA20948 rat pancreatic tumour membranes were 0.90 nM and 0.13 nM, respectively, compared with 0.35 nM for octreotide itself. Yields for the preparation of [ 131 I]IBO and [ 131 I]INO from N-succinimidyl 3-[ 131 I]iodobenzoate and N-succinimidyl 5-[ 131 I]iodopyridine-3- carboxylate, were 35-50%. In vitro assays with AR42J rat pancreatic tumour cells demonstrated considerably higher receptor-specific retention of cell-internalized radioiodine activity for [ 131 I]INO compared with [ 125 I]IBO. A tissue distribution study with both conjugates revealed low levels of activity in the thyroid, consistent with a low degree of deiodination of these radioiodinated peptide conjugates. (author)

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

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

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

  18. Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin.

    Science.gov (United States)

    van der Sluis, Rencia; Erasmus, Elardus

    2016-10-01

    Activation of fatty acids by the acyl-CoA synthetases (ACSs) is the vital first step in fatty acid metabolism. The enzymatic and physiological characterization of the human xenobiotic/medium chain fatty acid: CoA ligases (ACSMs) has been severely neglected even though xenobiotics, such as benzoate and salicylate, are detoxified through this pathway. This review will focus on the nomenclature and substrate specificity of the human ACSM ligases; the biochemical and enzymatic characterization of ACSM1 and ACSM2B; the high sequence homology of the ACSM2 genes (ACSM2A and ACSM2B) as well as what is currently known regarding disease association studies. Several discrepancies exist in the current literature that should be taken note of. For example, the single nucleotide polymorphisms (SNPs) reported to be associated with aspirin metabolism and multiple risk factors of metabolic syndrome are incorrect. Kinetic data on the substrate specificity of the human ACSM ligases are non-existent and currently no data exist on the influence of SNPs on the enzyme activity of these ligases. One of the biggest obstacles currently in the field is that glycine conjugation is continuously studied as a one-step process, which means that key regulatory factors of the two individual steps remain unknown.

  19. The domain-specific and temperature-dependent protein misfolding phenotype of variant medium-chain acyl-CoA dehydrogenase

    NARCIS (Netherlands)

    Jank, Johanna M.; Maier, Esther M.; Reiβ, Dunja D.; Haslbeck, Martin; Kemter, Kristina F.; Truger, Marietta S.; Sommerhoff, Christian P.; Ferdinandusse, Sacha; Wanders, Ronald J.; Gersting, Søren W.; Muntau, Ania C.

    2014-01-01

    The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a

  20. Central and peripheral des-acyl ghrelin regulates body temperature in rats.

    Science.gov (United States)

    Inoue, Yoshiyuki; Nakahara, Keiko; Maruyama, Keisuke; Suzuki, Yoshiharu; Hayashi, Yujiro; Kangawa, Kenji; Murakami, Noboru

    2013-01-04

    In the present study using rats, we demonstrated that central and peripheral administration of des-acyl ghrelin induced a decrease in the surface temperature of the back, and an increase in the surface temperature of the tail, although the effect of peripheral administration was less marked than that of central administration. Furthermore, these effects of centrally administered des-acyl ghrelin could not be prevented by pretreatment with [D-Lys3]-GHRP-6 GH secretagogue receptor 1a (GHS-R1a) antagonists. Moreover, these actions of des-acyl ghrelin on body temperature were inhibited by the parasympathetic nerve blocker methylscopolamine but not by the sympathetic nerve blocker timolol. Using immunohistochemistry, we confirmed that des-acyl ghrelin induced an increase of cFos expression in the median preoptic nucleus (MnPO). Additionally, we found that des-acyl ghrelin dilated the aorta and tail artery in vitro. These results indicate that centrally administered des-acyl ghrelin regulates body temperature via the parasympathetic nervous system by activating neurons in the MnPO through interactions with a specific receptor distinct from the GHS-R1a, and that peripherally administered des-acyl ghrelin acts on the central nervous system by passing through the blood-brain barrier, whereas it exerts a direct action on the peripheral vascular system. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride

    International Nuclear Information System (INIS)

    Li, Conghu; Tian, Zhenhua; Liu, Wentao; Li, Guoying

    2015-01-01

    The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen. - Highlights: • Acylated collagen retained the unique triple helix conformation. • Acylated collagen had stronger thermostability than native collagen. • Amide I was the most sensitive band to the temperature for acylated collagen. • Amide II was the most sensitive band to the temperature for native collagen. • Auto-peak at 1680 cm −1 for acylated collagen disappeared at higher temperature

  2. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  3. Fatty acyl-CoA reductases of birds

    Directory of Open Access Journals (Sweden)

    Hellenbrand Janine

    2011-12-01

    Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

  4. Fatty acyl-CoA reductases of birds

    Science.gov (United States)

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

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

    Science.gov (United States)

    Rowland, Owen; Domergue, Frédéric

    2012-09-01

    Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  6. Imaging N-acyl homoserine lactone quorum sensing in vivo

    DEFF Research Database (Denmark)

    Christensen, Louise Dahl; van Gennip, Maria; Jakobsen, Tim Holm

    2011-01-01

    In order to study N-acyl homoserine lactone (AHL)-based quorum sensing in vivo, we present a protocol using an Escherichia coli strain equipped with a luxR-based monitor system, which in the presence of exogenous AHL molecules expresses a green fluorescent protein (GFP). Lungs from mice challenged...

  7. Imaging N-acyl homoserine lactone quorum sensing in vivo

    DEFF Research Database (Denmark)

    Hultqvist, Louise Dahl; Alhede, Maria; Jakobsen, Tim Holm

    2018-01-01

    In order to study N-acyl homoserine lactone (AHL)-based quorum sensing in vivo, we present a protocol using an Escherichia coli strain equipped with a luxR-based monitor system, which in the presence of exogenous AHL molecules expresses a green fluorescent protein (GFP). Lungs from mice challenged...

  8. Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

    Science.gov (United States)

    Khandokar, Yogesh B; Srivastava, Parul; Cowieson, Nathan; Sarker, Subir; Aragao, David; Das, Shubagata; Smith, Kate M; Raidal, Shane R; Forwood, Jade K

    2017-12-15

    Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn 24 and Asp 39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg 93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Oxidative activation of dihydropyridine amides to reactive acyl donors

    DEFF Research Database (Denmark)

    Funder, Erik Daa; Trads, Julie Brender; Gothelf, Kurt Vesterager

    2015-01-01

    Amides of 1,4-dihydropyridine (DHP) are activated by oxidation for acyl transfer to amines, alcohols and thiols. In the reduced form the DHP amide is stable towards reaction with amines at room temperature. However, upon oxidation with DDQ the acyl donor is activated via a proposed pyridinium...

  10. Only Acyl Carrier Protein 1 (AcpP1 Functions in Pseudomonas aeruginosa Fatty Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Jin-Cheng Ma

    2017-11-01

    Full Text Available The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS, only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coliacpP mutant strain CY1877. And P. aeruginosaacpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL, but replacement of P. aeruginosaacpP1 with E. coliacpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coliacpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.

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

    Science.gov (United States)

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

    2014-07-01

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

  12. Rivastigmine Improves Appetite by Increasing the Plasma Acyl/Des-Acyl Ghrelin Ratio and Cortisol in Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Yoshiko Furiya

    2018-03-01

    Full Text Available Background: Weight loss accelerates cognitive decline and increases mortality in patients with dementia. While acetylcholinesterase (AChE inhibitors are known to cause appetite loss, we sometimes encounter patients in whom switching from donepezil (AChE inhibitor to rivastigmine (AChE and butyrylcholinesterase [BuChE] inhibitor improves appetite. Since BuChE inactivates ghrelin, a potent orexigenic hormone, we speculated that rivastigmine improves appetite by inhibiting BuChE-mediated ghrelin inactivation. Methods: The subjects were patients with mild to moderate Alzheimer disease treated with either rivastigmine patch (n = 11 or donepezil (n = 11 for 6 months. Before and after treatment, we evaluated appetite (0, decreased; 1, slightly decreased; 2, normal; 3, slightly increased; 4, increased, cognitive function, and blood biochemical variables, including various hormones. Results: Rivastigmine treatment significantly improved appetite (from 1.6 ± 0.5 to 2.6 ± 0.7, whereas donepezil treatment did not (from 2.0 ± 0.0 to 1.8 ± 0.4. Simultaneously, rivastigmine, but not donepezil, significantly decreased the serum cholinesterase activity (from 304.3 ± 60.5 to 246.8 ± 78.5 IU/L and increased the cortisol level (from 11.86 ± 3.12 to 14.61 ± 3.29 μg/dL and the acyl/des-acyl ghrelin ratio (from 4.03 ± 2.96 to 5.28 ± 2.72. The levels of leptin, insulin, total ghrel­in, and cognitive function were not significantly affected by either treatment. Conclusions: Our results suggest that compared with donepezil, rivastigmine has the advantage of improving appetite by increasing the acyl/des-acyl ghrelin ratio and cortisol level, thereby preventing weight loss.

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

    Science.gov (United States)

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

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

  14. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  15. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    modulator of the GABAA receptor in brain membranes. ACBP/DBI, or proteolytically derived polypeptides of ACBP/DBI, have also been implicated in the control of steroidogenesis in mitochondria and glucose-stimulated insulin secretion. Thus, it appears that ACBP/DBI is a remarkable, versatile protein. Now we....... There is a remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...... sites and to examine if transcripts from the ACBP/DBI gene were subject to alternative processing. In both brain and liver, transcription is initiated from two major and multiple minor initiation sites. No evidence for alternative splicing was obtained. The promoter region of the ACBP/DBI gene...

  16. Lipopolysaccharides with acylation defects potentiate TLR4 signaling and shape T cell responses.

    Directory of Open Access Journals (Sweden)

    Anna Martirosyan

    Full Text Available Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+ T and CD8(+ T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

  17. 1,5-Anhydro-D-fructose: regioselective acylation with fatty acids

    DEFF Research Database (Denmark)

    Lundt, Inge; Andersen, Søren Møller; Marcussen, Jan

    1999-01-01

    Regioselective acylation of 1,5-anhydro-D-fructose was performed with dodecanoic acid to give 1,5-anhydro-6-O-dodecanoyl-D-fructose, chemically in 50% yield and enzymatically in quantitative yield. Quantitative conversions were also obtained using hexadecanoic and octadecanoic acids as acyl donors...

  18. Enzymatic characterization of a human acyltransferase activity.

    Directory of Open Access Journals (Sweden)

    Akihiko Ozawa

    Full Text Available Non-histone protein acylation is increasingly recognized as an important posttranslational modification, but little is known as to the biochemical properties of protein serine acylating enzymes.We here report that we have identified a metal-stimulated serine octanoyltransferase activity in microsomes from human erythroleukemic (HEL cells. The HEL acylating enzyme was linear with respect to time and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc, and inhibited by chelating reagents. Hydroxylamine treatment removed most, but not all, of the attached radioactivity. A salt extract of microsomal membranes contained the major portion of enzyme activity, indicating that this acyltransferase is not an integral membrane protein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competition experiments, the acyltransferase was well inhibited by activated forms of fatty acids containing at least eight to fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptides des-acyl ghrelin and ACTH were also not acylated; however, des-acyl ghrelin containing the N-terminal tripeptide Gly-Ala-Met was acylated. Mutagenesis studies indicated a requirement for serine five residues from the amino terminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, properties preserved in the HEL cell enzyme purified through four sequential chromatographic steps.In conclusion, our data demonstrate the presence of a zinc-stimulated acyltransferase activity concentrated in the endoplasmic reticulum in HEL cells which is likely

  19. S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects.

    Science.gov (United States)

    Antinone, Sarah E; Ghadge, Ghanashyam D; Ostrow, Lyle W; Roos, Raymond P; Green, William N

    2017-01-25

    Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

  20. Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers

    NARCIS (Netherlands)

    Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

    2014-01-01

    ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned

  1. Acyl Chain Preference in Foam Cell Formation from Mouse Peritoneal Macrophages.

    Science.gov (United States)

    Fujiwara, Yuko; Hama, Kotaro; Tsukahara, Makoto; Izumi-Tsuzuki, Ryosuke; Nagai, Toru; Ohe-Yamada, Mihoko; Inoue, Keizo; Yokoyama, Kazuaki

    2018-01-01

    Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [ 14 C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [ 14 C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [ 14 C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.

  2. Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Krink-Koutsoubelis, Nicolas; Loechner, Anne C.; Lechner, Anna

    2018-01-01

    Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable...... compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S. cerevisiae. Our engineered strains provide......-CoA at 0.5 μM; and isovaleryl-CoA, n-butyryl-CoA, and n-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in S. cerevisiae. By providing this toolbox of acyl...

  3. Role of an Essential Acyl Coenzyme A Carboxylase in the Primary and Secondary Metabolism of Streptomyces coelicolor A3(2)

    Science.gov (United States)

    Rodríguez, E.; Banchio, C.; Diacovich, L.; Bibb, M. J.; Gramajo, H.

    2001-01-01

    Two genes, accB and accE, that form part of the same operon, were cloned from Streptomyces coelicolor A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of accB and accE in Escherichia coli and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an accB null mutant and the thiostrepton growth dependency of a tipAp accB conditional mutant confirmed that AccB is essential for S. coelicolor viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109–3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites. PMID:11526020

  4. Membrane binding of an acyl-lactoferricin B antimicrobial peptide from solid-state NMR experiments and molecular dynamics simulations.

    Science.gov (United States)

    Romo, Tod D; Bradney, Laura A; Greathouse, Denise V; Grossfield, Alan

    2011-08-01

    One approach to the growing health problem of antibiotic resistant bacteria is the development of antimicrobial peptides (AMPs) as alternative treatments. The mechanism by which these AMPs selectively attack the bacterial membrane is not well understood, but is believed to depend on differences in membrane lipid composition. N-acylation of the small amidated hexapeptide, RRWQWR-NH(2) (LfB6), derived from the 25 amino acid bovine lactoferricin (LfB25) can be an effective means to improve its antimicrobial properties. Here, we investigate the interactions of C6-LfB6, N-acylated with a 6 carbon fatty acid, with model lipid bilayers with two distinct compositions: 3:1 POPE:POPG (negatively charged) and POPC (zwitterionic). Results from solid-state (2)H and (31)P NMR experiments are compared with those from an ensemble of all-atom molecular dynamic simulations running in aggregate more than 8.6ms. (2)H NMR spectra reveal no change in the lipid acyl chain order when C6-LfB6 is bound to the negatively charged membrane and only a slight decrease in order when it is bound to the zwitterionic membrane. (31)P NMR spectra show no significant perturbation of the phosphate head groups of either lipid system in the presence of C6-LfB6. Molecular dynamic simulations show that for the negatively charged membrane, the peptide's arginines drive the initial association with the membrane, followed by attachment of the tryptophans at the membrane-water interface, and finally by the insertion of the C6 tails deep into the bilayer. In contrast, the C6 tail leads the association with the zwitterionic membrane, with the tryptophans and arginines associating with the membrane-water interface in roughly the same amount of time. We find similar patterns in the order parameters from our simulations. Moreover, we find in the simulations that the C6 tail can insert 1-2Å more deeply into the zwitterionic membrane and can exist in a wider range of angles than in the negatively charged membrane. We

  5. Purification of specific structured lipids by distillation: Effects on acyl migration

    DEFF Research Database (Denmark)

    Xu, Xuebing; Skands, A.; Adler-Nissen, Jens

    2001-01-01

    The cause and effects of acyl migration during the purification of specific structured lipids by distillation were studied in a conventional batch deodorizer with stripping steam. The mixture of specific structured lipids produced by lipase-catalyzed acidolysis between rapeseed oil and capric acid...... influenced the rate of acyl migration, and their combinations made the effect more severe. However, diacylglycerols were found to be the main reason for acyl migration. In the distillation of the specific structured lipid product mixture, distillation temperature and time were the main factors to determine...... the degree of acyl migration and the extent of separation of free fatty acids. The results indicate that more efficient separation technology should be used to improve the quality of the purified structured lipids. in order to reduce the distillation temperature, vacuum should be made as low as possible...

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

    International Nuclear Information System (INIS)

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

    1991-01-01

    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

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

    Science.gov (United States)

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

    2015-07-07

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

  8. The ACBP gene family in Rhodnius prolixus

    DEFF Research Database (Denmark)

    Majerowicz, David; Hannibal-Bach, Hans K; Castro, Rodolfo S C

    2016-01-01

    The acyl-CoA-binding proteins (ACBP) constitute a family of conserved proteins that bind acyl-CoA with high affinity and protect it from hydrolysis. Thus, ACBPs may have essential roles in basal cellular lipid metabolism. The genome of the insect Rhodnius prolixus encodes five ACBP genes similar...

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

    Science.gov (United States)

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

    2017-06-01

    Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. [Figure not available: see fulltext.

  10. Cholinesterase catalyzed hydrolysis of O-acyl derivatives of serotonin

    International Nuclear Information System (INIS)

    Makhaeva, G.F.; Suvorov, N.N.; Ginodman, L.N.; Antonov, V.K.; AN SSSR, Moscow. Inst. Bioorganicheskoj Khimii)

    1977-01-01

    Hydrolysis of O acyl serotonin derivatives containing the residues of monocarbon dicarbon and amino acids under the effect of horse serum butyryl cholinesterase and bull erythrocytic acetylcholinesterase has been studied. It has been established, that acetylcholinesterase hydrolizes O acetylserotonin only; butyrylcholinesterase hydrolizes all the compounds investigated, except for 5,5'-terephthaloildioxytriptamine. The kinetic parameters of hydrolysis were determined. O acyl serotonin derivatives turned out good substrates of butylrylcholinesterase; serotonin and 5.5'-terephtaloildioxytriptamine are effective competitine inhibitors of the enzyme. Estimating of resistance of O acyl serotonin derivatines to blood cholinesterase effect under physiological conditions shows that the compounds investigated with the exception of 5,5'-terephthaloildioxytriptamine must be quickly hydrolyzed under butyrylcholinesterase action. 5,5'-terephthaloildioxytriptamine is suggested as a radioprotective preparation with the prolonged effect, which agrees with the biological test results

  11. Bio-production of Baccatin III, an Important Precursor of Paclitaxel by a Cost-Effective Approach.

    Science.gov (United States)

    Lin, Shu-Ling; Wei, Tao; Lin, Jun-Fang; Guo, Li-Qiong; Wu, Guang-Pei; Wei, Jun-Bin; Huang, Jia-Jun; Ouyang, Ping-Lan

    2018-07-01

    Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.

  12. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    International Nuclear Information System (INIS)

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-01-01

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials

  13. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Bernhard [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany); Lecker, Laura S. M.; Zoltner, Martin [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Jaenicke, Elmar [Johannes Gutenberg-Universität, Jakob Welder Weg 26, 55128 Mainz (Germany); Schnell, Robert [Karolinska Institutet, 17 177 Stockholm (Sweden); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Brenk, Ruth, E-mail: w.n.hunter@dundee.ac.uk [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany)

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  14. Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions*

    Science.gov (United States)

    Lager, Ida; Yilmaz, Jenny Lindberg; Zhou, Xue-Rong; Jasieniecka, Katarzyna; Kazachkov, Michael; Wang, Peng; Zou, Jitao; Weselake, Randall; Smith, Mark A.; Bayon, Shen; Dyer, John M.; Shockey, Jay M.; Heinz, Ernst; Green, Allan; Banas, Antoni; Stymne, Sten

    2013-01-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism. PMID:24189065

  15. Improving surface functional properties of tofu whey-derived peptides by chemical modification with fatty acids.

    Science.gov (United States)

    Matemu, Athanasia Oswald; Katayama, Shigeru; Kayahara, Hisataka; Murasawa, Hisashi; Nakamura, Soichiro

    2012-04-01

    Effect of acylation with saturated fatty acids on surface functional properties of tofu whey-derived peptides was investigated. Tofu whey (TW) and soy proteins (7S, 11S, and acid-precipitated soy protein [APP]) were hydrolyzed by Protease M 'Amano' G, and resulting peptide mixtures were acylated with esterified fatty acids of different chain length (6C to 18C) to form a covalent linkage between the carboxyl group of fatty acid and the free amino groups of peptide. Acylation significantly (P properties of 7S, 11S, and APP peptides independent of fatty acid chain length. Acylation decreased water binding capacity although oil binding capacity of acylated tofu whey ultra filtered fraction (UFTW acids had shown significant higher surface hydrophobicity as in contrast with acylated UFTW acids can further affect functional properties of soy proteins. © 2012 Institute of Food Technologists®

  16. Acylation of cellular proteins with endogenously synthesized fatty acids

    International Nuclear Information System (INIS)

    Towler, D.; Glaser, L.

    1986-01-01

    A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [ 3 H]acetate, a general precursor of all fatty acids, using BC 3 H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [ 3 H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage

  17. LOCATION OF ACYL GROUPS ON TWO PARTLY ACYLATED GLYCOLIPIDS FROM STRAINS OF USTILAGO (SMUT FUNGI),

    Science.gov (United States)

    erythritol from Ustilago sp. (probably U. nuda (Jens.) Rostr. = U. tritici (Pers.) Rostr.) PRL-627 were acetalated with methyl vinyl ether, deacylated...Partly acylated ustilagic acids 8 (from Ustilago maydis (DC) Corda (= U. zeae Unger) PRL-119), consisting of partially esterified beta-cellobiosyl

  18. Synthesis of sulfadiazinyl acyl/aryl thiourea derivatives as calf intestinal alkaline phosphatase inhibitors, pharmacokinetic properties, lead optimization, Lineweaver-Burk plot evaluation and binding analysis.

    Science.gov (United States)

    Sajid-Ur-Rehman; Saeed, Aamer; Saddique, Gufran; Ali Channar, Pervaiz; Ali Larik, Fayaz; Abbas, Qamar; Hassan, Mubashir; Raza, Hussain; Fattah, Tanzeela Abdul; Seo, Sung-Yum

    2018-06-02

    To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a-4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a-4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a-4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC 50 0.251 ± 0.012 µM (standard KH 2 PO 4 4.317 ± 0.201 µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a-4j obey Lipinsk's rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues. Copyright © 2018. Published by Elsevier Ltd.

  19. Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin

    OpenAIRE

    Asakawa, A; Inui, A; Fujimiya, M; Sakamaki, R; Shinfuku, N; Ueta, Y; Meguid, M M; Kasuga, M

    2005-01-01

    Background/Aims: The gastric peptide ghrelin, an endogenous ligand for growth-hormone secretagogue receptor, has two major molecular forms: acylated ghrelin and desacyl ghrelin. Acylated ghrelin induces a positive energy balance, while desacyl ghrelin has been reported to be devoid of any endocrine activities. The authors examined the effects of desacyl ghrelin on energy balance.

  20. Acyl and silyl group effects in reactivity-based one-pot glycosylation: synthesis of embryonic stem cell surface carbohydrates Lc4 and IV(2)Fuc-Lc4.

    Science.gov (United States)

    Hsu, Yun; Lu, Xin-An; Zulueta, Medel Manuel L; Tsai, Chih-Ming; Lin, Kuo-I; Hung, Shang-Cheng; Wong, Chi-Huey

    2012-03-14

    Relative reactivity evaluations showed the graded arming of toluenyl thioglucosides by variously positioned silyl groups but not by their acyl counterparts. These findings were applied in reactivity-based one-pot assembly of linker-attached Lc(4) and IV(2)Fuc-Lc(4), which are components of human embryonic stem cell surface. The sugar-galectin-1 binding was also examined.

  1. THE LATEST ADVANCEMENTS IN THE ACYLATION REACTIONS VIA CROSS-DEHYDROGENATIVE COUPLING AND/OR METAL CATALYSTS

    Directory of Open Access Journals (Sweden)

    Soykan Ağar

    2017-12-01

    Full Text Available There are quite many examples in the scientific literature regarding the acylation reactions, especially the metal-catalyzed acylation reactions, metal-free acylation reactions, metal-catalyzed acylation via cross-dehydrogenative coupling (CDC reactions and metal-free acylation via cross-dehydrogenative coupling (CDC reactions. In this review paper, the most important examples of these domains were brought together and their mechanisms were exhibited in a clear, chronological format. Following these, the best example study towards green chemistry with a metal-free and high-yielding route was mentioned and discussed to demonstrate what has achieved in this field regarding the new acylation reaction mechanisms using the advantages of cross-dehydrogenative coupling (CDC reactions. The most prominent studies regarding these domains have been examined thoroughly and the latest progress in this field was explained in detail.

  2. Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae).

    Science.gov (United States)

    Kite, Geoffrey C; Rowe, Emily R; Lewis, Gwilym P; Veitch, Nigel C

    2011-04-01

    The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar

  3. Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis*

    Science.gov (United States)

    Capyk, Jenna K.; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C.; Eltis, Lindsay D.

    2011-01-01

    Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20–30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μm in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism. PMID:21987574

  4. Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis.

    Science.gov (United States)

    Capyk, Jenna K; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C; Eltis, Lindsay D

    2011-11-25

    Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k(cat)/K(m)) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K(m)(O(2)) was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ(1) ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.

  5. X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal

    Energy Technology Data Exchange (ETDEWEB)

    Hellberg, Kristina; Grimsrud, Paul A.; Kruse, Andrew C.; Banaszak, Leonard J.; Ohlendorf, Douglas H.; Bernlohr, David A. (UMM)

    2012-07-11

    Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long-chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4-hydroxy-2-nonenal (4-HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4-HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4-HNE have been solved to 1.9 {angstrom} and 2.3 {angstrom} resolution, respectively. While the 4-HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4-HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4-HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.

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

    International Nuclear Information System (INIS)

    Khan, A.; Javed, M.T.; Mahmood, F.; Hussain, R.

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Khan, A.; Javed, M. T.; Mahmood, F. [University of Agriculture, Faisalabad (Pakistan). Dept. of Pathology; Hussain, R. [The Islamia Univ. of Bahawalpur, Pakistan (Pakistan). Dept. of Veterinary and Animal Sciences

    2013-12-15

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

  8. Mangiferin treatment inhibits hepatic expression of acyl-coenzyme A:diacylglycerol acyltransferase-2 in fructose-fed spontaneously hypertensive rats: a link to amelioration of fatty liver

    Energy Technology Data Exchange (ETDEWEB)

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang [Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016 China (China); Chonan, Ritsu [Koei Kogyo Co., Ltd., Tokyo, 101-0063 Japan (Japan); Yamahara, Johji [Pharmafood Institute, Kyoto, 602-8136 Japan (Japan); Wang, Jianwei, E-mail: wangjianwei1968@gmail.com [Department of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016 China (China); Li, Yuhao, E-mail: yuhao@sitcm.edu.au [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences/Sydney Institute of Traditional Chinese Medicine, NSW 2000 Australia (Australia)

    2014-10-15

    Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. - Highlights: • We investigated the anti-steatotic effect of mangiferin (MA) in fructose-fed SHR. • MA (15 mg/kg/day for 7 weeks) ameliorated fructose-induced fatty liver in

  9. Mangiferin treatment inhibits hepatic expression of acyl-coenzyme A:diacylglycerol acyltransferase-2 in fructose-fed spontaneously hypertensive rats: a link to amelioration of fatty liver

    International Nuclear Information System (INIS)

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang; Chonan, Ritsu; Yamahara, Johji; Wang, Jianwei; Li, Yuhao

    2014-01-01

    Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. - Highlights: • We investigated the anti-steatotic effect of mangiferin (MA) in fructose-fed SHR. • MA (15 mg/kg/day for 7 weeks) ameliorated fructose-induced fatty liver in

  10. Purification, crystallization and preliminary X-ray diffraction analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus mutans strain UA159

    International Nuclear Information System (INIS)

    Kim, Tae-O; Im, Dong-Won; Jung, Ha Yun; Kwon, Seong Jung; Heo, Yong-Seok

    2012-01-01

    Enoyl-acyl carrier protein reductase (FabK) from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.40 Å. A triclosan-resistant flavoprotein termed FabK is the sole enoyl-acyl carrier protein reductase in Streptococcus pneumoniae and Streptococcus mutans. In this study, FabK from S. mutans strain UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.40 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P6 2 , with unit-cell parameters a = b = 105.79, c = 44.15 Å. The asymmetric unit contained one molecule, with a corresponding V M of 2.05 Å 3 Da −1 and a solvent content of 39.9%

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    get insight into these. Results We have created the CoaSim application as a flexible environment for Monte various types of genetic data under equilibrium and non-equilibrium coalescent variety of applications. Interaction with the tool is through the Guile version scripting language. Scheme scripts......Background Coalescent simulations are playing a large role in interpreting large scale intra- polymorphism surveys and for planning and evaluating association studies. Coalescent of data sets under different models can be compared to the actual data to test different evolutionary factors and thus...

  12. Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

    Science.gov (United States)

    Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

    2016-01-01

    Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

  13. Exploring Cooperative Effects in Oxidative NHC Catalysis: Regioselective Acylation of Carbohydrates.

    Science.gov (United States)

    Cramer, David L; Bera, Srikrishna; Studer, Armido

    2016-05-23

    The utility of oxidative NHC catalysis for both the regioselective and chemoselective functionalization of carbohydrates is explored. Chiral NHCs allow for the highly regioselective oxidative esterification of various carbohydrates using aldehydes as acylation precursors. The transformation was also shown to be amenable to both cis/trans diol isomers, free amino groups, and selective for specific sugar epimers in competition experiments. Efficiency and regioselectivity of the acylation can be improved upon using two different NHC catalysts that act cooperatively. The potential of the method is documented by the regioselective acylation of an amino-linked neodisaccharide. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Toward Green Acylation of (Heteroarenes: Palladium-Catalyzed Carbonylation of Olefins to Ketones

    Directory of Open Access Journals (Sweden)

    Jie Liu

    2017-11-01

    Full Text Available Green Friedel–Crafts acylation reactions belong to the most desired transformations in organic chemistry. The resulting ketones constitute important intermediates, building blocks, and functional molecules in organic synthesis as well as for the chemical industry. Over the past 60 years, advances in this topic have focused on how to make this reaction more economically and environmentally friendly by using green acylating conditions, such as stoichiometric acylations and catalytic homogeneous and heterogeneous acylations. However, currently well-established methodologies for their synthesis either produce significant amounts of waste or proceed under harsh conditions, limiting applications. Here, we present a new protocol for the straightforward and selective introduction of acyl groups into (hetero­arenes without directing groups by using available olefins with inexpensive CO. In the presence of commercial palladium catalysts, inter- and intramolecular carbonylative C–H functionalizations take place with good regio- and chemoselectivity. Compared to classical Friedel–Crafts chemistry, this novel methodology proceeds under mild reaction conditions. The general applicability of this methodology is demonstrated by the direct carbonylation of industrial feedstocks (ethylene and diisobutene as well as of natural products (eugenol and safrole. Furthermore, synthetic applications to drug molecules are showcased.

  15. Acute aerobic exercise differentially alters acylated ghrelin and perceived fullness in normal-weight and obese individuals.

    Science.gov (United States)

    Heden, Timothy D; Liu, Ying; Park, Youngmin; Dellsperger, Kevin C; Kanaley, Jill A

    2013-09-01

    Adiposity alters acylated ghrelin concentrations, but it is unknown whether adiposity alters the effect of exercise and feeding on acylated ghrelin responses. Therefore, the purpose of this study was to determine whether adiposity [normal-weight (NW) vs. obese (Ob)] influences the effect of exercise and feeding on acylated ghrelin, hunger, and fullness. Fourteen NW and 14 Ob individuals completed two trials in a randomized counterbalanced fashion, including a prior exercise trial (EX) and a no exercise trial (NoEX). During the EX trial, the participants performed 1 h of treadmill walking (55-60% peak O2 uptake) during the evening, 12 h before a 4-h standardized mixed meal test. Frequent blood samples were taken and analyzed for acylated ghrelin, and a visual analog scale was used to assess perceived hunger and fullness. In NW individuals, EX, compared with NoEX, reduced fasting acylated ghrelin concentrations by 18% (P = 0.03), and, in response to feeding, the change in acylated ghrelin (P = 0.02) was attenuated by 39%, but perceived hunger and fullness were unaltered. In Ob individuals, despite no changes in fasting or postprandial acylated ghrelin concentrations with EX, postprandial fullness was attenuated by 46% compared with NoEX (P = 0.05). In summary, exercise performed the night before a meal suppresses acylated ghrelin concentrations in NW individuals without altering perceived hunger or fullness. In Ob individuals, despite no changes in acylated ghrelin concentrations, EX reduced the fullness response to the test meal. Acylated ghrelin and perceived fullness responses are differently altered by acute aerobic exercise in NW and Ob individuals.

  16. Room-Temperature Alternative to the Arbuzov Reaction: The Reductive Deoxygenation of Acyl Phosphonates

    OpenAIRE

    Kedrowski, Sean M. A.; Dougherty, Dennis A.

    2010-01-01

    The reductive deoxygenation of acyl phosphonates using a Wolff−Kishner-like sequence is described. This transformation allows direct access to alkyl phosphonates from acyl phosphonates at room temperature. The method can be combined with acyl phosphonate synthesis into a one pot, four-step procedure for the conversion of carboxylic acids into alkyl phosphonates. The methodology works well for a variety of aliphatic acids and shows a functional group tolerance similar to that of other hydrazon...

  17. Studies on acylation of lysolecithin in chicken intestine

    International Nuclear Information System (INIS)

    Lokesh, B.R.; Madhava Rao, A.; Murthy, S.K.

    1976-01-01

    The enzymatic acylation of lysolecithin to lecithin is shown to occur in the brush border-free particulate fraction of the small intestines of neonatal chicken. It requires ATP, coenzyme A and Mg 2+ or Mn 2+ for maximal activity. The system is specific for oleic acid. The fatty acid composition at the α-position of lysolecithin does not seem to influence the rate of acylation. The fatty acid incorporated into lysolecithin is shown to occupy exclusively, the β-position. [ 32 P]lecithin and [1- 14 C]oleic acid has been used as tracers in the studies. (author)

  18. Quantum chemical study of penicillin: Reactions after acylation

    Science.gov (United States)

    Li, Rui; Feng, Dacheng; Zhu, Feng

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

  19. Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

    Directory of Open Access Journals (Sweden)

    Marta Palusinska-Szysz

    Full Text Available 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

  20. Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

    Science.gov (United States)

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

    2012-05-01

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

  1. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  2. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  3. Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset

    DEFF Research Database (Denmark)

    Smelt, A H; Poorthuis, B J; Onkenhout, W

    1998-01-01

    Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetrade......Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9......), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may...... be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L-carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis....

  4. Cis–Trans Configuration of Coumaric Acid Acylation Affects the Spectral and Colorimetric Properties of Anthocyanins

    Directory of Open Access Journals (Sweden)

    Gregory T. Sigurdson

    2018-03-01

    Full Text Available The color expression of anthocyanins can be affected by a variety of environmental factors and structural characteristics. Anthocyanin acylation (type and number of acids is known to be key, but the influence of acyl isomers (with unique stereochemistries remains to be explored. The objective of this study was to investigate the effects of cis–trans configuration of the acylating group on the spectral and colorimetric properties of anthocyanins. Petunidin-3-rutinoside-5-glucoside (Pt-3-rut-5-glu and Delphinidin-3-rutinoside-5-glucoside (Dp-3-rut-5-glu and their cis and trans coumaroylated derivatives were isolated from black goji and eggplant, diluted in pH 1–9 buffers, and analyzed spectrophotometrically (380–700 nm and colorimetrically (CIELAB during 72 h of storage (25 °C, dark. The stereochemistry of the acylating group strongly impacted the spectra, color, and stability of the Dp and Pt anthocyanins. Cis acylated pigments exhibited the greatest λmax in all pH, as much as 66 nm greater than their trans counterparts, showing bluer hues. Cis acylation seemed to reduce hydration across pH, increasing color intensity, while trans acylation generally improved color retention over time. Dp-3-cis-p-cou-rut-5-glu exhibited blue hues even in pH 5 (C*ab = 10, hab = 256° where anthocyanins are typically colorless. Cis or trans double bond configurations of the acylating group affected anthocyanin spectral and stability properties.

  5. Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution

    International Nuclear Information System (INIS)

    Subramanian, Sandhya; Abendroth, Jan; Phan, Isabelle Q. H.; Olsen, Christian; Staker, Bart L.; Napuli, A.; Van Voorhis, Wesley C.; Stacy, Robin; Myler, Peter J.

    2011-01-01

    The R. prowazekii 3-ketoacyl-(acyl-carrier-protein) reductase is similar to those from other prokaryotic pathogens but differs significantly from the mammalian orthologue, strengthening its case as a potential drug target. Rickettsia prowazekii, a parasitic Gram-negative bacterium, is in the second-highest biodefense category of pathogens of the National Institute of Allergy and Infectious Diseases, but only a handful of structures have been deposited in the PDB for this bacterium; to date, all of these have been solved by the SSGCID. Owing to its small genome (about 800 protein-coding genes), it relies on the host for many basic biosynthetic processes, hindering the identification of potential antipathogenic drug targets. However, like many bacteria and plants, its metabolism does depend upon the type II fatty-acid synthesis (FAS) pathway for lipogenesis, whereas the predominant form of fatty-acid biosynthesis in humans is via the type I pathway. Here, the structure of the third enzyme in the FAS pathway, 3-ketoacyl-(acyl-carrier-protein) reductase, is reported at a resolution of 2.25 Å. Its fold is highly similar to those of the existing structures from some well characterized pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei, but differs significantly from the analogous mammalian structure. Hence, drugs known to target the enzymes of pathogenic bacteria may serve as potential leads against Rickettsia, which is responsible for spotted fever and typhus and is found throughout the world

  6. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides.

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-17

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a 'piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (K d =39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  7. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

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

    International Nuclear Information System (INIS)

    Janikowska, K; Rachoń, J; Makowiec, S

    2014-01-01

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

  9. Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

    International Nuclear Information System (INIS)

    Friedmann, P.S.; Wren, F.E.; Matthews, J.N.

    1990-01-01

    Epidermal melanocytes (MC) synthesize melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melanogenesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E1, E2, D2, F2 alpha, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradiated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P less than 0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanogenesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C

  10. Crystal Structure of Rat Carnitine Palmitoyltransferase II (CPT-II)

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao,Y.; Jogl, G.; Esser, V.; Tong, L.

    2006-01-01

    Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the {beta}-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9 Angstroms resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.

  11. Predicting protein-binding RNA nucleotides with consideration of binding partners.

    Science.gov (United States)

    Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

    2015-06-01

    In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

  12. Cardiovascular and metabolic risk profile and acylation-stimulating protein levels in children with Prader-Willi syndrome and effects of growth hormone treatment

    NARCIS (Netherlands)

    R.F.A. de Lind van Wijngaarden (Roderick); K. Cianflone (Katherine); Y. Gao; R.W.J. Leunissen (Ralph); A.C.S. Hokken-Koelega (Anita)

    2010-01-01

    textabstractContext: Reports on the cardiovascular and metabolic risk profile in children with Prader-Willi syndrome (PWS) and the effects of GH treatment are scarce. Acylation-stimulating protein (ASP) stimulates glucose uptake and triglyceride storage in adipose tissue. Objectives: The aim was to

  13. Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

    Science.gov (United States)

    Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

    2016-02-26

    We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Acylated flavonol glycosides from Larix needles

    NARCIS (Netherlands)

    Niemann, Gerard J.

    2006-01-01

    Kaempferol-3-p-coumarylglucoside (KCG) was isolated from ether fractions of acetone-extracted freeze-dried needles of all larch species investigated. In each case, KCG was found as one of the main flavonoids, whereas often a variety of closely related, acylated flavonoids was present in either

  15. A simple, effective, green method for regioselective 3-acylation of unprotected indoles

    DEFF Research Database (Denmark)

    Tran, Phuong Huong; Tran, Hai N.; Hansen, Poul Erik

    2015-01-01

    A fast and green method is developed for regioselective acylation of indoles in the 3-position without the need for protection of the NH position. The method is based on Friedel-Crafts acylation using acid anhydrides. The method has been optimized, and Y(OTf)3 in catalytic amounts is found...

  16. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

    Directory of Open Access Journals (Sweden)

    Katsumi Iizuka

    2017-02-01

    Full Text Available Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase, fructolysis (Glut5, ketohexokinase, and lipogenesis (acetyl CoA carboxylase, fatty acid synthase. ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  17. Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate1[OPEN

    Science.gov (United States)

    Free, Heather C.A.; Smith, Bronwen G.

    2018-01-01

    Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. PMID:29724771

  18. Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis.

    Science.gov (United States)

    Suh, M C; Schultz, D J; Ohlrogge, J B

    1999-03-01

    Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.

  19. Attempts to Synthesize 3-acyl-4-hydroxycoumarins from Meldrum’s acid -- and Related Chemistry

    OpenAIRE

    Ye, Fengbin; Tropp, Kristin; Yu, Yiting

    2007-01-01

    We start our synthetic work with the acylation of Meldrum’s acid to get three different 5-acyl Meldrum’s acids. These compounds are attacked by various nucleophiles containing different hetero atoms to obtain β-ketoesters, β-ketoamides and the corresponding β-keto-phosphorus compounds respectively. New β-ketoamides could be synthesized and characterized. The reaction of acylated Meldrum’s acid and diphenylphosphine did not lead to the expected β-keto-phosphide compound, but the resulting prod...

  20. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  1. Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia

    DEFF Research Database (Denmark)

    Moesgaard, B.; Hansen, Harald S.; Jaroszewski, J.W.

    1999-01-01

    -phospho(N-acyl)-ethanolamine (NAPE(PLAS)), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N-acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs...

  2. Enantioselective N-Heterocyclic Carbene Catalysis via the Dienyl Acyl Azolium.

    Science.gov (United States)

    Gillard, Rachel M; Fernando, Jared E M; Lupton, David W

    2018-04-16

    Herein we report the enantioselective N-heterocyclic carbene catalyzed (4+2) annulation of the dienyl acyl azolium with enolates. The reaction exploits readily accessible acyl fluorides and TMS enol ethers to give a range of highly enantio- and diastereo-enriched cyclohexenes (most >97:3 er and >20:1 dr). The reaction was found to require high nucleophilicity NHC catalysts with mechanistic studies supporting a stepwise 1,6-addition/β-lactonization. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin

    DEFF Research Database (Denmark)

    Rissanen, Sami; Grzybek, Michal; Orłowski, Adam

    2017-01-01

    membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane...

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

    Science.gov (United States)

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

    2012-01-01

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

  5. Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from a Drug Acyl Glucuronide Metabolite: Multiple Albumin Adductions in Diclofenac Patients

    Science.gov (United States)

    Hammond, Thomas G.; Meng, Xiaoli; Jenkins, Rosalind E.; Maggs, James L.; Castelazo, Anahi Santoyo; Regan, Sophie L.; Bennett, Stuart N. L.; Earnshaw, Caroline J.; Aithal, Guruprasad P.; Pande, Ira; Kenna, J. Gerry; Stachulski, Andrew V.; Park, B. Kevin

    2014-01-01

    Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-related hypersensitivities had either a single drug-derived adduct or one of five combinations of 2–8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein’s 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug’s AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association. PMID:24902585

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

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  7. Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

    Science.gov (United States)

    Hiramine, Yasushi; Tanabe, Toshizumi

    2011-06-01

    Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

  8. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  9. Suppression of acyl migration in enzymatic production of structured lipids through temperature programming

    DEFF Research Database (Denmark)

    Yang, Tiankui; Fruekilde, Maj-Britt; Xu, Xuebing

    2005-01-01

    Acyl migration in the glycerol backbone often leads to the increase of by-products in the enzymatic production of specific structured lipids. Acyl migration is a thermodynamic process and is very difficult to stop fully in actual reactions. The objective of this study was to investigate...

  10. Reaction of tantalum-alkyne complexes with isocyanates or acyl cyanides

    International Nuclear Information System (INIS)

    Kataoka, Yasutaka; Oguchi, Yoshiyuki; Yoshizumi, Kazuyuki; Miwatashi, Seiji; Takai, Kazuhiko; Utimoto, Kiitiro

    1992-01-01

    Treatment of alkynes with low-valent tantalum derived from TiCl 5 and zinc produces tantalum-alkyne complexes (not isolated), which react in situ with phenyl isocyanate (or butyl isocyanate) to give (E)-α, β-unsaturated amides stereoselectively. The tantalum-alkyne complexes also react with acyl cyanides in the presence of BF 3 ·OEt 2 to give α-cyanohydrins. In both reactions, filtration of the reaction mixture containing the tantalum-alkyne complexes before addition of isocyanates (or acyl cyanides) is indispensable to obtain good yields. (author)

  11. Acute effect of exercise intensity and duration on acylated ghrelin and hunger in men.

    Science.gov (United States)

    Broom, David R; Miyashita, Masashi; Wasse, Lucy K; Pulsford, Richard; King, James A; Thackray, Alice E; Stensel, David J

    2017-03-01

    Acute exercise transiently suppresses the orexigenic gut hormone acylated ghrelin, but the extent to which exercise intensity and duration determine this response is not fully understood. The effects of manipulating exercise intensity and duration on acylated ghrelin concentrations and hunger were examined in two experiments. In experiment one, nine healthy males completed three, 4-h conditions (control, moderate-intensity running (MOD) and vigorous-intensity running (VIG)), with an energy expenditure of ~2.5 MJ induced in both MOD (55-min running at 52% peak oxygen uptake (V.O 2peak )) and VIG (36-min running at 75% V.O 2peak ). In experiment two, nine healthy males completed three, 9-h conditions (control, 45-min running (EX45) and 90-min running (EX90)). Exercise was performed at 70% V.O 2peak In both experiments, participants consumed standardised meals, and acylated ghrelin concentrations and hunger were quantified at predetermined intervals. In experiment one, delta acylated ghrelin concentrations were lower than control in MOD (ES = 0.44, P = 0.01) and VIG (ES = 0.98, P Hunger ratings were similar across the conditions (P = 0.35). In experiment two, delta acylated ghrelin concentrations were lower than control in EX45 (ES = 0.77, P Hunger ratings were lower than control in EX45 (ES = 0.20, P = 0.01) and EX90 (ES = 0.27, P = 0.001); EX45 and EX90 were similar (ES = 0.07, P = 0.34). Hunger and delta acylated ghrelin concentrations remained suppressed at 1.5 h in EX90 but not EX45. In conclusion, exercise intensity, and to a lesser extent duration, are determinants of the acylated ghrelin response to acute exercise. © 2017 Society for Endocrinology.

  12. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

  13. Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat.

    Science.gov (United States)

    Grillo, M P; Benet, L Z

    2001-08-01

    Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl-N-acyl

  14. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  15. Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

    Science.gov (United States)

    Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

    2013-01-01

    Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

  16. Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: relationship with cholinesterase.

    Science.gov (United States)

    Tham, Elaine; Liu, Jianhua; Innis, Sheila; Thompson, David; Gaylinn, Bruce D; Bogarin, Roberto; Haim, Alon; Thorner, Michael O; Chanoine, Jean-Pierre

    2009-05-01

    Acylated (octanoylated) ghrelin stimulates food intake and growth hormone secretion and is deacylated into desacyl ghrelin by butyrylcholinesterase. Acylated and desacyl ghrelin both promote adipogenesis. Ghrelin concentrations decrease with hyperglycemia and hyperinsulinism. We hypothesized that 1) acylated ghrelin increases during pregnancy, contributing positively to energy balance, but is lower in women with gestational diabetes and 2) butyrylcholinesterase activity is inversely correlated with acylated ghrelin concentrations. In a first group of subjects, using two-site sandwich ghrelin assays that specifically detect full-length forms, we investigated women with and without gestational diabetes (n = 14/group) during pregnancy and after delivery. We examined whether changes in ghrelin during a test meal were correlated with changes in pituitary growth hormone [assessed through calculation of the area under the curve (AUC) during the test meal]. In postpartum subjects, the percent of total ghrelin that is acylated was four to five times higher than previously observed using single antibody assays. During pregnancy, acylated ghrelin concentrations (mean +/- SE) were lower compared with the postpartum period throughout the meal (AUC 1.2 +/- 0.2 vs. 10.2 +/- 1.9 ng.ml(-1).90 min(-1), P < 0.001). In the postpartum, acylated ghrelin and growth hormone were positively correlated (r = 0.50, P = 0.007). Desacyl (but not acylated) ghrelin was increased in subjects with gestational diabetes during and after pregnancy (AUC 15.4 +/- 1.9 vs. 8.6 +/- 1.2 ng.ml(-1).90 min(-1), P = 0.005). In a second group of subjects (n = 13), acylated ghrelin was similarly suppressed during pregnancy. Circulating octanoate concentrations (3.1 +/- 0.5 vs. 4.5 +/- 0.6 microg/ml, P = 0.029) and cholinesterase activity (705 +/- 33 vs. 1,013 +/- 56 U/ml, P < 0.001) were lower during pregnancy compared with the postpartum period. In conclusion, acylated ghrelin markedly decreases during pregnancy

  17. Veronica: Acylated flavone glycosides as chemosystematic markers

    DEFF Research Database (Denmark)

    Albach, Dirk C.; Grayer, Renée J.; Kite, Geoffrey C.

    2005-01-01

    HPLC/DAD and LCeMS of an extract of Veronica spicata subgenus Pseudolysimachium, Plantaginaceae) revealed the presence of six 6-hydroxyluteolin glycosides acylated with phenolic acids, three of which are new compounds and which we called spicosides. A flavonoid survey of seven more species...

  18. Medium-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Waddell, Leigh; Wiley, Veronica; Carpenter, Kevin

    2006-01-01

    The fatty acid oxidation disorder most commonly identified by tandem mass spectrometry newborn screening is the potentially fatal medium-chain acyl-CoA dehydrogenase deficiency (MCAD). In clinically presenting cases, 80% are homozygous for the common mutation, c.985A > G and 18% heterozygous. We ...

  19. Proghrelin peptides: Desacyl ghrelin is a powerful inhibitor of acylated ghrelin, likely to impair physiological effects of acyl ghrelin but not of obestatin A study of pancreatic polypeptide secretion from mouse islets

    DEFF Research Database (Denmark)

    Kumar, Rajesh; Salehi, Albert; Rehfeld, Jens F

    2010-01-01

    Proghrelin, produced by the ghrelin (A-like) cells of the gastric mucosa, gives rise to cleavage products, including desacyl ghrelin, acyl ghrelin and obestatin. The products are thought to be secreted concomitantly. In an earlier study we found acyl ghrelin and obestatin, but not desacyl ghrelin...

  20. Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

    Science.gov (United States)

    Jiang, Yanfang; Morgan-Kiss, Rachael M; Campbell, John W; Chan, Chi Ho; Cronan, John E

    2010-02-02

    Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

  1. Structural requirements of acylated Gly-l-Ala-d-Glu analogs for activation of the innate immune receptor NOD2.

    Science.gov (United States)

    Gobec, Martina; Mlinarič-Raščan, Irena; Dolenc, Marija Sollner; Jakopin, Žiga

    2016-06-30

    The fragment of bacterial peptidoglycan muramyl dipeptide (MDP) has long been known for its adjuvant activity, however the underlying mechanism of this action has only recently been elucidated. It is ascribed to its agonist action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). In spite of the pressing need for novel adjuvants for human use, this discovery is hampered, by not knowing the structural requirements underlying the immunostimulatory activity. We have investigated how minor modifications of hit compound acyl Gly-L-Ala-D-Glu derivative I modulate the molecular recognition by NOD2. A series of novel desmuramyldipeptides has been designed and synthesized leading to the identification of compound 16, in which the sugar moiety is replaced by a 6-phenylindole moiety, that exhibits the strongest NOD2 activation to date sans the carbohydrate moiety. The results have enabled a deeper understanding of the structural requirements of desmuramylpeptides for NOD2 activation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

    Science.gov (United States)

    Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

    2016-03-14

    Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

  3. Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.

    Science.gov (United States)

    Hara, Yoshinori; Seki, Masahide; Matsuoka, Satoshi; Hara, Hiroshi; Yamashita, Atsushi; Matsumoto, Kouji

    2008-12-01

    The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

  4. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  5. pHP-Tethered N-Acyl Carbamate: A Photocage for Nicotinamide.

    Science.gov (United States)

    Salahi, Farbod; Purohit, Vatsal; Ferraudi, Guillermo; Stauffacher, Cynthia; Wiest, Olaf; Helquist, Paul

    2018-05-04

    The synthesis of a new photocaged nicotinamide having an N-acyl carbamate linker and a p-hydroxyphenacyl (pHP) chromophore is described. The photophysical and photochemical studies showed an absorption maximum at λ = 330 nm and a quantum yield for release of 11% that are dependent upon both pH and solvent. While the acyl carbamate releases nicotinamide efficiently, a simpler amide linker was inert to photocleavage. This photocaged nicotinamide has significant advantages with respect to quantum yield, absorbance wavelength, rate of release, and solubility that make it the first practical example of a photocaged amide.

  6. Ground-State Distortion in N-Acyl-tert-butyl-carbamates (Boc) and N-Acyl-tosylamides (Ts): Twisted Amides of Relevance to Amide N-C Cross-Coupling.

    Science.gov (United States)

    Szostak, Roman; Shi, Shicheng; Meng, Guangrong; Lalancette, Roger; Szostak, Michal

    2016-09-02

    Amide N-C(O) bonds are generally unreactive in cross-coupling reactions employing low-valent transition metals due to nN → π*C═O resonance. Herein we demonstrate that N-acyl-tert-butyl-carbamates (Boc) and N-acyl-tosylamides (Ts), two classes of acyclic amides that have recently enabled the development of elusive amide bond N-C cross-coupling reactions with organometallic reagents, are intrinsically twisted around the N-C(O) axis. The data have important implications for the design of new amide cross-coupling reactions with the N-C(O) amide bond cleavage as a key step.

  7. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  8. The development of a decision analytic model of changes in mean deviation in people with glaucoma: the COA model.

    Science.gov (United States)

    Kymes, Steven M; Lambert, Dennis L; Lee, Paul P; Musch, David C; Siegfried, Carla J; Kotak, Sameer V; Stwalley, Dustin L; Fain, Joel; Johnson, Chris; Gordon, Mae O

    2012-07-01

    To create and validate a statistical model predicting progression of primary open-angle glaucoma (POAG) assessed by loss of visual field as measured in mean deviation (MD) using 3 landmark studies of glaucoma progression and treatment. A Markov decision analytic model using patient level data described longitudinal MD changes over 7 years. Patient-level data from the Collaborative Initial Glaucoma Treatment Study (n = 607), the Ocular Hypertension Treatment Study (OHTS; n = 148; only those who developed POAG in the first 5 years of OHTS) and Advanced Glaucoma Intervention Study (n = 591), the COA model. We developed a Markov model with transition matrices stratified by current MD, age, race, and intraocular pressure categories and used a microsimulation approach to estimate change in MD over 7 years. Internal validation compared model prediction for 7 years to actual MD for COA participants. External validation used a cohort of glaucoma patients drawn from university clinical practices. Change in visual field as measured in MD in decibels (dB). Regressing the actual MD against the predicted produced an R(2) of 0.68 for the right eye and 0.63 for the left. The model predicted ending MD for right eyes of 65% of participants and for 63% of left eyes within 3 dB of actual results at 7 years. In external validation the model had an R(2) of 0.79 in the right eye and 0.77 in the left at 5 years. The COA model is a validated tool for clinicians, patients, and health policy makers seeking to understand longitudinal changes in MD in people with glaucoma. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  9. Mild and Highly Efficient Copper(I Inspired Acylation of Alcohols and Polyols

    Directory of Open Access Journals (Sweden)

    Enoch A. Mensah

    2017-01-01

    Full Text Available A new and highly efficient method mediated by tetrakis(acetonitrilecopper(I triflate for activating both simple and highly hindered anhydrides in the acylation of alcohols and polyols is described. This new acylation method is mild and mostly proceeds at room temperature with low catalyst loading. The method is versatile and has been extended to a wide variety of different alcohol substrates to afford the corresponding ester products in good to excellent yields.

  10. Diversity and dispersal of a ubiquitous protein family: acyl-CoA dehydrogenases.

    Science.gov (United States)

    Shen, Yao-Qing; Lang, B Franz; Burger, Gertraud

    2009-09-01

    Acyl-CoA dehydrogenases (ACADs), which are key enzymes in fatty acid and amino acid catabolism, form a large, pan-taxonomic protein family with at least 13 distinct subfamilies. Yet most reported ACAD members have no subfamily assigned, and little is known about the taxonomic distribution and evolution of the subfamilies. In completely sequenced genomes from approximately 210 species (eukaryotes, bacteria and archaea), we detect ACAD subfamilies by rigorous ortholog identification combining sequence similarity search with phylogeny. We then construct taxonomic subfamily-distribution profiles and build phylogenetic trees with orthologous proteins. Subfamily profiles provide unparalleled insight into the organisms' energy sources based on genome sequence alone and further predict enzyme substrate specificity, thus generating explicit working hypotheses for targeted biochemical experimentation. Eukaryotic ACAD subfamilies are traditionally considered as mitochondrial proteins, but we found evidence that in fungi one subfamily is located in peroxisomes and participates in a distinct beta-oxidation pathway. Finally, we discern horizontal transfer, duplication, loss and secondary acquisition of ACAD genes during evolution of this family. Through these unorthodox expansion strategies, the ACAD family is proficient in utilizing a large range of fatty acids and amino acids-strategies that could have shaped the evolutionary history of many other ancient protein families.

  11. Synthesis of 1-isopropyl-3-acyl-5-methyl-benzimidazolone Derivatives and Their Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Shaopeng Wei

    2013-03-01

    Full Text Available A series of N-acylated analogues of 1-isopropyl-3-acyl-5-methyl-benzimidazolone were synthesized. Bioassay results indicated that analogues 5-07 and 5-19 exhibited the most potency against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Analogues 5-02, 5-07, 5-12, 5-15, 5-19, 5-20 and 5-25 could effectively inhibit the spore germination of Botrytis cinerea. The relationship between structure and their antimicrobial activity (SAR has also been discussed according to aliphatic acids and aromatic acids derivatives, respectively. This implied that the N-acylated derivatives of 5-methyl-benzimidazolone might be potential antimicrobial agents.

  12. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    International Nuclear Information System (INIS)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

    1991-01-01

    Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-07-01

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

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

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Knudsen, J

    1997-01-01

    (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations...

  15. Liberdade e coação no direito de Kant

    Directory of Open Access Journals (Sweden)

    Pinheiro, Celso de Moraes

    2007-01-01

    Full Text Available Kant divide a filosofia moral em duas partes: Ética e Teoria da Justiça. Cada uma é compota de diferentes descrições de deveres e direitos. A ética contém deveres e direitos internos, voluntários e não-coercitivos. A teoria da justiça contém deveres e direitos externos e coercitivos. Os dois tipos de deveres e direitos são definidos em sua relação um com o outro. O que distingue os deveres éticos, ou deveres de virtude, dos deveres jurídicos, é que a compulsão externa para o dever de virtude é baseado na livre coerção própria. Assim, a finalidade deste artigo é pesquisar a noção de dever, e a relação entre dever, liberdade e coação

  16. Density functional theory studies on the nano-scaled composites consisted of graphene and acyl hydrazone molecules

    Science.gov (United States)

    Ren, J. L.; Zhou, L.; Lv, Z. C.; Ding, C. H.; Wu, Y. H.; Bai, H. C.

    2016-07-01

    Graphene, which is the first obtained single atomic layer 2D materials, has drawn a great of concern in nano biotechnology due to the unique property. On one hand, acyl hydrazone compounds belonging to the Schif bases have aroused considerable attention in medicine, pharmacy, and analytical reagent. However, few understanding about the interaction between graphene and acyl hydrazone molecules is now available. And such investigations are much crucial for the applications of these new nano-scaled composites. The current work revealed theoretical investigations on the nano-scaled composites built by acyl hydrazone molecules loaded on the surface of graphene. The relative energy, electronic property and the interaction between the counterparts of graphene/acyl hydrazone composites are investigated based on the density functional theory calculations. According to the obtained adsorption energy, the formation of the nano-scaled composite from the isolated graphene and acyl hydrazone molecule is exothermic, and thus it is energetically favorable to form these nano composites in viewpoint of total energy change. The frontier molecular orbital for the nano composite is mainly distributed at the graphene part, leading to that the energy levels of the frontier molecular orbital of the nano composites are very close to that of isolated graphene. Moreover, the counterpart interaction for the graphene/acyl hydrazone composites is also explored based on the discussions of orbital hybridization, charge redistribution and Van der Waals interaction.

  17. Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Li, Zhijie; Zhai, Yujia; Fang, Junnan; Zhou, Qiangjun; Geng, Yunqi; Sun, Fei

    2010-01-01

    Very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been crystallized in space group C2 and its X-ray diffraction data set has been collected to 1.6 Å resolution. Unlike other VLCADs that were reported to form dimers, the purified cVLCAD was found as a homotetrameric protein according to static light-scattering measurements. Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid β-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12 mg ml −1 ) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100 mM Tris–HCl pH 8.0, 150 mM sodium chloride, 200 mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3 Å, α = γ = 90.0, β = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient V M of 2.76 Å 3 Da −1 and a solvent content of 55%

  18. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  19. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  20. Serum Levels of Acyl-Carnitines along the Continuum from Normal to Alzheimer's Dementia

    Science.gov (United States)

    Sapere, Nadia; La Marca, Giancarlo; Angiolillo, Antonella; Vitale, Michela; Corbi, Graziamaria; Scapagnini, Giovanni; Intrieri, Mariano; Russo, Claudio

    2016-01-01

    This study aimed to determine the serum levels of free L-carnitine, acetyl-L-carnitine and 34 acyl-L-carnitine in healthy subjects and in patients with or at risk of Alzheimer’s disease. Twenty-nine patients with probable Alzheimer’s disease, 18 with mild cognitive impairment of the amnestic type, 24 with subjective memory complaint and 46 healthy subjects were enrolled in the study, and the levels of carnitine and acyl-carnitines were measured by tandem mass spectrometry. The concentrations of acetyl-L-carnitine progressively decreased passing from healthy subjects group (mean±SD, 5.6±1.3 μmol/L) to subjective memory complaint (4.3±0.9 μmol/L), mild cognitive impairment (4.0±0.53 μmol/L), up to Alzheimer’s disease (3.5±0.6 μmol/L) group (p<0.001). The differences were significant for the comparisons: healthy subjects vs. subjective memory complaint, mild cognitive impairment or Alzheimer’s disease group; and subjective memory complaint vs. Alzheimer’s disease group. Other acyl-carnitines, such as malonyl-, 3-hydroxyisovaleryl-, hexenoyl-, decanoyl-, dodecanoyl-, dodecenoyl-, myristoyl-, tetradecenoyl-, hexadecenoyl-, stearoyl-, oleyl- and linoleyl-L-carnitine, showed a similar decreasing trend, passing from healthy subjects to patients at risk of or with Alzheimer’s disease. These results suggest that serum acetyl-L-carnitine and other acyl-L-carnitine levels decrease along the continuum from healthy subjects to subjective memory complaint and mild cognitive impairment subjects, up to patients with Alzheimer’s disease, and that the metabolism of some acyl-carnitines is finely connected among them. These findings also suggest that the serum levels of acetyl-L-carnitine and other acyl-L-carnitines could help to identify the patients before the phenotype conversion to Alzheimer’s disease and the patients who would benefit from the treatment with acetyl-L-carnitine. However, further validation on a larger number of samples in a longitudinal

  1. Serum Levels of Acyl-Carnitines along the Continuum from Normal to Alzheimer's Dementia.

    Directory of Open Access Journals (Sweden)

    Adriana Cristofano

    Full Text Available This study aimed to determine the serum levels of free L-carnitine, acetyl-L-carnitine and 34 acyl-L-carnitine in healthy subjects and in patients with or at risk of Alzheimer's disease. Twenty-nine patients with probable Alzheimer's disease, 18 with mild cognitive impairment of the amnestic type, 24 with subjective memory complaint and 46 healthy subjects were enrolled in the study, and the levels of carnitine and acyl-carnitines were measured by tandem mass spectrometry. The concentrations of acetyl-L-carnitine progressively decreased passing from healthy subjects group (mean±SD, 5.6±1.3 μmol/L to subjective memory complaint (4.3±0.9 μmol/L, mild cognitive impairment (4.0±0.53 μmol/L, up to Alzheimer's disease (3.5±0.6 μmol/L group (p<0.001. The differences were significant for the comparisons: healthy subjects vs. subjective memory complaint, mild cognitive impairment or Alzheimer's disease group; and subjective memory complaint vs. Alzheimer's disease group. Other acyl-carnitines, such as malonyl-, 3-hydroxyisovaleryl-, hexenoyl-, decanoyl-, dodecanoyl-, dodecenoyl-, myristoyl-, tetradecenoyl-, hexadecenoyl-, stearoyl-, oleyl- and linoleyl-L-carnitine, showed a similar decreasing trend, passing from healthy subjects to patients at risk of or with Alzheimer's disease. These results suggest that serum acetyl-L-carnitine and other acyl-L-carnitine levels decrease along the continuum from healthy subjects to subjective memory complaint and mild cognitive impairment subjects, up to patients with Alzheimer's disease, and that the metabolism of some acyl-carnitines is finely connected among them. These findings also suggest that the serum levels of acetyl-L-carnitine and other acyl-L-carnitines could help to identify the patients before the phenotype conversion to Alzheimer's disease and the patients who would benefit from the treatment with acetyl-L-carnitine. However, further validation on a larger number of samples in a longitudinal

  2. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  3. The occurrence of gibberellin-binding protein(s) in pea

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Z.H.

    1988-01-01

    In vitro gibberellin (GA) binding properties of a cytosol fraction from epicotyls of dwarf pea (Pisum sativum L. cv. Progress No. 9) and tall pea (Pisum sativum L. cv. Alaska) were investigated using ({sup 3}H)GA{sub 4} in a DEAE filter paper assay at 0-3 C. The binding obtained is saturable, reversible, and temperature labile in dwarf pea, and has a half-life of dissociation of 5-6 min. By varying the concentration of ({sup 3}H)GA{sub 4} in the incubation medium the Kd was estimated to be 120-140 nM in dwarf pea and 70 nM in tall pea. The number of binding sites (n) was estimated to be 0.66 and 0.43 pmole mg{sup {minus}1} soluble protein in dwarf pea and in tall pea, respectively. In competition binding assays, biologically active GAs, such as GA{sub 3} and GA{sub 4} could reduce the level of ({sup 3}H)GA{sub 4} binding much more than the biologically inactive GA{sub 4} methyl ester and epi-GA{sub 4}. Changes in gibberellin-binding protein(s) were studied during seed germination. While the Kd of the binding protein(s) for ({sup 3}H)GA{sub 4} remained the same, there was a marked increase in the number of binding sites from 24 h soaked seed to 8-day old seedlings. Also, the Kd and the number of binding sites in the GA-responsive apical part and in the nonresponsive basal part in the epicotyl were similar. The effect of light on gibberellin-binding protein in dwarf pea was also studied. The GA-binding protein in dwarf pea was partially purified by gel filtration and ion exchange chromatography.

  4. Lipase-catalyzed biodiesel synthesis with different acyl acceptors

    Directory of Open Access Journals (Sweden)

    Ognjanović Nevena D.

    2008-01-01

    Full Text Available Biodiesel is an alternative fuel for diesel engine that is environmentally acceptable. Conventionally, biodiesel is produced by transesterification of triglycerides and short alcohols in the presence of an acid or an alkaline catalyst. There are several problems associated with this kind of production that can be resolved by using lipase as the biocatalyst. The aim of the present work was to investigate novel acyl acceptors for biodiesel production. 2-Propanol and n-butanol have a less negative effect on lipase stability, and they also improve low temperature properties of the fuel. However, excess alcohol leads to inactivation of the enzyme, and glycerol, a major byproduct, can block the immobilized enzyme, resulting in low enzymatic activity. This problem was solved by using methyl acetate as acyl acceptor. Triacetylglycerol is produced instead of glycerol, and it has no negative effect on the activity of the lipase.

  5. Proghrelin peptides: Desacyl ghrelin is a powerful inhibitor of acylated ghrelin, likely to impair physiological effects of acyl ghrelin but not of obestatin A study of pancreatic polypeptide secretion from mouse islets

    DEFF Research Database (Denmark)

    Kumar, Rajesh; Salehi, Albert; Rehfeld, Jens F

    2010-01-01

    Proghrelin, produced by the ghrelin (A-like) cells of the gastric mucosa, gives rise to cleavage products, including desacyl ghrelin, acyl ghrelin and obestatin. The products are thought to be secreted concomitantly. In an earlier study we found acyl ghrelin and obestatin, but not desacyl ghrelin......, to suppress the release of hormones from isolated islets of mouse and rat pancreas....

  6. The effect of BAY o 2752 on bile acid absorption and cholesterol esterification

    International Nuclear Information System (INIS)

    Harnett, K.M.

    1988-01-01

    BAY o 2752 [N,N-(1,11-undecandiyl)bis(2,3-dihydro-2-methyl-1H-indole-1-carboxamide)] has been demonstrated to inhibit intestinal cholesterol absorption in rats. Studies were carried out on male Wistar rats to determine if this drug alters intestinal bile acid absorption or cholesterol esterification by acyl CoA: cholesterol acyltransferase (ACAT) or cholesterol ester hydrolase (CEH). BAY o 2752 did not affect intestinal absorption of taurocholic acid (TC) from ileal segments perfused in vivo with a tragacanth suspension in phosphate buffer containing NaCl, TC, and 24- 14 C-TC as determined by the excretory rate of radioactivity in bile. BAY o 2752 also did not affect the uptake of TC into ileal everted sacs incubated in stirred, gassed Krebs-Ringer bicarbonate buffer with 1 mM TC, 24- 14 C-TC and 3 H-inulin. BAY o 2752 also did not bind TC; TG, in a filtrate of the above solutions remained at 92-98% of control

  7. The effect of BAY o 2752 on bile acid absorption and cholesterol esterification

    Energy Technology Data Exchange (ETDEWEB)

    Harnett, K.M.

    1988-01-01

    BAY o 2752 (N,N-(1,11-undecandiyl)bis(2,3-dihydro-2-methyl-1H-indole-1-carboxamide)) has been demonstrated to inhibit intestinal cholesterol absorption in rats. Studies were carried out on male Wistar rats to determine if this drug alters intestinal bile acid absorption or cholesterol esterification by acyl CoA: cholesterol acyltransferase (ACAT) or cholesterol ester hydrolase (CEH). BAY o 2752 did not affect intestinal absorption of taurocholic acid (TC) from ileal segments perfused in vivo with a tragacanth suspension in phosphate buffer containing NaCl, TC, and 24-{sup 14}C-TC as determined by the excretory rate of radioactivity in bile. BAY o 2752 also did not affect the uptake of TC into ileal everted sacs incubated in stirred, gassed Krebs-Ringer bicarbonate buffer with 1 mM TC, 24-{sup 14}C-TC and {sup 3}H-inulin. BAY o 2752 also did not bind TC; TG, in a filtrate of the above solutions remained at 92-98% of control.

  8. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  9. Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala.

    Science.gov (United States)

    Srivastava, Sameer; Vishwakarma, Rishi K; Arafat, Yasir Ali; Gupta, Sushim K; Khan, Bashir M

    2015-04-01

    Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

  10. Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

    Science.gov (United States)

    Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

    1984-03-01

    N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

  11. Molecular Mechanisms of Insulin Resistance in Humans and Their Potential Links With Mitochondrial Dysfunction

    OpenAIRE

    Morino, Katsutaro; Petersen, Kitt Falk; Shulman, Gerald I.

    2006-01-01

    Recent studies using magnetic resonance spectroscopy have shown that decreased insulin-stimulated muscle glycogen synthesis due to a defect in insulin-stimulated glucose transport activity is a major factor in the pathogenesis of type 2 diabetes. The molecular mechanism underlying defective insulin-stimulated glucose transport activity can be attributed to increases in intramyocellular lipid metabolites such as fatty acyl CoAs and diacylglycerol, which in turn activate a serine/threonine kina...

  12. Fatty acid oxidation disorders as primary cause of sudden and unexpected death in infants and young children

    DEFF Research Database (Denmark)

    Banner, Jytte; Kølvraa, S; Gregersen, N

    1997-01-01

    Disorders of fatty acid metabolism are known to be responsible for cases of sudden and unexpected death in infancy. At least 14 disorders are known at present. 120 cases of sudden infant death syndrome (SIDS) had been examined for a prevalent mutation (G985) causing medium chain acyl Co......A dehydrogenase deficiency, which is inherited in an autosomal recessive mode. No over-representation of either homozygous or heterozygous cases was found....

  13. 40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

    Science.gov (United States)

    2010-07-01

    ...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

  14. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  15. Biochemical, structural and molecular dynamics analyses of the potential virulence factor RipA from Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rodrigo Torres

    Full Text Available Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation operon (ripA, ripB and ripC is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the

  16. Des-Acyl Ghrelin and Ghrelin O-Acyltransferase Regulate Hypothalamic-Pituitary-Adrenal Axis Activation and Anxiety in Response to Acute Stress

    NARCIS (Netherlands)

    Stark, R.; Santos, V.V.; Geenen, B.; Cabral, A.; Dinan, T.; Bayliss, J.A.; Lockie, S.H.; Reichenbach, A.; Lemus, M.B.; Perello, M.; Spencer, S.J.; Kozicz, L.T.; Andrews, Z.B.

    2016-01-01

    Ghrelin exists in two forms in circulation, acyl ghrelin and des-acyl ghrelin, both of which have distinct and fundamental roles in a variety of physiological functions. Despite this fact, a large proportion of papers simply measure and refer to plasma ghrelin without specifying the acylation

  17. ORF Alignment: NT_037436 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available hila melanogaster] sp|P42281|ACBP_DROME ... Acyl-CoA-binding protein homolog (ACBP) (Diazepam ... ... ... binding inhibitor homolog) (DBI) gb|AAA21650.1| ... diazepam binding inhibitor gb|AAA21649.1| diazep

  18. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  19. Synthesis and Bioactivity of Pyrazole Acyl Thiourea Derivatives

    Directory of Open Access Journals (Sweden)

    Jian Wu

    2012-05-01

    Full Text Available Sixteen novel pyrazole acyl thiourea derivatives 6 were synthesized from monomethylhydrazine (phenylhydrazine and ethyl acetoacetate. The key 5-chloro-3-methyl-1-substituted-1H-pyrazole-4-carbonyl chloride intermediates 4 were first generated in four steps through cyclization, formylation, oxidation and acylation. Thess were then reacted with ammonium thiocyanate in the presence of PEG-400 to afford 5-chloro-3-methyl-1-substituted-1H-pyrazole-4-carbonyl isothiocyanates 5. Subsequent reaction with fluorinated aromatic amines resulted in the formation of the title compounds. The synthesized compound were unequivocally characterized by IR, 1H-NMR, 13C-NMR and elemental analysis and some of the synthesized compounds displayed good antifungal activities against Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica and anti-TMV activity in preliminary antifungal activity tests.

  20. Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

    Science.gov (United States)

    Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

  1. THE EFFECTS OF EXERCISE ON FOOD INTAKE AND HUNGER: RELATIONSHIP WITH ACYLATED GHRELIN AND LEPTIN

    Directory of Open Access Journals (Sweden)

    Serife Vatansever-Ozen

    2011-06-01

    Full Text Available This study investigated the effects of a long bout of aerobic exercise on hunger and energy intake and circulating levels of leptin and acylated ghrelin. Ten healthy male subjects undertook two, 4 h trials in a randomized crossover design. In the exercise trial subjects ran for 105 min at 50% of maximal oxygen uptake and the last 15 min at 70% of maximal oxygen uptake followed by a 120 min rest period. In the control trial, subjects rested for 4 h. Subjects consumed a buffet test meal at 180 min during each trial. Hunger ratings, acylated ghrelin, leptin, glucose and insulin concentrations were measured at 0, 1, 2, 3 and 4 h. No differences were found at baseline values for hunger, acylated ghrelin, leptin, insulin and glucose for both trials (p > 0.05. The estimated energy expenditure of the exercise trial was 1550 ± 136 kcal. Exercise did not change subsequent absolute energy intake, but produced a significant decrease (p < 0.05 in relative energy intake. A two-way ANOVA revealed a significant (p < 0. 05 interaction effect for hunger and acylated ghrelin. In conclusion, this exercise regimen had a positive effect on reducing appetite which is related to reduced acylated ghrelin responses over time. This finding lends support for a role of exercise in weight management

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

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  3. Production of specific-structured lipids by enzymatic interesterification: elucidation of acyl migration by response surface design

    DEFF Research Database (Denmark)

    Xu, Xuebing; Skands, Anja; Høy, Carl-Erik

    1998-01-01

    Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of by-products. In this paper, the elucidation of acyl...

  4. Extracellular and intracellular steroid binding proteins

    International Nuclear Information System (INIS)

    Wagner, R.K.

    1978-01-01

    Steroid hormone binding proteins can be measured, after the removal of endogenous steroids, as specific complexes with radio-labelled hormones. In this study all the requirements for a quantitative determination of steroid hormone binding proteins are defined. For different methods, agargel electrophoresis, density gradient centrifugation, equilibrium dialysis and polyacrylamide electrophoresis have been evaluated. Agar electrophoresis at low temperature was found to be the simplest and most useful procedure. With this method the dissociation rates of high affinity complexes can be assessed and absolute binding protein concentrations can be determined. The dissociation rates of the oestradiol-oestrogen receptor complex and the R-5020-progestin receptor complex are low (1-2% per h run time.) In contrast, that of complexes between androgen receptor and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one (DHT), progestin receptor and progesterone, corticosteroid binding globulin (CBG) and cortisol or progesterone and sex hormone binding globulin (SHBG) and DHT were hign (16-27% per h run time). Target tissue extracts (cytosols) contain, besides soluble tissue proteins, large amounts of plasma proteins. The extent of this plasma contamination can be determined by measuring the albumin concentration in cytosols by immunodiffusion. In cytosols of 4 different human target tissues the albumin content varied from 20-30% corresponding to an even higher whole plasma concentration. Steroid binding plasma proteins, such as CBG and SHBG are constituents of this containment. (author)

  5. Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).

    Science.gov (United States)

    Gottfried, D S; Manjula, B N; Malavalli, A; Acharya, A S; Friedman, J M

    1999-08-31

    HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

  6. Development of RI protein chip system for measurement of ADMA as risk factor of liver disease, cardiovascular disease and diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Park, Gil Hong; An, Sin Ae; Choi, Hyun Mi; Cheong, Kyung Ah; Chang, Yeon Soo [Korea University, Seoul (Korea, Republic of)

    2010-05-15

    ADMA (asymmetric dimethylarginine) is an endogenous competitive NOS (nitric oxide synthase) inhibitor. Elevation of ADMA level in body fluid is related to various diseases including diabetes mellitus, atherosclerosis, hypercholesterolemia, chronic heart failure, and hypertension. Our goal was to elucidate the role for ADMA and protein arginine methylation in the pathogenesis of diabetes and develop RI protein chip system for easy determination of ADMA levels in blood or tissues. As a result, ADMA regulated by protein arginine methylation, DDAH1, and NOS was demonstrated to play some role in the pathogenesis of diabetes mellitus, and arginine methylation of some proteins indicated their possible involvement in the change in ADMA levels. The concept of the RI protein chip system was devised such that firstly, ADMA was acylated by radio-labelled succinic acid, and followed by binding of the complex to anti-acyl ADMA-specific antibody coated on chip. Acylation condition of ADMA with radio-labelled succinic acid was established

  7. Development of RI protein chip system for measurement of ADMA as risk factor of liver disease, cardiovascular disease and diabetes

    International Nuclear Information System (INIS)

    Park, Gil Hong; An, Sin Ae; Choi, Hyun Mi; Cheong, Kyung Ah; Chang, Yeon Soo

    2010-05-01

    ADMA (asymmetric dimethylarginine) is an endogenous competitive NOS (nitric oxide synthase) inhibitor. Elevation of ADMA level in body fluid is related to various diseases including diabetes mellitus, atherosclerosis, hypercholesterolemia, chronic heart failure, and hypertension. Our goal was to elucidate the role for ADMA and protein arginine methylation in the pathogenesis of diabetes and develop RI protein chip system for easy determination of ADMA levels in blood or tissues. As a result, ADMA regulated by protein arginine methylation, DDAH1, and NOS was demonstrated to play some role in the pathogenesis of diabetes mellitus, and arginine methylation of some proteins indicated their possible involvement in the change in ADMA levels. The concept of the RI protein chip system was devised such that firstly, ADMA was acylated by radio-labelled succinic acid, and followed by binding of the complex to anti-acyl ADMA-specific antibody coated on chip. Acylation condition of ADMA with radio-labelled succinic acid was established

  8. Asymmetric Chemoenzymatic Reductive Acylation of Ketones by a Combined Iron-Catalyzed Hydrogenation-Racemization and Enzymatic Resolution Cascade

    KAUST Repository

    El-Sepelgy, Osama; Brzozowska, Aleksandra; Rueping, Magnus

    2017-01-01

    . By merging the iron-catalyzed redox reactions with enantioselective enzymatic acylations a wide range of benzylic, aliphatic and (hetero)aromatic ketones, as well as diketones, were reductively acylated. The corresponding products were isolated with high

  9. Binding events of (S )-N -(3-oxo-octanoyl)-homoserine lactone with agrobacterium tumefaciens mutant cells studied by saturation transfer difference NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cabeca, Luis Fernando; Pomini, Armando Mateus; Cruz, Pedro Luiz R.; Marsaioli, Anita J. [University of Campinas (UNICAMP), SP (Brazil). Chemistry Inst.

    2011-07-01

    Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. (author)

  10. Fragment-based quantum mechanical calculation of protein-protein binding affinities.

    Science.gov (United States)

    Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao

    2018-04-29

    The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  11. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  12. Selective Acylation Enhances Membrane Charge Sensitivity of the Antimicrobial Peptide Mastoparan-X

    DEFF Research Database (Denmark)

    Etzerodt, Thomas Povl; Henriksen, Jonas Rosager; Rasmussen, Palle

    2011-01-01

    and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared...... to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other...

  13. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-01-01

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

  14. Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation.

    Science.gov (United States)

    Sánchez-García, Alicia; Moreno-Pérez, Antonio J; Muro-Pastor, Alicia M; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2010-06-01

    Acyl-acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. A Turkish Patient With Succinyl-CoA:3-Oxoacid CoA Transferase Deficiency Mimicking Diabetic Ketoacidosis

    Directory of Open Access Journals (Sweden)

    Sahin Erdol MD

    2016-05-01

    Full Text Available Succinyl-CoA:3-oxoacid CoA transferase (SCOT deficiency is an autosomal recessive disorder of ketone body utilization that is clinically characterized with intermittent ketoacidosis crises. We report here the second Turkish case with SCOT deficiency. She experienced 3 ketoacidotic episodes: The first ketoacidotic crisis mimicked diabetic ketoacidosis because of the associated hyperglycemia. Among patients with SCOT deficiency, the blood glucose levels at the first crises were variable, and this case had the highest ever reported blood glucose level. She is a compound heterozygote with 2 novel mutations, c.517A>G (K173E and c.1543A>G (M515V, in exons 5 and 17 of the OXCT1 gene, respectively. In patient’s fibroblasts, SCOT activity was deficient and, by immunoblot analysis, SCOT protein was much reduced. The patient attained normal development and had no permanent ketosis. The accurate diagnosis of SCOT deficiency in this case had a vital impact on the management strategy and outcome.

  16. The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

    Science.gov (United States)

    Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

    2017-12-05

    Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C-14-peptides

    DEFF Research Database (Denmark)

    Pedersen, T.B.; Kaasgaard, Thomas; Jensen, M.O.

    2005-01-01

    The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated...... peptide, which is a synthetic decapeptide N-terminally linked to a C-14 acyl chain (C-14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C-14-peptide on the lipid bilayer...... gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C-14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10...

  18. Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation

    Science.gov (United States)

    Raboune, Siham; Stuart, Jordyn M.; Leishman, Emma; Takacs, Sara M.; Rhodes, Brandon; Basnet, Arjun; Jameyfield, Evan; McHugh, Douglas; Widlanski, Theodore; Bradshaw, Heather B.

    2014-01-01

    A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation. PMID:25136293

  19. Sequence Classification: 891238 [

    Lifescience Database Archive (English)

    Full Text Available oA-binding protein, transports newly synthesized acyl-CoA esters from fatty acid synthetase (Fas1p-Fas2p) to...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|6321474|ref|NP_011551.1| Acyl-C

  20. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  1. Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export.

    Science.gov (United States)

    White, Phillip J; Lapworth, Amanda L; An, Jie; Wang, Liping; McGarrah, Robert W; Stevens, Robert D; Ilkayeva, Olga; George, Tabitha; Muehlbauer, Michael J; Bain, James R; Trimmer, Jeff K; Brosnan, M Julia; Rolph, Timothy P; Newgard, Christopher B

    2016-07-01

    A branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Zucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Our data are consistent with a model wherein elevated circulating BCAA contribute to development of

  2. Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export

    Directory of Open Access Journals (Sweden)

    Phillip J. White

    2016-07-01

    Full Text Available Objective: A branched-chain amino acid (BCAA-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Methods: Zucker-lean rats (ZLR and Zucker-fatty rats (ZFR were fed either a custom control, low fat (LF diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val were reduced by 45% (LF-RES. We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. Results: LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH, was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Conclusions: Our data are consistent with a model wherein

  3. Acylation Reactions over Zeolites and Mesoporous Catalysts

    Czech Academy of Sciences Publication Activity Database

    Voláková, Martina; Vitvarová, Dana; Čejka, Jiří

    2009-01-01

    Roč. 2, č. 6 (2009), s. 486-499 ISSN 1864-5631 R&D Projects: GA ČR GA104/07/0383; GA ČR GD203/08/H032; GA MPO FT-TA5/005 Institutional research plan: CEZ:AV0Z40400503 Keywords : acylation * ketones * mesoporous materials * shape-selectivity * zeolites Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.767, year: 2009

  4. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

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

    Energy Technology Data Exchange (ETDEWEB)

    Manna, Suvendu; Saha, Prosenjit [Materials Science Centre, IIT Kharagpur, WB 721302 (India); Roy, Debasis, E-mail: debasis@civil.iitkgp.ernet.in [Department of Civil Engineering, IIT Kharagpur, WB 721302 (India); Sen, Ramkrishna [Department of Biotechnology, IIT Kharagpur, WB 721302 (India); Adhikari, Basudam [Materials Science Centre, IIT Kharagpur, WB 721302 (India)

    2015-11-30

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

  6. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

    Science.gov (United States)

    Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

    2008-04-01

    Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

  7. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  8. Acyl coenzyme A thioesterase 7 regulates neuronal fatty acid metabolism to prevent neurotoxicity.

    Science.gov (United States)

    Ellis, Jessica M; Wong, G William; Wolfgang, Michael J

    2013-05-01

    Numerous neurological diseases are associated with dysregulated lipid metabolism; however, the basic metabolic control of fatty acid metabolism in neurons remains enigmatic. Here we have shown that neurons have abundant expression and activity of the long-chain cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase 7 (ACOT7) to regulate lipid retention and metabolism. Unbiased and targeted metabolomic analysis of fasted mice with a conditional knockout of ACOT7 in the nervous system, Acot7(N-/-), revealed increased fatty acid flux into multiple long-chain acyl-CoA-dependent pathways. The alterations in brain fatty acid metabolism were concomitant with a loss of lean mass, hypermetabolism, hepatic steatosis, dyslipidemia, and behavioral hyperexcitability in Acot7(N-/-) mice. These failures in adaptive energy metabolism are common in neurodegenerative diseases. In agreement, Acot7(N-/-) mice exhibit neurological dysfunction and neurodegeneration. These data show that ACOT7 counterregulates fatty acid metabolism in neurons and protects against neurotoxicity.

  9. Endotoxin Structures in the Psychrophiles Psychromonas marina and Psychrobacter cryohalolentis Contain Distinctive Acyl Features

    Directory of Open Access Journals (Sweden)

    Charles R. Sweet

    2014-07-01

    Full Text Available Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide, a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2- units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

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

    Science.gov (United States)

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

    2012-01-01

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

  11. Relationships between acylated ghrelin with growth hormone, insulin resistance, lipid profile, and cardio respiratory function in lean and obese men

    Directory of Open Access Journals (Sweden)

    Hasan Matin Homaee

    2011-01-01

    Conclusions: Obese and lean inactive young men had different levels of acylated ghrelin, GH, insulin, insulin resistance index, cardiorespiratory function and body fat percent. Body fat percent, insulin, and GH levels appear to be best determinant factors of acylated ghrelin levels. Also, in both obese and lean young men, higher levels of cardiovascular function were associated with higher levels of acylated ghrelin.

  12. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...

  13. Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice

    DEFF Research Database (Denmark)

    Kragh, Peter M; Pedersen, Christina B; Schmidt, Stine P

    2007-01-01

    Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A....... The transgenic mice were mated with an SCAD-deficient mouse strain (BALB/cByJ) and, in the second generation, three mouse lines were obtained without endogenous SCAD expression but harboring hSCAD-wt, hSCAD-319C > T, and hSCAD-625G > A transgenes, respectively. All three lines had expression of the transgene...... developed for any of the lines transgenic for the hSCAD folding variants. The indicated remarkable efficiency of the mouse protein quality control system in the degradation of SCAD folding variants should be further substantiated and investigated, since it might indicate ways to prevent disease...

  14. Effect of room temperature ionic liquid structure on the enzymatic acylation of flavonoids

    DEFF Research Database (Denmark)

    Lue, Bena-Marie; Guo, Zheng; Xu, Xuebing

    2010-01-01

    Enzymatic acylation reactions of flavonoids (rutin, esculin) with long chain fatty acids (palmitic, oleic acids) were carried out in 14 different ionic liquid media containing a range of cation and anion structures. Classification of RTILs according to flavonoid solubility (using COSMO...... must be struck that maximized flavonoid solubility with minimum negative impact on lipase activity. The process also benefitted from an increased reaction temperature which may have helped to reduced mass transfer limitations. Keywords: Room temperature ionic liquids (RTILs); Biosynthesis; Acylation......; Flavonoids; Lipase; Long chain fatty acids...

  15. An insight on acyl migration in solvent-free ethanolysis of model triglycerides using Novozym 435.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. GTP-binding proteins in rat liver nuclear envelopes

    International Nuclear Information System (INIS)

    Rubins, J.B.; Benditt, J.O.; Dickey, B.F.; Riedel, N.

    1990-01-01

    Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membranes

  17. Expression of Lipid Metabolism-Related Proteins Differs between Invasive Lobular Carcinoma and Invasive Ductal Carcinoma.

    Science.gov (United States)

    Cha, Yoon Jin; Kim, Hye Min; Koo, Ja Seung

    2017-01-23

    We comparatively investigated the expression and clinical implications of lipid metabolism-related proteins in invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) of the breast. A total of 584 breast cancers (108 ILC and 476 IDC) were subjected to tissue microarray and immunohistochemical analysis for lipid metabolism-related proteins including hormone-sensitive lipase (HSL), perilipin A, fatty acid binding protein (FABP)4, carnitine palmitoyltransferase (CPT)-1, acyl-CoA oxidase 1, and fatty acid synthetase (FASN). HSL, perilipin A, and FABP4 expression (all p invasive cancers, HSL and FABP4 were highly expressed in luminal A-type ILC ( p cancers, HSL and FABP4 were more highly expressed in ILC ( p < 0.001). Univariate analysis found associations of shorter disease-free survival with CPT-1 positivity ( p = 0.004) and acyl-CoA oxidase 1 positivity ( p = 0.032) and of shorter overall survival with acyl-CoA oxidase 1 positivity ( p = 0.027). In conclusion, ILC and IDC exhibited different immunohistochemical lipid metabolism-related protein expression profiles. Notably, ILC exhibited high HSL and FABP4 and low perilipin A expression.

  18. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  19. RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

    Science.gov (United States)

    Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

    2017-05-01

    Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

  20. Gas-phase acylation of aminopropyl-silica gel in the synthesis of some chemically bonded silica materials for analytical applications

    International Nuclear Information System (INIS)

    Basiuk, Vladimir; Khil'chevskaya, E.G.

    1991-01-01

    Gas-phase acylation of aminopropyl-silica gel with aliphatic dicarboxylic (succinic, adipic and sebacic) and 4-aminobenzoic acids is proposed as a rapid and efficient one-step method for the synthesis of carboxyalkyl- and 4-aminophenylamidopropyl-silica gels, usually used as zwitterion exchangers for liquid chromatography and matrices for multi-step syntheses of silica-bound aromatic azo reagents for the sorption and chromatographic separation of metal ions. Acylation degrees of 59-90% are achieved after 0.5 h. IR spectra of the acylation products and near-UV-visible spectra for bonded aromatic azo compounds, based on 4-aminobenzamidopropyl-silica gel, are presented. Some positive and negative aspects of the gas-phase acylation are discussed. (author). 34 refs.; 2 figs.; 2 tabs

  1. Acylated 2-(N-arylaminomethylene)benzo[b]thiophene-3(2H)-Ones: Molecular Switches with Varying Migrants and Substituents

    International Nuclear Information System (INIS)

    Dubonosov, A.D.; Rybalkin, V.P.; Tsukanov, A.V.; Minkin, V.I.; Popova, L.L.; Revinsky, Y.V.; Bren, V.A.; Minkin, V.I.

    2009-01-01

    Synthesis and properties of photo chromic acylated 2-(N-arylaminomethylene)benzo[b]thiophene-3(2H)-ones are described. Their structure largely depends on the nature of acyl migrant and in a less degree on N-aryl substituent.

  2. N-Cinnamoylation of Antimalarial Classics: Effects of Using Acyl Groups Other than Cinnamoyl toward Dual-Stage Antimalarials.

    Science.gov (United States)

    Gomes, Ana; Machado, Marta; Lobo, Lis; Nogueira, Fátima; Prudêncio, Miguel; Teixeira, Cátia; Gomes, Paula

    2015-08-01

    In a follow-up study to our reports of N-cinnamoylated chloroquine and quinacrine analogues as promising dual-stage antimalarial leads with high in vitro potency against both blood-stage Plasmodium falciparum and liver-stage Plasmodium berghei, we decided to investigate the effect of replacing the cinnamoyl moiety with other acyl groups. Thus, a series of N-acylated analogues were synthesized, and their activities against blood- and liver-stage Plasmodium spp. were assessed along with their in vitro cytotoxicities. Although the new N-acylated analogues were found to be somewhat less active and more cytotoxic than their N-cinnamoylated counterparts, they equally displayed nanomolar activities in vitro against blood-stage drug-sensitive and drug-resistant P. falciparum, and significant in vitro liver-stage activity against P. berghei. Therefore, it is demonstrated that simple N-acylated surrogates of classical antimalarial drugs are promising dual-stage antimalarial leads. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

    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

  4. ETFDH mutations as a major cause of riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency

    DEFF Research Database (Denmark)

    Olsen, Rikke K J; Olpin, Simon E; Andresen, Brage S

    2007-01-01

    Multiple acyl-CoA dehydrogenation deficiency (MADD) is a disorder of fatty acid, amino acid and choline metabolism that can result from defects in two flavoproteins, electron transfer flavoprotein (ETF) or ETF: ubiquinone oxidoreductase (ETF:QO). Some patients respond to pharmacological doses......; several had previously suffered cyclical vomiting. Urine organic acid and plasma acyl-carnitine profiles indicated MADD. Clinical and biochemical parameters were either totally or partly corrected after riboflavin treatment. All patients had mutations in the gene for ETF:QO. In one patient, we show...... that the ETF:QO mutations are associated with a riboflavin-sensitive impairment of ETF:QO activity. This patient also had partial deficiencies of flavin-dependent acyl-CoA dehydrogenases and respiratory chain complexes, most of which were restored to control levels after riboflavin treatment. Low activities...

  5. The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular Utilization

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Black, P N; Zhao, X D

    2001-01-01

    CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization...

  6. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein

    NARCIS (Netherlands)

    Hateboer, G.; Timmers, H.T.M.; Rustgi, A.K.; Billaud, Marc; Veer, L.J. Van 't; Bernards, R.A.

    1993-01-01

    Using a protein binding assay, we show that the amino-teminal 204 amino acids of the c-Myc protein interact di y with a key component of the basal p tdon factor TFID, the TATA box-binding protein (TBP). Essentialy the same region of the c-Myc protein alo binds the product of the retinoblatoma

  7. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

  8. Inhibitory effect on in vitro LDL oxidation and HMG Co-A reductase activity of the liquid-liquid partitioned fractions of Hericium erinaceus (Bull.) Persoon (lion's mane mushroom).

    Science.gov (United States)

    Rahman, Mohammad Azizur; Abdullah, Noorlidah; Aminudin, Norhaniza

    2014-01-01

    Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  9. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  10. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  11. Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Nielsen, Kristian Fog; Dalsgaard, Inger

    2007-01-01

    produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-l-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-l-homoserine lactone (3-oxo-C9-HSL), were...

  12. Multiple protonation equilibria in electrostatics of protein-protein binding.

    Science.gov (United States)

    Piłat, Zofia; Antosiewicz, Jan M

    2008-11-27

    All proteins contain groups capable of exchanging protons with their environment. We present here an approach, based on a rigorous thermodynamic cycle and the partition functions for energy levels characterizing protonation states of the associating proteins and their complex, to compute the electrostatic pH-dependent contribution to the free energy of protein-protein binding. The computed electrostatic binding free energies include the pH of the solution as the variable of state, mutual "polarization" of associating proteins reflected as changes in the distribution of their protonation states upon binding and fluctuations between available protonation states. The only fixed property of both proteins is the conformation; the structure of the monomers is kept in the same conformation as they have in the complex structure. As a reference, we use the electrostatic binding free energies obtained from the traditional Poisson-Boltzmann model, computed for a single macromolecular conformation fixed in a given protonation state, appropriate for given solution conditions. The new approach was tested for 12 protein-protein complexes. It is shown that explicit inclusion of protonation degrees of freedom might lead to a substantially different estimation of the electrostatic contribution to the binding free energy than that based on the traditional Poisson-Boltzmann model. This has important implications for the balancing of different contributions to the energetics of protein-protein binding and other related problems, for example, the choice of protein models for Brownian dynamics simulations of their association. Our procedure can be generalized to include conformational degrees of freedom by combining it with molecular dynamics simulations at constant pH. Unfortunately, in practice, a prohibitive factor is an enormous requirement for computer time and power. However, there may be some hope for solving this problem by combining existing constant pH molecular dynamics

  13. Asymmetric Chemoenzymatic Reductive Acylation of Ketones by a Combined Iron-Catalyzed Hydrogenation-Racemization and Enzymatic Resolution Cascade

    KAUST Repository

    El-Sepelgy, Osama

    2017-02-28

    A general and practical process for the conversion of prochiral ketones into the corresponding chiral acetates has been realized. An iron carbonyl complex is reported to catalyze the hydrogenation-dehydrogenation-hydrogenation of prochiral ketones. By merging the iron-catalyzed redox reactions with enantioselective enzymatic acylations a wide range of benzylic, aliphatic and (hetero)aromatic ketones, as well as diketones, were reductively acylated. The corresponding products were isolated with high yields and enantioselectivities. The use of an iron catalyst together with molecular hydrogen as the hydrogen donor and readily available ethyl acetate as acyl donor make this cascade process highly interesting in terms of both economic value and environmental credentials.

  14. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Science.gov (United States)

    2010-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1...., chloride (PMN P-01-646; CAS No. 391232-99-8) is subject to reporting under this section for the significant...

  15. Transitional change in rat fetal cell proliferation in response to ghrelin and des-acyl ghrelin during the last stage of pregnancy

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Yoshiyuki; Nakahara, Keiko [Department of Veterinary Physiology, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192 (Japan); Kangawa, Kenji [Department of Biochemistry, National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565-8565 (Japan); Murakami, Noboru, E-mail: a0d201u@cc.miyazaki-u.ac.jp [Department of Veterinary Physiology, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192 (Japan)

    2010-03-12

    Expression of mRNA for the ghrelin receptor, GHS-R1a, was detected in various peripheral and central tissues of fetal rats, including skin, bone, heart, liver, gut, brain and spinal cord, on embryonic day (ED)15 and ED17. However, its expression in skin, bone, heart and liver, but not in gut, brain and spinal cord, became relatively weak on ED19 and disappeared after birth (ND2). Ghrelin and des-acyl ghrelin facilitated the proliferation of cultured fetal (ED17, 19), but not neonatal (ND2), skin cells. On the other hand, with regard to cells from the spinal cord and hypothalamus, the proliferative effect of ghrelin continued after birth, whereas the effect of des-acyl ghrelin on neurogenesis in these tissues was lost at the ED19 fetal and ND2 neonatal stages. Immunohistochemistry revealed that the cells in the hypothalamus induced to proliferate by ghrelin at the ND2 stage were positive for nestin and glial fibrillary acidic protein. These results suggest that in the period immediately prior to, and after birth, rat fetal cells showing proliferation in response to ghrelin and des-acyl ghrelin are at a transitional stage characterized by alteration of the expression of GHS-R1a and an undefined des-acyl ghrelin receptor, their responsiveness varying among different tissues.

  16. Transitional change in rat fetal cell proliferation in response to ghrelin and des-acyl ghrelin during the last stage of pregnancy

    International Nuclear Information System (INIS)

    Inoue, Yoshiyuki; Nakahara, Keiko; Kangawa, Kenji; Murakami, Noboru

    2010-01-01

    Expression of mRNA for the ghrelin receptor, GHS-R1a, was detected in various peripheral and central tissues of fetal rats, including skin, bone, heart, liver, gut, brain and spinal cord, on embryonic day (ED)15 and ED17. However, its expression in skin, bone, heart and liver, but not in gut, brain and spinal cord, became relatively weak on ED19 and disappeared after birth (ND2). Ghrelin and des-acyl ghrelin facilitated the proliferation of cultured fetal (ED17, 19), but not neonatal (ND2), skin cells. On the other hand, with regard to cells from the spinal cord and hypothalamus, the proliferative effect of ghrelin continued after birth, whereas the effect of des-acyl ghrelin on neurogenesis in these tissues was lost at the ED19 fetal and ND2 neonatal stages. Immunohistochemistry revealed that the cells in the hypothalamus induced to proliferate by ghrelin at the ND2 stage were positive for nestin and glial fibrillary acidic protein. These results suggest that in the period immediately prior to, and after birth, rat fetal cells showing proliferation in response to ghrelin and des-acyl ghrelin are at a transitional stage characterized by alteration of the expression of GHS-R1a and an undefined des-acyl ghrelin receptor, their responsiveness varying among different tissues.

  17. Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

    DEFF Research Database (Denmark)

    Mullaney, Brendan C; Blind, Raymond D; Lemieux, George A

    2010-01-01

    Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3...... mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine...... program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans....

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

    Directory of Open Access Journals (Sweden)

    Steffen Kawelke

    Full Text Available 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.

  19. Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

    Science.gov (United States)

    Kawashima, Y; Hirose, A; Kozuka, H

    1986-01-01

    Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed. PMID:2874791

  20. Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice

    DEFF Research Database (Denmark)

    Gallego, Sandra F; Sprenger, Richard R; Neess, Ditte

    2017-01-01

    The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier a...

  1. Efficient identification of phosphatidylserine-binding proteins by ORF phage display

    International Nuclear Information System (INIS)

    Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

    2009-01-01

    To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

  2. Improved Synthesis of 1-O-Acyl-β-d-Glucopyranose Tetraacetates

    Directory of Open Access Journals (Sweden)

    Yu Chen

    2017-04-01

    Full Text Available An improved synthesis of 1-O-acyl glucosyl esters that avoids the use of expensive Ag reagents as well as the hydrolysis of unstable glucosyl bromides is reported. Notably, β-configuration products were obtained exclusively in good yields.

  3. Characterization of binding of N'-nitrosonornicotine to protein

    International Nuclear Information System (INIS)

    Hughes, M.F.

    1986-01-01

    The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of [ 14 C]NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding to liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N 2 or CO:O 2 (8:2) significantly decreased the NADPH-dependent binding of [ 14 C]NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of [ 14 C]NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation

  4. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  5. A Simple, Rapid and Mild One Pot Synthesis of Benzene Ring Acylated and Demethylated Analogues of Harmine under Solvent-free Conditions

    Directory of Open Access Journals (Sweden)

    Bina S. Siddiqui

    2008-08-01

    Full Text Available A simple, rapid, solvent-free, room temperature one pot synthesis of benzene ring acylated and demethylated analogues of harmine using acyl halides/acid anhydrides and AlCl3 has been developed. Eight different acyl halides/acid anhydrides were used in the synthesis. The resulting mixture of products was separated by column chromatography to afford 10- and 12-monoacyl analogues, along with 10,12-diacyl-11-hydroxy products. In five cases the corresponding 10-acyl-11-hydroxy analogues were also obtained. Yields from the eight syntheses (29 products in total were in the 6-34% range and all compounds were fully characterized.

  6. Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency

    DEFF Research Database (Denmark)

    Pedersen, Christina Bak; Bross, P.; Winter, V.S.

    2003-01-01

    and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate...... proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.......Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding...

  7. Discovery of Nanomolar Desmuramylpeptide Agonists of the Innate Immune Receptor Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Possessing Immunostimulatory Properties.

    Science.gov (United States)

    Gobec, Martina; Tomašič, Tihomir; Štimac, Adela; Frkanec, Ruža; Trontelj, Jurij; Anderluh, Marko; Mlinarič-Raščan, Irena; Jakopin, Žiga

    2018-04-12

    Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants, and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis, and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.

  8. Synthesis of N-Acylated Amino Acid Surfactant from L-Proline and Palmitoyl Chloride

    International Nuclear Information System (INIS)

    Meutia Fadhilah Hasibuan; Mohd Wahid Samsudin; Rahimi Mohd Yusop; Suria Ramli

    2015-01-01

    A biodegradable, less toxic and environmentally friendly N-acylated amino acid surfactant was prepared from the amino acid L-proline and palmitoyl chloride through acylation reaction using the Schotten-Baumann reaction condition. The reaction result was a white flake form and the percentage of the crude yield was 72 % with melting point in range of 52 - 58 degree Celsius. Functional group of amide which was detected using Fourier Transform Infrared method showed the presence of N-palmitoyl proline. The purity analysis using High Performance Liquid Chromatography and Thin Layer Chromatography showed the result was a mixture compound. (author)

  9. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel

    2013-05-13

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  10. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel; Harb, Jamil; Qudeimat, Enas

    2013-01-01

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  11. Anti-tumor effects of novel 5-O-acyl plumbagins based on the inhibition of mammalian DNA replicative polymerase activity.

    Directory of Open Access Journals (Sweden)

    Moe Kawamura

    Full Text Available We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone inhibits the activity of human mitochondrial DNA polymerase γ (pol γ. In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins. These chemically modified plumbagins enhanced mammalian pol inhibition and their cytotoxic activity. Plumbagin conjugated with chains consisting of more than C18-unsaturated fatty acids strongly inhibited the activities of calf pol α and human pol γ. Plumbagin conjugated with oleic acid (C18:1-acyl plumbagin showed the strongest suppression of human colon carcinoma (HCT116 cell proliferation among the ten synthesized 5-O-acyl plumbagins. The inhibitory activity on pol α, a DNA replicative pol, by these compounds showed high correlation with their cancer cell proliferation suppressive activity. C18:1-Acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. This compound inhibited the proliferation of various human cancer cell lines, and was the cytotoxic inhibitor showing strongest inhibition towards HT-29 colon cancer cells (LD50 = 2.9 µM among the nine cell lines tested. In an in vivo anti-tumor assay conducted on nude mice bearing solid tumors of HT-29 cells, C18:1-acyl plumbagin was shown to be a promising tumor suppressor. These data indicate that novel 5-O-acyl plumbagins act as anti-cancer agents based on mammalian DNA replicative pol α inhibition. Moreover, the results suggest that acylation of plumbagin is an effective chemical modification to improve the anti-cancer activity of vitamin K3 derivatives, such as plumbagin.

  12. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  13. Improved detection of calcium-binding proteins in polyacrylamide gels

    International Nuclear Information System (INIS)

    Anthony, F.A.; Babitch, J.A.

    1984-01-01

    The authors refined the method of Schibeci and Martonosi (1980) to enhance detection of calcium-binding proteins in polyacrylamide gels using 45 Ca 2+ . Their efforts have produced a method which is shorter, has 40-fold greater sensitivity over the previous method, and will detect 'EF hand'-containing calcium-binding proteins in polyacrylamide gels below the 0.5 μg level. In addition this method will detect at least one example from every described class of calcium-binding protein, including lectins and γ-carboxyglutamic acid containing calcium-binding proteins. The method should be useful for detecting calcium-binding proteins which may trigger neurotransmitter release. (Auth.)

  14. Effects of clofibric acid on the biliary excretion of benoxaprofen glucuronide and taurine conjugate in rats.

    Science.gov (United States)

    Okada, K; Kanoh, H; Mohri, K

    2011-10-01

    Benoxaprofen (BOP) is a 2-methyl propionic acid derivative with anti-inflammatory activity. BOP has an asymmetric carbon, and receives chiral inversion from R to S in vivo. BOP is metabolized to glucuronide (BOP-G) and taurine conjugate (BOP-T). The configuration of BOP-G is mainly S, and that of BOP-T is R. Chiral inversion of R to S of the propionic acid moiety and amino acid conjugation of carboxyl compounds proceed via an acyl CoA intermediate. It is known that fibrates, used in hyperlipidemia, induce acyl CoA synthetase and increase CoA concentration. We administered racemic BOP (10 mg/kg body weight) to rats (CFA+) pre-administered clofibric acid (CFA, 280 mg/kg/day), and studied BOP, BOP-G, and BOP-T enantiomer concentrations in plasma and bile up to 12 h after administration. The findings were compared with those in rats (CFA-) that had not received CFA. Furthermore, we studied the amounts of BOP-G enantiomer produced by glucuronidation in vitro using microsomes pretreated with CFA. The amounts of (S)-BOP-G in CFA+ rats were 2.7-fold larger than that in CFA- rats. Although (R)-BOP-T was excreted in CFA- rats, BOP-T could not be detected in CFA+ rats. Plasma clearance values of racemic BOP and (S)-BOP in CFA+ rats were 5-fold and 6-fold larger than those in CFA- rats, respectively. (S)-BOP-G formation activities were higher than (R)-BOP-G formation activities in both CFA+and CFA- microsomes. These findings suggest that CFA increases biliary excretion of (S)-BOP-G and facilitates plasma elimination of BOP, and further suggests that CFA predominantly induces chiral inversion to S rather than metabolic reaction to (R)-BOP-T, resulting in an increase of (S)-BOP-G.

  15. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  16. Synthesis and biological evaluation of S-acyl-3-thiopropyl prodrugs of N-phosphonoacetyl-L-aspartate (PALA).

    Science.gov (United States)

    Gagnard, Valérie; Leydet, Alain; Le Mellay, Véronique; Aubenque, Marielle; Morère, Alain; Montero, Jean-Louis

    2003-10-01

    The synthesis of new prodrugs of PALA characterised by the presence of S-acyl-3-thiopropyl, as enzyme-labile groups on the phosphonate moiety of PALA, is reported. The cytotoxic activities of PALA prodrugs were determined against human cell line (SW1573 lung carcinoma cells). A number of prodrugs bearing S-pivaloyl as acyl groups displayed cytotoxic activity in the same order of magnitude of PALA.

  17. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  18. Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP-/- mice

    DEFF Research Database (Denmark)

    Sørensen, Signe Bek; Neess, Ditte; Dixen, Karen

    2015-01-01

    of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene......Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed...

  19. Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization

    Energy Technology Data Exchange (ETDEWEB)

    Nahoum, Virginie; Lipski, Alexandra; Quillard, Fabien; Guichou, Jean-François [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France); Boublik, Yvan [CNRS, UMR5237, Centre de Recherche de Biochimie Macromoléculaire (CRBM), 34293 Montpellier (France); Pérez, Efrèn [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Germain, Pierre [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), BP 10142, 67404 Illkirch CEDEX (France); Lera, Angel R. de [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Bourguet, William, E-mail: bourguet@cbs.cnrs.fr [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France)

    2008-07-01

    Attempts have been made to crystallize the ligand-binding domain of the human retinoid X receptor in complex with a variety of newly synthesized ligands. An inverse correlation was observed between the ‘crystallizability’ and the structural dynamics of the various receptor–ligand complexes. Crystallization trials of the human retinoid X receptor α ligand-binding domain (RXRα LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRα LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRα LBD–ligand–CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

  20. Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

    Science.gov (United States)

    Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

    Science.gov (United States)

    Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

    1995-02-10

    The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

  2. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  3. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

    Science.gov (United States)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

    2017-09-15

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  4. Clinical aspects of short-chain acyl-CoA dehydrogenase deficiency

    NARCIS (Netherlands)

    Maldegem, B.T.; Wanders, R.J.A.; Wijburg, F.A.

    2010-01-01

    Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation. SCADD is biochemically characterized by increased C4-carnitine in plasma and ethylmalonic acid in urine. The diagnosis of SCADD is confirmed by DNA analysis showing

  5. Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches.

    Science.gov (United States)

    Gobin-Limballe, Stéphanie; McAndrew, Ryan P; Djouadi, Fatima; Kim, Jung-Ja; Bastin, Jean

    2010-05-01

    Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ss-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme-FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer-monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach. Copyright 2010 Elsevier B.V. All rights reserved.

  6. Decreased hepatic contents of coenzyme A molecular species in mice after subchronic mild social defeat stress.

    Science.gov (United States)

    Kubota, Yoshifumi; Goto, Tatsuhiko; Hagiya, Yuki; Chohnan, Shigeru; Toyoda, Atsushi

    2016-01-01

    Social stress may precipitate psychiatric disorders such as depression, which is related to the occurrence of the metabolic syndrome, including obesity and type 2 diabetes. We have evaluated the effects of social stress on central and peripheral metabolism using a model of depression in mice. In the present study, we focused on coenzyme A (CoA) molecular species [i.e. non-esterified CoA (CoASH), acetyl-CoA and malonyl-CoA] which play important roles in numerous metabolic pathways, and we analyzed changes in expression of these molecules in the hypothalamus and liver of adult male mice (C57BL/6J) subjected to 10 days of subchronic mild social defeat stress (sCSDS) with ICR mice as aggressors. Mice (n = 12) exposed to showed hyperphagia- and polydipsia-like symptoms and increased body weight gain compared with control mice which were not affected by exposure to ICR mice (n = 12). To elucidate the underlying metabolic features in the sCSDS model, acetyl-CoA, malonyl-CoA and CoASH tissue levels were analyzed using the acyl-CoA cycling method. The levels of hypothalamic malonyl-CoA, which decreases feeding behavior, were not influenced by sCSDS. However, sCSDS reduced levels of acetyl-CoA, malonyl-CoA and total CoA (sum of the three CoA molecular species) in the liver. Hence, hyperphagia-like symptoms in sCSDS mice evidently occurred independently of hypothalamic malonyl-CoA, but might consequently lead to down-regulation of hepatic CoA via altered expression of nudix hydrolase 7. Future studies should investigate the molecular mechanism(s) underlying the down-regulation of liver CoA pools in sCSDS mice.

  7. Chromate Binding and Removal by the Molybdate-Binding Protein ModA.

    Science.gov (United States)

    Karpus, Jason; Bosscher, Michael; Ajiboye, Ifedayo; Zhang, Liang; He, Chuan

    2017-04-04

    Effective and cheap methods and techniques for the safe removal of hexavalent chromate from the environment are in increasingly high demand. High concentrations of hexavalent chromate have been shown to have numerous harmful effects on human biology. We show that the E. coli molybdate-binding protein ModA is a genetically encoded tool capable of removing chromate from aqueous solutions. Although previously reported to not bind chromate, we show that ModA binds chromate tightly and is capable of removing chromate to levels well below current US federal standards. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. CC1, a novel crenarchaeal DNA binding protein.

    Science.gov (United States)

    Luo, Xiao; Schwarz-Linek, Uli; Botting, Catherine H; Hensel, Reinhard; Siebers, Bettina; White, Malcolm F

    2007-01-01

    The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

  9. Diketones and ketoesters synthesis by acylation of substituted trimethylsilyl lithio-malonates

    International Nuclear Information System (INIS)

    Mayani, Mbutyabo

    1983-01-01

    The acylation of trimethylsilyl substituted lithio malonates with dicarbonyl-dichlorides and diacid monoester chlorides gives, after a simple hydrolysis by water, various diketones and ketoesters. The yields are generally good. The method is easy. (author) [fr

  10. DEPENDENCE BETWEEN ACYLATION DEGREE AND SPECIFIC LYSIS ACTITIVY OF THE PSEUDOMONAS AERUGINOSA М6 BACTERIOPHAGE

    Directory of Open Access Journals (Sweden)

    Martynov A. V.

    2016-06-01

    Full Text Available Introduction. The purpose of this work is to study the influence of various degrees of protein’s acylation on lytic activity and other biological properties of Pseudomonas M6 bacteriophage. Materials and methods. The subject of the study were samples of the Pseudomonas O 12 bacteriophage, hereinafter named the M6 phage. Pure phage lines were obtained from individual standard-morphology phage plaques (plaque-forming units, or PFUs after ten passes over the indicator strain. In subsequent experiments, M6 phage samples were selected that had a sufficient level of purity (homogeneous in the morphology of virions that inactivated the homologic antiphage serum by no less than 99.0% and the heterologic antibacterial serum by no more than 1%, and a titer no lower than 10 PFU per ml (PFU/ml. Titration of the phages was conducted using the standard bilayer agar. The crude protein content in the phage suspensions was determined using the spectroscopic method (at 280 and 260 nm. Succinylation of the phage samples was conducted according to the method developed by T.J. Molenaar. The phage’s lytic activity spectrum was determined through spot tests on 224 strains of the Pseudomonas genus and 106 strains of other genera (correspondingly, of strains Enterobacter -40, Escherichia -10, Citrobacter -8, Hafnia -7, Serratia -6, Shigella -10, Salmonella -8, Proteus -7 through the use of a phage suspension containing 10 5 - 10 6 PFU/ml. (This concentration of infectious phage corpuscles corresponds with the critical distribution of the M6 phage. The results of the research were subjected to statistical processing using single-factor dispersion analysis. Results and discussion. One hundred percent phage protein acylation leads to a loss of adsorption ability on sensitive cells and is accompanied by a significant (more than 103 times decrease in the level of specific lytic activity in the phage preparations. The succinylation type being studied does not cause

  11. Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids.

    Science.gov (United States)

    Huo, Tianyao; Ferruzzi, Mario G; Schwartz, Steven J; Failla, Mark L

    2007-10-31

    A carotenoid-rich salad meal with varying amounts and types of triglycerides (TG) was digested using simulated gastric and small intestinal conditions. Xanthophylls (lutein and zeaxanthin) and carotenes (alpha-carotene, beta-carotene, and lycopene) in chyme and micelle fraction were quantified to determine digestive stability and efficiency of micellarization (bioaccessibility). Micellarization of lutein (+zeaxanthin) exceeded that of alpha- and beta-carotenes, which was greater than that of lycopene for all test conditions. Micellarization of carotenes, but not lutein (+zeaxanthin), was enhanced (P structured TG (c18:1 > c8:0 > c4:0). The degree of unsaturation of c18 fatty acyl chains in TG added to the salad purée did not significantly alter the efficiency of micellarization of carotenoids. Relatively low amounts of triolein and canola oil (0.5-1%) were required for maximum micellarization of carotenes, but more oil (approximately 2.5%) was required when TG with medium chain saturated fatty acyl groups (e.g., trioctanoin and coconut oil) was added to the salad. Uptake of lutein and beta-carotene by Caco-2 cells also was examined by exposing cells to micelles generated during the simulated digestion of salad purée with either triolein or trioctanoin. Cell accumulation of beta-carotene was independent of fatty acyl composition of micelles, whereas lutein uptake was slightly, but significantly, increased from samples with digested triolein compared to trioctanoin. The results show that the in vitro transfer of alpha-carotene, beta-carotene, and lycopene from chyme to mixed micelles during digestion requires minimal (0.5-1%) lipid content in the meal and is affected by the length of fatty acyl chains but not the degree of unsaturation in TG. In contrast, fatty acyl chain length has limited if any impact on carotenoid uptake by small intestinal epithelial cells. These data suggest that the amount of TG in a typical meal does not limit the bioaccessibility of

  12. N-Acylated and d Enantiomer Derivatives of a Nonamer Core Peptide of Lactoferricin B Showing Improved Antimicrobial Activity

    OpenAIRE

    Wakabayashi, Hiroyuki; Matsumoto, Hiroshi; Hashimoto, Koichi; Teraguchi, Susumu; Takase, Mitsunori; Hayasawa, Hirotoshi

    1999-01-01

    N-acylated or d enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

  13. N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.

    Science.gov (United States)

    Wakabayashi, H; Matsumoto, H; Hashimoto, K; Teraguchi, S; Takase, M; Hayasawa, H

    1999-05-01

    N-acylated or D enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

  14. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  15. Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels?

    Science.gov (United States)

    Özcan, Behiye; Neggers, Sebastian J C M M; Miller, Anne Reifel; Yang, Hsiu-Chiung; Lucaites, Virginia; Abribat, Thierry; Allas, Soraya; Huisman, Martin; Visser, Jenny A; Themmen, Axel P N; Sijbrands, Eric J G; Delhanty, Patric J D; van der Lely, Aart Jan

    2014-06-01

    The objective of this study was to assess the effects of a continuous overnight infusion of des-acyl ghrelin (DAG) on acylated ghrelin (AG) levels and glucose and insulin responses to a standard breakfast meal (SBM) in eight overweight patients with type 2 diabetes. Furthermore, in the same patients and two additional subjects, the effects of DAG infusion on AG concentrations and insulin sensitivity during a hyperinsulinemic-euglycemic clamp (HEC) were assessed. A double-blind, placebo-controlled cross-over study design was implemented, using overnight continuous infusions of 3 and 10  μg DAG/kg per h and placebo to study the effects on a SBM. During a HEC, we studied the insulin sensitivity. We observed that, compared with placebo, overnight DAG administration significantly decreased postprandial glucose levels, both during continuous glucose monitoring and at peak serum glucose levels. The degree of improvement in glycemia was correlated with baseline plasma AG concentrations. Concurrently, DAG infusion significantly decreased fasting and postprandial AG levels. During the HEC, 2.5  h of DAG infusion markedly decreased AG levels, and the M-index, a measure of insulin sensitivity, was significantly improved in the six subjects in whom we were able to attain steady-state euglycemia. DAG administration was not accompanied by many side effects when compared with placebo. DAG administration improves glycemic control in obese subjects with type 2 diabetes through the suppression of AG levels. DAG is a good candidate for the development of compounds in the treatment of metabolic disorders or other conditions with a disturbed AG:DAG ratio, such as type 2 diabetes mellitus or Prader-Willi syndrome. © 2014 European Society of Endocrinology.

  16. Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy.

    Science.gov (United States)

    Westermann, C M; Dorland, L; Votion, D M; de Sain-van der Velden, M G M; Wijnberg, I D; Wanders, R J A; Spliet, W G M; Testerink, N; Berger, R; Ruiter, J P N; van der Kolk, J H

    2008-05-01

    The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

  17. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  18. Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

    Science.gov (United States)

    Nieto Penalver, Carlos G; Morin, Danièle; Cantet, Franck; Saurel, Olivier; Milon, Alain; Vorholt, Julia A

    2006-01-23

    Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

  19. The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

    Science.gov (United States)

    Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

    2017-10-01

    The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

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

    Directory of Open Access Journals (Sweden)

    Jang Eun Lee

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

  1. Acylation of aromatic alcohols and phenols over InCl3 ...

    Indian Academy of Sciences (India)

    Unknown

    toluene sulphonic acid,5 ZnCl2,6 COCl2,7 Sc(OTf)3. 8 or Bi(OTf)3. 9] catalyst ... tion of benzene and other aromatic compounds.12,13. In this communication, we ... val of solvent from the reaction mixture by distillation. The acylated products ...

  2. Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif

    Directory of Open Access Journals (Sweden)

    Chang Jan-Gowth

    2011-10-01

    Full Text Available Abstract Background Multiple acyl-coenzyme A dehydrogenase deficiency (MADD is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. Results High resolution melting (HRM analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser and the hotspot mutation (p.Ala84Thr from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD simulations and normal mode analysis (NMA, we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Conclusions Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

  3. Abstracts of Plenary Lectures and Posters. International Symposium of the Structure and Function of Plant Lipids (7th) held in Davis, California on July 27-August 1, 1986,

    Science.gov (United States)

    1986-08-01

    IAA) on pea (Pisum sativum L.) stems have shown that within one hour of treatment a decrease of incorporation of [l 4 Cjcholine into...the results were similar as obtained in the in vivo experiment. 94 PYL-CoA ELONGATION SYSTEI4S IN Allium porrum MICROSC14ES. R. Lessire, J.J...demonstrated that C1 s-CoA and C20 -CoA are the best substrates for exogenous acyl-CoA elongation, using Allium porrur epidermal cell microscrmes as the

  4. Acylation, Diastereoselective Alkylation, and Cleavage of an Oxazolidinone Chiral Auxiliary: A Multistep Asymmetric Synthesis Experiment for Advanced Undergraduates

    Science.gov (United States)

    Smith, Thomas E.; Richardson, David P.; Truran, George A.; Belecki, Katherine; Onishi, Megumi

    2008-01-01

    An introduction to the concepts and experimental techniques of diastereoselective synthesis using a chiral auxiliary is described. The 4-benzyl-2-oxazolidinone chiral auxiliary developed by Evans is acylated with propionic anhydride under mild conditions using DMAP as an acyl transfer catalyst. Deprotonation with NaN(TMS)[subscript 2] at -78…

  5. Diagnostic utility of alpha-methylacyl CoA racemase (P504S) on prostate needle biopsy.

    Science.gov (United States)

    Jiang, Zhong; Woda, Bruce A

    2004-11-01

    Alpha-methylacyl CoA racemase (AMACR), also known as P504S, was identified by the analysis of cDNA library subtraction in conjunction with high throughput microarray screening from prostate tissue and has been proven to be one of the very few biomarkers that can distinguish cancer from benign cells with high sensitivity and specificity for prostate carcinoma. It is a successful example of the translation of molecular findings into clinical practice. This review focuses on the study of AMACR (P504S) expression in small focal prostate cancer and atypical small acinar proliferation (ASAP) on needle biopsies and emphasizes the utility of AMACR (P504S) in routine surgical pathology practice. We also discuss the potential pitfalls and caveats in the interpretation of immunostaining results.

  6. Structural organization of the human short-chain acyl-CoA dehydrogenase gene

    DEFF Research Database (Denmark)

    Corydon, M J; Andresen, B S; Bross, P

    1997-01-01

    Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid beta-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion...... shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C....... The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees....

  7. Kinetic study on the inhibition of xanthine oxidase by acylated derivatives of flavonoids synthesised enzymatically.

    Science.gov (United States)

    de Araújo, Maria Elisa Melo Branco; Franco, Yollanda Edwirges Moreira; Alberto, Thiago Grando; Messias, Marcia Cristina Fernandes; Leme, Camila Wielewski; Sawaya, Alexandra Christine Helena Frankland; Carvalho, Patricia de Oliveira

    2017-12-01

    Studies have reported that flavonoids inhibit xanthine oxidase (XO) activity; however, poor solubility and stability in lipophilic media limit their bioavailability and applications. This study evaluated the kinetic parameters of XO inhibition and partition coefficients of flavonoid esters biosynthesised from hesperidin, naringin, and rutin via enzymatic acylation with hexanoic, octanoic, decanoic, lauric, and oleic acids catalysed by Candida antarctica lipase B (CALB). Quantitative determination by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) showed higher conversion yields (%) for naringin and rutin esters using acyl donors with 8C and 10C. Rutin decanoate had higher partition coefficients (0.95), and naringin octanoate and naringin decanoate showed greater inhibitory effects on XO (IC 50 of 110.35 and 117.51 μM, respectively). Kinetic analysis showed significant differences (p flavonoids before and after acylation regarding K m values, whereas the values for V max were the same, implying the competitive nature of XO inhibition.

  8. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of ...

  9. Correlation of changes in rate of sterol synthesis with changes in HMG CoA reductase activity in cultured lens epithelial cells

    International Nuclear Information System (INIS)

    Cenedella, R.J.; Hitchener, W.R.

    1986-01-01

    In the present study, the authors correlated changes in HMG CoA reductase activity with changes in relative rates of sterol synthesis measured from either 3 H 2 O or 1- 14 C-acetate for bovine lens epithelial cells cultured in the presence or absence of lipoproteins. Enzyme activity and rates of incorporation of 3 H 2 O or 1- 14 C-acetate into digitonin precipitable sterols were measured in cells on the 4th day of subculture in DMEM containing 9% whole calf serum (WM) or 9% lipoprotein deficient serum (LDM). In three experiments, HMG CoA reductase activity (U/10 6 cells) averaged 2.2 +/- 0.1 times greater for cells grown in LDM than WM. Sterol synthesis averaged 3.0 +/- 0.4 times greater when measured with 3 H 2 O and 4.0 +/- 1.1 times greater when measured with 14 C-acetate. Thus, 3 H 2 O and 14 C-acetate appear to be comparable substrates for estimating changes in relative rates of sterol synthesis by cultured cells. The larger increases in rates of sterol synthesis than in reductase activity in response to decreased cholesterol could reflect stimulation at additional metabolic steps in the cholesterol pathway beyond mevalonic acid

  10. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius; Thomas, Ludivine; Serano, Natalia Lorena Gorron; Lilley, Kathryn S.; Gehring, Christoph A

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently

  11. Insight into cofactor recognition in arylamine N-acetyltransferase enzymes

    DEFF Research Database (Denmark)

    Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe

    2015-01-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes that catalyze the acetyl-CoA-dependent acetylation of arylamines. To better understand the mode of binding of the cofactor by this family of enzymes, the structure of Mesorhizobium loti NAT1 [(RHILO)NAT1] was determined...... for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover......, it supports the notion that the presence of the `mammalian/eukaryotic insertion loop' in certain NAT enzymes impacts the mode of binding CoA by imposing structural constraints....

  12. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  13. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  14. Carbapenems and SHV-1 β-Lactamase Form Different Acyl-Enzyme Populations in Crystals and Solution

    Science.gov (United States)

    Kalp, Matthew; Carey, Paul R.

    2009-01-01

    The reactions between single crystals of the SHV-1 β-lactamase enzyme and the carbapenems, meropenem, imipenem and ertapenem, have been studied by Raman microscopy. Aided by quantum mechanical calculations, major populations of two acyl-enzyme species, a labile Δ2-pyrroline and a more tightly bound Δ1-pyrroline, have been identified for all three compounds. These isomers differ only in the position of the double bond about the carbapenem nucleus. This discovery is consonant with X-ray crystallographic findings that also identified two populations for meropenem bound in SHV-1: one with the acyl C=O group in the oxyanion hole and the second with the acyl group rotated 180 degrees compared to its expected position [Nukaga, M., Bethel, C. R., Thomson, J. M., Hujer, A. M., Distler, A. M., Anderson, V. E., Knox, J. R., and Bonomo, R. A. (2008) Journal of the American Chemical Society]. When crystals of the Δ1 and Δ2 containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Δ2 species was observed by kinetic Raman experiments. However, no change in the Δ1 population was observed over 1 hour, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Δ1 species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Δ2 species corresponds to the form with the carbonyl inside the oxyanion hole. Soak-in and soak-out Raman experiments also demonstrated that tautomeric exchange between the Δ1 and Δ2 forms does not occur on the crystalline enzyme. When meropenem or ertapenem were reacted with SHV-1 in solution, the Raman difference spectra demonstrated that only a major population corresponding to the Δ1 acyl-enzyme could be detected. The 1003 cm-1 mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal Raman intensity standard and the ratio of its intensity to that of the 1600 cm-1 feature of Δ1 provides an

  15. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  16. DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas

    Science.gov (United States)

    Tahir, Shaista; Badshah, Amin; Hussain, Raja Azadar; Tahir, Muhammad Nawaz; Tabassum, Saira; Patujo, Jahangir Ali; Rauf, Muhammad Khawar

    2015-11-01

    Four new nitrosubstituted acylthioureas i.e. 1-acetyl-3-(4-nitrophenyl)thiourea (TU1), 1-acetyl-3-(2-methyl-4-nitrophenyl)thiourea (TU2), 1-acetyl-3-(2-methoxy-4-nitrophenyl)thiourea (TU3) and 1-acetyl-3-(4-chloro-3-nitrophenyl)thiourea (TU4) have been synthesized and characterized (by C13 and H1 nuclear magnetic resonance, Fourier transform infrared spectroscopy and single crystal X-ray diffraction). As a preliminary investigation of the anti-cancer potencies of the said compounds, DNA interaction studies have been carried out using cyclic voltammetry and UV-vis spectroscopy along with verification from computational studies. The drug-DNA binding constants are found to be in the order, KTU3 9.04 × 106 M-1 > KTU4 8.57 × 106 M-1 > KTU2 6.05 × 106 M-1 > KTU1 1.16 × 106 M-1. Furthermore, the antioxidant, cytotoxic, antibacterial and antifungal activities have been carried out against DPPH (1,1-diphenyl-2-dipicrylhydrazyl), Brine shrimp eggs, gram positive (Micrococcus luteus, Staphylococcus aureus) and gram negative (Bordetella bronchiseptica, Salmonella typhimurium, Enterobacter aerogens) and fungal cultures (Aspergillus fumigatus, Mucor species, Aspergillus niger, Aspergillus flavus) respectively.

  17. Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

    Science.gov (United States)

    Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

    2011-12-01

    In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

  18. delta 6 Hexadecenoic acid is synthesized by the activity of a soluble delta 6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.

    Science.gov (United States)

    Cahoon, E B; Cranmer, A M; Shanklin, J; Ohlrogge, J B

    1994-11-04

    delta 6 Hexadecenoic acid (16:1 delta 6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble delta 6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, delta 6 16:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in delta 9 stearoyl (18:0)- and delta 4 16:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor delta 9 18:0-ACP desaturase and 57% identity with the mature coriander delta 4 16:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the delta 6 desaturation of 16:0-ACP. These results demonstrate that 16:1 delta 6 in T. alata endosperm is formed by the activity of a soluble delta 6 16:0-ACP desaturase that is structurally related to the delta 9 18:0- and delta 4 16:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

  19. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull. Persoon (Lion’s Mane Mushroom

    Directory of Open Access Journals (Sweden)

    Mohammad Azizur Rahman

    2014-01-01

    Full Text Available Oxidation of low-density lipoprotein (LDL has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM, hexane (HEX, dichloromethane (DCM, ethyl acetate (EA, and aqueous residue (AQ. The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins for the formation of conjugated diene (CD at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL of thiobarbituric acid reactive substances (TBARS at 1 mg/mL. It also mostly inhibited (59.91% the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  20. Synthesis of 1-indanones through the intramolecular Friedel-Crafts acylation reaction using NbCl5 as Lewis acid

    International Nuclear Information System (INIS)

    Polo, Ellen Christine; Silva-Filho, Luiz Carlos da; Silva, Gil Valdo Jose da; Constantino, Mauricio Gomes

    2008-01-01

    The intramolecular Friedel-Crafts acylation reaction of 3-arylpropanoic acids to give 1-indanones can be effected in good yields under mild conditions (room temperature) by using niobium pentachloride. Our results indicate that NbCl 5 acts both as reagent (to transform carboxylic acids into acyl chlorides) and as catalyst in the Friedel-Crafts cyclization. (author)

  1. Antileishmanial Activity of Aldonamides and N-Acyl-Diamine Derivatives

    Directory of Open Access Journals (Sweden)

    Elaine S. Coimbra

    2008-01-01

    Full Text Available A number of lipophilic N-acyl-diamines and aldonamides have been synthesized and tested for their in vitro antiproliferative activity against Leishmania amazonensis and L. chagasi. Ribonamides, having one amino group, displayed good to moderate inhibition of parasite growth. The best result was obtained for compounds 10 and 15 with IC50 against L. chagasi below 5 μM.

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

    Directory of Open Access Journals (Sweden)

    Surang Chankhamhaengdecha

    2013-01-01

    Full Text Available 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.

  3. Novel regulator of acylated ghrelin, CF801, reduces weight gain, rebound feeding after a fast, and adiposity in mice

    Directory of Open Access Journals (Sweden)

    Martin K Wellman

    2015-09-01

    Full Text Available Ghrelin is a 28 amino-acid hormonal peptide that is intimately related to the regulation of food intake and body weight. Once secreted, ghrelin binds to the growth hormone secretagogue receptor-1a (GHSR-1a, the only known receptor for ghrelin and is capable of activating a number of signaling cascades ultimately resulting in an increase in food intake and adiposity. Because ghrelin has been linked to overeating and the development of obesity, a number of pharmacological interventions have been generated in order to interfere with either the activation of ghrelin or interrupting ghrelin signaling as a means to reducing appetite and decrease weight gain. Here we present a novel peptide, CF801, capable of reducing circulating acylated ghrelin levels and subsequent body weight gain and adiposity. To this end, we show that IP administration of CF801 is sufficient to reduce circulating plasma acylated ghrelin levels. Acutely, intraperitoneal injections of CF801 resulted in decreased rebound feeding after an overnight fast. When delivered chronically decreased weight gain and adiposity without affecting caloric intake. CF801, however, did cause a change in diet preference, decreasing preference for a high fat diet and increasing preference for regular chow diet. Given the complexity of ghrelin receptor function, we propose that CF801 along with other compounds that regulate ghrelin secretion may prove to be a beneficial tool in the study of the ghrelin system, and potential targets for ghrelin based obesity treatments without altering the function of ghrelin receptors.

  4. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  5. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  6. Factor VII and protein C are phosphatidic acid-binding proteins.

    Science.gov (United States)

    Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

    2013-08-20

    Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

  7. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping