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Sample records for acyl enzyme intermediate

  1. Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Bagonis, Maria; Kershaw, Nadia J; Domene, Carmen; Claridge, Timothy D W; Wharton, Christopher W; Schofield, Christopher J

    2009-01-21

    Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated

  2. Folylpolyglutamate synthetase: direct evidence for an acyl phosphate intermediate in the enzyme-catalyzed reaction

    SciTech Connect

    Banerjee, R.; McGuire, J.J.; Shane, B.; Coward, J.K.

    1986-05-01

    The nature of the intermediate in the reaction catalyzed by folylpoly-..gamma..-glutamate synthetase (FPGS) has been investigated. Incubation of ..cap alpha..,..gamma..-(/sup 18/O)methotrexate with ATP, glutamate, and FPGS resulted in the formation of (/sup 18/O)phosphate, thus providing strong evidence for the formation of a ..gamma..-glutamyl phosphate during catalysis. The inorganic phosphate formed in the enzyme-catalyzed reaction was separated from other products and substrates by chromatography on DEAE-cellulose, then converted to the trimethyl ester, and analyzed by mass spectroscopy. Stoichiometric formation of (/sup 18/O)phosphate was observed in the case of the E. coli enzyme, isolated from a transformant containing the cloned FPGS-dihydrofolate synthetase (folC) gene. In addition, /sup 31/P-NMR analysis of the phosphate isolated from the reaction using E. coli FPGS showed the expected /sup 18/O-isotopic perturbations due to both singly bonded and doubly bonded P-/sup 18/O species. Similar experiments were carried out with FPGS isolated from hog liver. In this case, the small amounts of pure enzyme available precluded use of the NMR technique. However, mass spectral analysis of the derivatized phosphate product revealed the presence of (/sup 18/O)-trimethyl phosphate, thus indicating that the reaction catalyzed by the mammalian enzyme also proceeds via an acyl phosphate intermediate.

  3. Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad

    SciTech Connect

    Ruzzini, Antonio C.; Ghosh, Subhangi; Horsman, Geoff P.; Foster, Leonard J.; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2012-03-14

    Meta-cleavage product (MCP) hydrolases are members of the {alpha}/{beta}-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 {angstrom} resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of {sup 18}O into the benzoate produced during hydrolysis in H{sub 2}{sup 18}O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES{sup red}, previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.

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

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

  5. Acetic anhydride: an intermediate analogue in the acyl-exchange reaction of citramalate lyase.

    PubMed

    Buckel, W

    1976-04-15

    1. Reactivation of deacetyl citramalate lyase by acetic anhydride proceeds through an enzyme-anhydride complex prior to actual acetylation. The reaction is inhibited by citramalate which is competitive with acetic anhydride. 2. A corresponding complex is an intermediate in the carboxymethylation of deacetyl enzyme by iodoacetate. However, the inhibition of this reaction by S-citramalate appears to be non-competitive with iodoacetate. 3. The results lead to the conclusion that acetic anhydride can be regarded as a structural analogue of citramalic acetic anhydride, the proposed intermediate in the acyl exchange reaction on citramalate lyase. 4. The formation of 6-citryl thiolester from the 1-thiolester via the cyclic citric anhydride provides a chemicla model for enzymic acyl exchange. 5. The data suggest that anhydrides are of general importance in acyl exchange reactions of thiolesters. PMID:1278157

  6. Acyl Silicates and Acyl Aluminates as Activated Intermediates in Peptide Formation on Clays

    NASA Astrophysics Data System (ADS)

    White, David H.; Kennedy, Robert M.; Macklin, John

    1984-12-01

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

  7. Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis.

    PubMed

    Fodor, Krisztián; Harmat, Veronika; Neutze, Richard; Szilágyi, László; Gráf, László; Katona, Gergely

    2006-02-21

    Atomic resolution (intermediate structures revealed that the strength of the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conserved hydrogen bonds of antiparallel beta-sheet between the enzyme and the substrate become significantly shorter in the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish) trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 A resolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcine pancreatic elastase at 0.95 A resolution) presumably synchronously with the nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizes the energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilic attack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bond shortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of the catalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transition from the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102 and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization for acyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significant deviation from planarity is not observed in the reactive bonds of either the Michaelis complex or the acyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted toward the oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effect of the oxyanion hole. PMID:16475800

  8. The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal.

    PubMed

    Cronan, John E

    2014-06-01

    ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4'-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP-enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4'-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping. PMID:24825445

  9. Acylation of N-p-Toluenesulfonylpyrrole Under Friedel-Crafts Conditions. Evidence for Organoaluminum Intermediates

    PubMed Central

    Huffman, John W.; Smith, Valerie J.; Padgett, Lea W.

    2008-01-01

    The Friedel-Crafts acylation of N-p-toluenesulfonylpyrrole under Friedel-Crafts conditions has been reinvestigated. Evidence is presented in support of the hypothesis that when AlCl3 is used as the Lewis acid, acylation proceeds via reaction of an organoaluminum intermediate with the acyl halide. This leads to the production of the 3-acyl derivative as the major product. With weaker Lewis acids (EtAlCl2, Et2AlCl) or less than one equivalent of AlCl3 the relative amount of 2-acyl product is increased. A mechanistic rationalization is presented to explain these data. PMID:19247425

  10. Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates

    SciTech Connect

    Banerjee, R.V.; Shane, B.; McGuire, J.J.; Coward, J.K.

    1988-12-13

    The transfer of /sup 17/O and/or /sup 18/O from (COOH-/sup 17/O or -/sup 18/O) enriched substrates to inorganic phosphate (P/sub i/) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-/sup 18/O-labeled folate, methotrexate, and dihydropteroate, in addition to (/sup 17/O)-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. P/sub i/ was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for /sup 17/O and /sup 18/O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate /sup 18/O-enriched carboxyl group to P/sub i/ occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of P/sub i/ obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that /sup 18/O transfer had occurred.

  11. Sonochemical enzyme-catalyzed regioselective acylation of flavonoid glycosides.

    PubMed

    Ziaullah; Rupasinghe, H P Vasantha

    2016-04-01

    This work compares a highly efficient and alternative method of sonication-assisted lipase catalyzed acylation of quercetin-3-O-glucoside and phloretin-2'-glucoside, using Candida antarctica lipase B (Novozyme 435(®)), with a range of fatty acids. In this study, sonication-assisted irradiation coupled with stirring has been found to be more efficient and economical than conventional reaction conditions. Sonication-assisted acylation accelerated the reactions and reduced the time required by 4-5 folds. PMID:26829593

  12. Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes.

    PubMed

    Bavdek, Andrej; Vazquez, Hector M; Conzelmann, Andreas

    2015-11-01

    Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [(3)H]lyso-phosphatidic acid inside liposomes after [(3)H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [(3)H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [(3)H]lyso-phosphatidic acid or of dephosphorylated [(3)H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required. PMID:26325346

  13. Facile Detection of Acyl- and Peptidyl- intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions During Tandem Mass Spectrometry

    PubMed Central

    Dorrestein, Pieter C.; Bumpus, Stefanie B.; Calderone, Christopher T.; Garneau-Tsodikova, Sylvie; Aron, Zachary D.; Straight, Paul D.; Kolter, Roberto; Walsh, Christopher T.; Kelleher, Neil L.

    2008-01-01

    With the emergence of drug resistance and the genomic revolution there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultra large enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas phase carrier domains, facile elimination occurs in benchtop and Fourier-Transform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This “Top Down” approach quickly illuminated diverse acyl-intermediates on the carrier domains of the nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2H or 18O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS/PKS projects with benchtop mass spectrometers. PMID:17042494

  14. Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In higher plants, the superfamily of carboxyl-CoA ligases and related proteins, collectively called acyl activating enzymes (AAEs), has evolved to provide enzymes for many pathways of primary and secondary metabolism and for the conjugation of hormones to amino acids. Across the superfamily there is...

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

    PubMed

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

    2015-12-01

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

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

    PubMed

    Rowland, Owen; Domergue, Frédéric

    2012-09-01

    Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols. PMID:22794916

  17. Exogenous myristic acid can be partially degraded prior to activation to form acyl-acyl carrier protein intermediates and lipid A in Vibrio harveyi.

    PubMed Central

    Shen, Z; Byers, D M

    1994-01-01

    To study the involvement of acyl carrier protein (ACP) in the metabolism of exogenous fatty acids in Vibrio harveyi, cultures were incubated in minimal medium with [9,10-3H]myristic acid, and labeled proteins were analyzed by gel electrophoresis. Labeled acyl-ACP was positively identified by immunoprecipitation with anti-V. harveyi ACP serum and comigration with acyl-ACP standards and [3H]beta-alanine-labeled bands on both sodium dodecyl sulfate- and urea-polyacrylamide gels. Surprisingly, most of the acyl-ACP label corresponded to fatty acid chain lengths of less than 14 carbons: C14, C12, C10, and C8 represented 33, 40, 14, and 8% of total [3H]14:0-derived acyl-ACPs, respectively, in a dark mutant (M17) of V. harveyi which lacks myristoyl-ACP esterase activity; however, labeled 14:0-ACP was absent in the wild-type strain. 14:0- and 12:0-ACP were also the predominant species labeled in complex medium. In contrast, short-chain acyl-ACPs (< or = C6) were the major labeled derivatives when V. harveyi was incubated with [3H]acetate, indicating that acyl-ACP labeling with [3H]14:0 in vivo is not due to the total degradation of [3H]14:0 to [3H]acetyl coenzyme A followed by resynthesis. Cerulenin increased the mass of medium- to long-chain acyl-ACPs (> or = C8) labeled with [3H]beta-alanine fivefold, while total incorporation of [3H]14:0 was not affected, although a shift to shorter chain lengths was noted. Additional bands which comigrated with acyl-ACP on sodium dodecyl sulfate gels were identified as lipopolysaccharide by acid hydrolysis and thin-layer chromatography. The levels of incorporation of [3H] 14:0 into acyl-ACP and lipopolysaccharide were 2 and 15%, respectively, of that into phospholipid by 10 min. Our results indicate that in contrast to the situation in Escherichia coli, exogenous fatty acids can be activated to acyl-ACP intermediates after partial degradation in V. harveyi and can effectively label products (i.e., lipid A) that require ACP as an acyl

  18. Direct evidence for an acyl phosphate intermediate in the folylpoly-. gamma. -glutamate synthetase and dihydrofolate synthetase-catalyzed reactions

    SciTech Connect

    Banerjee, R.

    1987-01-01

    The mechanism of the reactions catalyzed by two enzymes, namely dihydrofolate synthetase (DHFS) and folylpoly-..gamma..-glutamate synthetase (FPGS), has been investigated. The nature of the intermediate in each of the two reactions was monitored simultaneously in the multifunctional enzyme, FPGS/DHFS from E. coli. The latter was isolated from a transformant containing the cloned FPGS/DHFS gene. Incubation of (/sup 18/O)-H/sub 2/Pte and (/sup 17/O)-glutamate with ATP and the enzyme, resulted in the formation of (/sup 18/O)- and (/sup 17/O)-P/sub i/, thus providing strong evidence for the formation of an acyl phosphate species during catalysis of each reaction. The inorganic phosphate formed in the enzyme-catalyzed reaction was purified by chromatography on DEAE-cellulose, then converted to the trimethyl ester and analyzed by mass spectroscopy /sup 17/O NMR and /sup 31/P NMR. Stoichiometric formation of (/sup 17/O)- and (/sup 18/O)-Pi was observed. /sup 31/P NMR analysis showed the expected /sup 18/O-induced isotopic perturbations. The presence of (/sup 17/O)-trimethyl phosphate was revealed by /sup 17/O NMR. The mechanism of the FPGS-catalyzed reaction was also investigated with the antifolate (/sup 18/O)-methotrexate.

  19. Deacylation Mechanism and Kinetics of Acyl-Enzyme Complex of Class C β-Lactamase and Cephalothin.

    PubMed

    Tripathi, Ravi; Nair, Nisanth N

    2016-03-17

    Understanding the molecular details of antibiotic resistance by the bacterial enzymes β-lactamases is vital for the development of novel antibiotics and inhibitors. In this spirit, the detailed mechanism of deacylation of the acyl-enzyme complex formed by cephalothin and class C β-lactamase is investigated here using hybrid quantum-mechanical/molecular-mechanical molecular dynamics methods. The roles of various active-site residues and substrate in the deacylation reaction are elucidated. We identify the base that activates the hydrolyzing water molecule and the residue that protonates the catalytic serine (Ser64). Conformational changes in the active sites and proton transfers that potentiate the efficiency of the deacylation reaction are presented. We have also characterized the oxyanion holes and other H-bonding interactions that stabilize the reaction intermediates. Together with the kinetic and mechanistic details of the acylation reaction, we analyze the complete mechanism and the overall kinetics of the drug hydrolysis. Finally, the apparent rate-determining step in the drug hydrolysis is scrutinized. PMID:26918257

  20. Regioselective enzymatic acylation of methyl shikimate. Influence of acyl chain length and solvent polarity on enzyme specificity.

    PubMed

    Armesto, Nuria; Ferrero, Miguel; Fernández, Susana; Gotor, Vicente

    2002-07-12

    Candida antarctica lipase A (CAL-A) selectively catalyzes the acylation at the secondary C-4 hydroxyl group of methyl shikimate (2), which possesses three secondary hydroxyl groups, the C-3 allylic one being chemically more reactive. The effect both of the acyl group of the acylating agents and of the solvent polarity has been studied. The selectivity of CAL-A is almost complete with acyl donors that possess short chains. However, when acyl donors have longer chains, better results are obtained by C. antarctica lipase B (CAL-B). PMID:12098318

  1. Lipopolysaccharide differentially decreases plasma acyl and desacyl ghrelin levels in rats: potential role of the circulating ghrelin acylating enzyme GOAT

    PubMed Central

    Stengel, Andreas; Goebel, Miriam; Wang, Lixin; Reeve, Joseph R.; Taché, Yvette; Lambrecht, Nils W.G.

    2014-01-01

    Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100μg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2h, 5h and 7h (p<0.01) post injection compared to vehicle and recovered at 24h. At 2h there was a significantly greater decrease of AG (-53%) than DG (-28%) resulting in a decreased AG/DG ratio (1:5, p <0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p <0.001), whereas gastric GOAT protein was slightly increased by 10% (p<0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5-2h period post LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2h is associated with reduced circulating GOAT. PMID:20599577

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

    PubMed Central

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

    2013-01-01

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

  3. Utilization of a depsipeptide substrate for trapping acyl—enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases

    PubMed Central

    Rasmussen, James R.; Strominger, Jack L.

    1978-01-01

    The penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus catalyzed the hydrolysis of the D-lactic acid residue from the depsipeptide diacetyl-L-lysyl-D-alanyl-D-lactic acid. The ester substrate was hydrolyzed faster than the peptide analogue, diacetyl-L-lysyl-D-alanyl-D-alanine, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, presumably because acylation (k2), which is the rate-limiting step of the peptidase reaction, occurred more rapidly during cleavage of the ester bond than during cleavage of the amide bond. No rate acceleration was observed with the S. aureus carboxypeptidase for which deacylation (k3) is already the rate-determining step with the peptide substrate. The efficiency of utilization of the depsipeptide (Vmax/Km) was greatly enhanced (19- to 147-fold) for all three enzymes. After incubation of the B. subtilis carboxypeptidase and [14C]diacetyl-L-lysyl-D-alanyl-D-lactic acid at pH 5.0 and lowering of the pH to 3.0, a radioactive acyl-enzyme intermediate containing 0.43 mol of substrate per mol of enzyme was isolated by Sephadex G-50 chromatography. After acetone precipitation, the acyl group of the denatured acyl-enzyme complex appeared to be bound to the protein by an ester bond. Acyl enzymes were also detected for the S. aureus and E. coli carboxypeptidases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography of enzyme incubated with [14C]depsipeptide and precipitated with acetone. Images PMID:415311

  4. Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent.

    PubMed

    Plou, Francisco J; Cruces, M Angeles; Ferrer, Manuel; Fuentes, Gloria; Pastor, Eitel; Bernabé, Manuel; Christensen, Morten; Comelles, Francisco; Parra, José L; Ballesteros, Antonio

    2002-06-13

    Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters versus sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesise different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalysed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process. PMID:12142143

  5. Acyl-Carbon Bond Cleaving Cytochrome P450 Enzymes: CYP17A1, CYP19A1 and CYP51A1.

    PubMed

    Akhtar, Muhammad; Wright, J Neville

    2015-01-01

    Cytochrome P450 (P450 or CYP) enzymes in their resting state contain the heme-iron in a high-spin FeIII state. Binding of a substrate to a P450 enzyme allows transfer of the first electron, producing a Fe(II) species that reacts with oxygen to generate a low-spin iron superoxide intermediate (FeIII-O-O•) ready to accept the second electron to produce an iron peroxy anion intermediate (a, FeIII-O-O-). In classical monooxygenation reactions, the peroxy anion upon protonation fragments to form the reactive Compound I intermediate (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). However, when the substrate projects a carbonyl functionality, of the type b, at the active site as is the case for reactions catalyzed by CYP17A1, CYP19A1 and CYP51A1, the peroxy anion (FeIII-O-O-) is trapped, yielding a tetrahedral intermediate (c) that fragments to an acyl-carbon cleavage product (d plus an acid). Analogous acyl-carbon cleavage reactions are also catalyzed by certain hepatic P450s and CYP125A1 from Mycobacterium tuberculosis. A further improvisation on the theme is provided by aldehyde deformylases that convert long-chain aliphatic aldehydes to hydrocarbons. CYP17A1 is involved in the biosynthesis of corticoids as well as androgens. The flux toward these two classes of hormones seems to be regulated by cytochrome b 5, at the level of the acyl-carbon cleavage reaction. It is this regulation of CYP17A1 that provides a safety mechanism, ensuring that during corticoid biosynthesis, which requires 17α-hydroxylation by CYP17A1, androgen formation is avoided (Fig. 4.1). PMID:26002733

  6. Series mechanism of enzyme deactivation. Characterization of intermediate forms.

    PubMed

    Gianfreda, L; Marrucci, G; Grizzuti, N; Greco, G

    1985-06-01

    Acid phosphatase (E.C. 3.1.3.2) undergoes complex thermal deactivation phenomena, as revealed by the two-slope pattern of the enzyme logarithmic-specific-activity versus time curves. The native enzyme first decays toward an equilibrium distribution of less, but still active, intermediate structures and these, in turn, undergo a final degradation to a completely inactive form. The effect of the experimental conditions at which the enzyme is kept during the deactivation process on the characteristics of these intermediate enzymatic structures has been investigated. The kinetic parameters of p-nitro-phenyl phosphate hydrolysis, as catalyzed by some of these intermediate forms, have been determined and the results compared to those obtained with the native enzyme. PMID:18553749

  7. Variant of human enzyme sequesters reactive intermediate.

    PubMed

    Ewalt, Karla L; Yang, Xiang-Lei; Otero, Francella J; Liu, Jianming; Slike, Bonnie; Schimmel, Paul

    2005-03-22

    In cellular environments, coupled hydrolytic reactions are used to force efficient product formation in enzyme-catalyzed reactions. In the first step of protein synthesis, aminoacyl-tRNA synthetases react with amino acid and ATP to form an enzyme-bound adenylate that, in the next step, reacts with tRNA to form aminoacyl-tRNA. The reaction liberates pyrophosphate (PP(i)) which, in turn, can be hydrolyzed by pyrophosphatase to drive efficient aminoacylation. A potential polymorphic variant of human tryptophanyl-tRNA synthetase is shown here to sequester tryptophanyl adenylate. The bound adenylate does not react efficiently with the liberated PP(i) that normally competes with tRNA to resynthesize ATP and free amino acid. Structural analysis of this variant showed that residues needed for binding ATP phosphates and thus PP(i) were reoriented from their conformations in the structure of the more common sequence variant. Significantly, the reorientation does not affect reaction with tRNA, so that efficient aminoacylation is achieved. PMID:15766249

  8. Enzyme clustering accelerates processing of intermediates through metabolic channeling.

    PubMed

    Castellana, Michele; Wilson, Maxwell Z; Xu, Yifan; Joshi, Preeti; Cristea, Ileana M; Rabinowitz, Joshua D; Gitai, Zemer; Wingreen, Ned S

    2014-10-01

    We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation. PMID:25262299

  9. Enzyme clustering accelerates processing of intermediates through metabolic channeling

    PubMed Central

    Castellana, Michele; Wilson, Maxwell Z.; Xu, Yifan; Joshi, Preeti; Cristea, Ileana M.; Rabinowitz, Joshua D.; Gitai, Zemer; Wingreen, Ned S.

    2015-01-01

    We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation. PMID:25262299

  10. Mechanism of action of clostridial glycine reductase: Isolation and characterization of a covalent acetyl enzyme intermediate

    SciTech Connect

    Arkowitz, R.A.; Abeles, R.H. )

    1991-04-23

    Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + P{sub i} + 2e{sup {minus}} {yields} acetyl phosphate + NH{sub 4}{sup +}. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. The authors now demonstrate that protein C catalyzes exchange of ({sup 32}P)P{sub i} into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, they have isolated acetyl protein C and shown that it is qualitatively, catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with P{sub i} to give acetyl phosphate. When ({sup 14}C)acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. Treatment with KBH{sub 4} removes all the radioactivity associated with protein C, resulting in the formation of ({sup 14}C)ethanol. They conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from ({sup 3}H)H{sub 2}O into acetyl phosphate. This exchange reaction supports the proposal that an enol of the acetyl enzyme is an intermediate in the reaction sequence.

  11. Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad

    PubMed Central

    Buller, Andrew R.; Townsend, Craig A.

    2013-01-01

    The study of proteolysis lies at the heart of our understanding of biocatalysis, enzyme evolution, and drug development. To understand the degree of natural variation in protease active sites, we systematically evaluated simple active site features from all serine, cysteine and threonine proteases of independent lineage. This convergent evolutionary analysis revealed several interrelated and previously unrecognized relationships. The reactive rotamer of the nucleophile determines which neighboring amide can be used in the local oxyanion hole. Each rotamer–oxyanion hole combination limits the location of the moiety facilitating proton transfer and, combined together, fixes the stereochemistry of catalysis. All proteases that use an acyl-enzyme mechanism naturally divide into two classes according to which face of the peptide substrate is attacked during catalysis. We show that each class is subject to unique structural constraints that have governed the convergent evolution of enzyme structure. Using this framework, we show that the γ-methyl of Thr causes an intrinsic steric clash that precludes its use as the nucleophile in the traditional catalytic triad. This constraint is released upon autoproteolysis and we propose a molecular basis for the increased enzymatic efficiency introduced by the γ-methyl of Thr. Finally, we identify several classes of natural products whose mode of action is sensitive to the division according to the face of attack identified here. This analysis of protease structure and function unifies 50 y of biocatalysis research, providing a framework for the continued study of enzyme evolution and the development of inhibitors with increased selectivity. PMID:23382230

  12. Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans▿

    PubMed Central

    Meng, Yu; Ingram-Smith, Cheryl; Cooper, Leroy L.; Smith, Kerry S.

    2010-01-01

    Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme. PMID:20851904

  13. Carbapenems and SHV-1 β-Lactamase Form Different Acyl-Enzyme Populations in Crystals and Solution

    PubMed Central

    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

  14. Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes

    PubMed Central

    2015-01-01

    Conspectus A [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5′-deoxyadenosyl radical (5′-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5′-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5′-dA• to the product radical l-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set

  15. 1-alkenyl-2-acyl glycerol is an intermediate in myocardial plasmenylcholine biosynthesis

    SciTech Connect

    Ford, D.; Gross, R.

    1987-05-01

    The present study was undertaken to identify the metabolic pathway(s) responsible for myocardial plasmenylcholine biosynthesis. Rabbit myocardium contained .46 +/- .09 nmol/g wet wight of 1-alkenyl-2-acyl glycerol (AAG) which predominantly consisted of 16:0 molecular species at the sn-1 position. Incubation of rabbit myocardial microsomes (RMM) with (/sup 14/C)CDP-choline (/sup 14/C-CDPC) resulted in the rapid incorporation of radiolabeled choline into the choline glycerophospholipid pool. RP-HPLC separation of molecular species demonstrated that nearly equal amounts of radiolabel were incorporated into plasmenylcholine and phosphatidylcholine subclasses despite the fact that RMM contained 21 times the mass of diacyl glycerol as compared to AAG. RMM incorporation of /sup 14/C-CDPC into choline glycerophospholipids was substantially greater than incorporation of (/sup 14/C) phosphorylcholine or (/sup 14/C) choline. RMM incorporation of /sup 14/C-CDPC into plasmalogen molecular species was stimulated two fold by 500 ..mu..M CMP. Taken together, these results demonstrate that rabbit myocardium contains substantial quantities of AAG and that endogenous AAG is an efficient precursor of myocardial plasmenylcholine.

  16. Enzymatic regioselective acylation of nucleosides in biomass-derived 2-methyltetrahydrofuran: kinetic study and enzyme substrate recognition.

    PubMed

    Gao, Wen-Li; Li, Ning; Zong, Min-Hua

    2013-03-10

    Enzymatic regioselective acylation of pyrimidine nucleosides was mediated by immobilized lipase from Penicillium expansum in 2-methyltetrahydrofuran (MeTHF), a bio-solvent derived from biomass. Despite of the moderate dissolution ability of MeTHF toward nucleosides, the initial enzymatic reaction rate was much higher in this eco-friendly solvent than in other commonly used organic solvents. This could be explained by the lower apparent activation energy of the enzymatic reaction (24.5 vs. 43.3-57.1kJ/mol) and the higher catalytic efficiency of the enzyme (Vmax/Km, 5.8 vs. 1.1-2.9h(-1)) in MeTHF. The enzymatic acylation of a group of ribonucleosides afforded the desirable 5'-esters with the conversions of 96-99% and 5'-regioselectivities of 96 to >99%. In enzymatic acylation of 2'-deoxynucleosides, however, 5'-regioselectivities showed a clear dependence on the 5-substituents present in the base moiety although the substrate conversions reached >98% within 1-3h. In the cases of 2',3'-dideoxynucleoside analogs, the reaction rate decreased markedly due to the lack of 3'-hydroxyl. PMID:23337886

  17. Fluoride inhibition of inorganic pyrophosphatase. II. Isolation and characterization of a covalent intermediate between enzyme and entire substrate molecule.

    PubMed

    Baykov, A A; Bakuleva, N P; Nazarova, T I; Avaeva, S M

    1977-03-15

    A presumed pyrophosphoryl-enzyme intermediate of the reaction catalyzed by bakers' yeast inorganic pyrophosphatase pyrophosphate phosphohydrolase, EC 3.6.1.1) has been isolated using fluoride-mediated inactivation of the enzyme during catalysis. The analysis of the F--inactivated pyrophosphatase revealed the presence of one molecule of PPi and one atom of fluoride per active site. The incubation of the inactivated enzyme at 25 degrees C and pH 7.2 resulted in gradual recovery of catalytic activity and concomitant removal of PPi by a first-order reaction with tau1/2 of 1 h. The digestion of the F--treated pyrophosphatase with pepsin yielded phosphorous-containing peptides, which were reduced with NaBH4 and gave homoserine and homoserine lactone after acid hydrolysis. This suggests that the PPi residue is linked to the protein through a bond of an acyl phosphate type involving the beta-COOH function of aspartic acid. Together with the results of the kinetic studies of fluoride inhibition of pyrophosphatase reported in accompanying papers, these findings strongly indicate that the enzyme-substrate compound stabilized by fluoride is a transient of the catalytic reaction. PMID:843537

  18. Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

    PubMed Central

    Fice, D; Shen, Z; Byers, D M

    1993-01-01

    A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism. Images PMID:8384617

  19. Characterization of a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8: a bacterial enzyme catalyzing the reduction of fatty acyl-CoA to fatty alcohol.

    PubMed

    Willis, Robert M; Wahlen, Bradley D; Seefeldt, Lance C; Barney, Brett M

    2011-12-01

    Fatty alcohols are of interest as a renewable feedstock to replace petroleum compounds used as fuels, in cosmetics, and in pharmaceuticals. One biological approach to the production of fatty alcohols involves the sequential action of two bacterial enzymes: (i) reduction of a fatty acyl-CoA to the corresponding fatty aldehyde catalyzed by a fatty acyl-CoA reductase, followed by (ii) reduction of the fatty aldehyde to the corresponding fatty alcohol catalyzed by a fatty aldehyde reductase. Here, we identify, purify, and characterize a novel bacterial enzyme from Marinobacter aquaeolei VT8 that catalyzes the reduction of fatty acyl-CoA by four electrons to the corresponding fatty alcohol, eliminating the need for a separate fatty aldehyde reductase. The enzyme is shown to reduce fatty acyl-CoAs ranging from C8:0 to C20:4 to the corresponding fatty alcohols, with the highest rate found for palmitoyl-CoA (C16:0). The dependence of the rate of reduction of palmitoyl-CoA on substrate concentration was cooperative, with an apparent K(m) ~ 4 μM, V(max) ~ 200 nmol NADP(+) min(-1) (mg protein)(-1), and n ~ 3. The enzyme also reduced a range of fatty aldehydes with decanal having the highest activity. The substrate cis-11-hexadecenal was reduced in a cooperative manner with an apparent K(m) of ~50 μM, V(max) of ~8 μmol NADP(+) min(-1) (mg protein)(-1), and n ~ 2. PMID:22035211

  20. High Level QM/MM Modeling of the Formation of the Tetrahedral Intermediate in the Acylation of Wild Type and K73A Mutant TEM-1 Class A β-Lactamase

    NASA Astrophysics Data System (ADS)

    Hermann, Johannes C.; Pradon, Juliette; Harvey, Jeremy N.; Mulholland, Adrian J.

    2009-09-01

    The breakdown of β-lactam antibiotics by β-lactamases is the most important resistance mechanism of Gram negative bacteria against these drugs. The reaction mechanism of class A β-lactamases, the most widespread family of these enzymes, consists of two main steps: acylation of an active site serine by the antibiotic, followed by deacylation and release of the cleaved compound. We have investigated the first step in acylation (the formation of the tetrahedral intermediate) for the reaction of benzylpenicillin in the TEM-1 enzyme using high level combined quantum mechanics/molecular mechanics (QM/MM) methods. Structures were optimized at the B3LYP/6-31+G(d)/CHARMM27 level, with energies for key points calculated up to the ab initio SCS-MP2/aug-cc-pVTZ/CHARMM27 level. The results support a mechanism in which Glu166 removes a proton (via an intervening water molecule) from Ser70, which in turn attacks the β-lactam of the antibiotic. Depending on the method used, the calculated barriers range from 3 to 12 kcal mol-1 for this step, consistent with experimental data. We have also modeled this reaction step in a model of the K73A mutant enzyme. The barrier to reaction in this mutant model is found to be slightly higher: the results indicate that Lys73 stabilizes the transition state, in particular deprotonated Ser70, lowering the barrier by about 1.7 kcal mol-1. This finding may help to explain the conservation of Lys73, in addition to the role we have previously found for it in the later stages of the reaction ( Hermann et al. Org. Biomol. Chem. 2006, 4, 206 - 210 ).

  1. Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis

    PubMed Central

    2011-01-01

    Background Triacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. Results We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. Conclusions In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa

  2. Hydrogen-bonding in enzyme catalysis. Fourier-transform infrared detection of ground-state electronic strain in acyl-chymotrypsins and analysis of the kinetic consequences.

    PubMed Central

    White, A J; Wharton, C W

    1990-01-01

    I.r. difference spectra are presented for 3-(indol-3-yl)acryloyl-, cinnamoyl-, 3-(5-methylthien-2-yl)acryloyl-, dehydrocinnamoyl- and dihydrocinnamoyl-chymotrypsins at low pH, where the acyl-enzymes are catalytically inactive. At least two absorption bands are seen in each case in the ester carbonyl stretching region of the spectrum. Cinnamoyl-chymotrypsin substituted at the carbonyl carbon atom with 13C was prepared. A difference spectrum in which 13C-substituted acyl-enzyme was subtracted from [12C]acyl-enzyme shows two bands in the ester carbonyl region and thus confirms the assignment of the features to the single ester carbonyl group. The frequencies of the ester carbonyl bands are interpreted in terms of differential hydrogen-bonding. In each case a lower-frequency relatively narrow band is assigned to a productive potentially reactive binding mode in which the carbonyl oxygen atom is inserted in the oxyanion hole of the enzyme active centre. The higher-frequency band, which is broader, is assigned to a non-productive binding mode in each case, where a water molecule bridges from the carbonyl oxygen atom to His-57; this mode is equivalent to the crystallographically determined structure of 3-(indol-3-yl)acryloyl-chymotrypsin, i.e. the Henderson structure. A difference spectrum of dihydrocinnamoyl-chymotrypsin taken at higher pH shows resolution of a feature centred upon 1731 cm-1, which is assigned to a non-bonded conformer in which the carbonyl oxygen atom is not hydrogen-bonded. Perturbation of the protein spectrum in the presence of acyl groups is interpreted in terms of enhanced structural rigidity. It is reported that the ester carbonyl region of the difference spectrum of cinnamoyl-subtilisin is complicated by overlap of features that arise from protein perturbation. Measurements of carbonyl absorption frequencies in a number of solvents of the methyl esters of the acyl groups used to make acyl-enzymes have permitted determination of the apparent

  3. A Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Acyl Hydrolase Activities1[W][OA

    PubMed Central

    Vijayaraj, Panneerselvam; Jashal, Charnitkaur B.; Vijayakumar, Anitha; Rani, Sapa Hima; Venkata Rao, D.K.; Rajasekharan, Ram

    2012-01-01

    Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX4D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions. PMID:22915575

  4. Quantum chemical study of penicillin: Reactions after acylation

    NASA Astrophysics Data System (ADS)

    Li, Rui; Feng, Dacheng; Zhu, Feng

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

  5. Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase.

    PubMed

    Hilbi, H; Dimroth, P

    1994-01-01

    Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na+ and less efficiently by Li+ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent Km for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent Ki of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase. PMID:18251085

  6. Genomic analysis reveals versatile organisms for quorum quenching enzymes: acyl-homoserine lactone-acylase and -lactonase.

    PubMed

    Kalia, Vipin Chandra; Raju, Sajan C; Purohit, Hemant J

    2011-01-01

    Microbial virulence and their resistance to multiple drugs have obliged researchers to look for novel drug targets. Virulence of pathogenic microbes is regulated by signal molecules such as acylated homoserine lactone (AHL) produced during a cell density dependent phenomenon of quorum sensing (QS). In contrast, certain microbes produce AHL-lactonases and -acylases to degrade QS signals, also termed as quorum quenching. Mining sequenced genome databases has revealed organisms possessing conserved domains for AHL-lactonases and -acylases: i) Streptomyces (Actinobacteria), ii) Deinococcus (Deinococcus-Thermus), iii) Hyphomonas (α-Proteobacteria), iv) Ralstonia (β-Proteobacteria), v) Photorhabdus (γ-Proteobacteria), and certain marine gamma proteobacterium. Presence of genes for both the enzymes within an organism was observed in the following: i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. These observations are supported by the presence motifs for lactonase and acylase in these strains. Phylogenetic analysis and multiple sequence alignment of the gene sequences for AHL-lactonases and -acylases have revealed consensus sequences which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Quorum quenching can be exploited to prevent food spoilage, bacterial infections and bioremediation. PMID:21660112

  7. Genomic Analysis Reveals Versatile Organisms for Quorum Quenching Enzymes: Acyl-Homoserine Lactone-Acylase and -Lactonase

    PubMed Central

    Kalia, Vipin Chandra; Raju, Sajan C; Purohit, Hemant J

    2011-01-01

    Microbial virulence and their resistance to multiple drugs have obliged researchers to look for novel drug targets. Virulence of pathogenic microbes is regulated by signal molecules such as acylated homoserine lactone (AHL) produced during a cell density dependent phenomenon of quorum sensing (QS). In contrast, certain microbes produce AHL-lactonases and -acylases to degrade QS signals, also termed as quorum quenching. Mining sequenced genome databases has revealed organisms possessing conserved domains for AHL-lactonases and –acylases: i) Streptomyces (Actinobacteria), ii) Deinococcus (Deinococcus-Thermus), iii) Hyphomonas (α-Proteobacteria), iv) Ralstonia (β-Proteobacteria), v) Photorhabdus (γ-Proteobacteria), and certain marine gamma proteobacterium. Presence of genes for both the enzymes within an organism was observed in the following: i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. These observations are supported by the presence motifs for lactonase and acylase in these strains. Phylogenetic analysis and multiple sequence alignment of the gene sequences for AHL-lactonases and –acylases have revealed consensus sequences which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Quorum quenching can be exploited to prevent food spoilage, bacterial infections and bioremediation. PMID:21660112

  8. Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate*

    PubMed Central

    Withers, Saunia; Lu, Fachuang; Kim, Hoon; Zhu, Yimin; Ralph, John; Wilkerson, Curtis G.

    2012-01-01

    Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be “monomer” precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters. PMID:22267741

  9. Electron nuclear double resonance determined structures of enzyme reaction intermediates: structural evidence for substrate destabilization

    NASA Astrophysics Data System (ADS)

    Makinen, Marvin W.

    1998-12-01

    Angle selective ENDOR of nitroxyl spin-labels is briefly reviewed to illustrate the methodology of structure analysis developed in our laboratory for characterizing catalytically competent intermediates of enzyme catalyzed reactions. ENDOR structure determination of a reaction intermediate of α-chymotrypsin formed with a kinetically specific spin-labeled substrate and of an enzyme-inhibitor complex formed with a spin-labeled transition-state inhibitor analog is briefly described. Both spin-labeled molecules bound in the active site of the enzyme are found in torsionally distorted conformations. It is suggested that this torsionally distorted state in which the bound ligand is of higher potential energy than in the ground state conformation reflects substrate destabilization in the course of the enzyme catalyzed reaction.

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

    PubMed

    Lee, Wook; Engels, Bernd

    2013-07-11

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

  11. ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro

    PubMed Central

    Lin, Chung-Chih; Love, Harold D.; Gushue, Jennifer N.; Bergeron, John J.M.; Ostermann, Joachim

    1999-01-01

    Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions. PMID:10613904

  12. Construction of a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates

    PubMed Central

    Jiang, Wei; Fang, Baishan

    2016-01-01

    Systems that can regulate and coordinate the expression of multiple enzymes for metabolic regulation and synthesis of important drug intermediates are poorly explored. In this work, a strategy for constructing a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates was developed and evaluated by connecting protein-protein expressions, regulating the strength of ribosome binding sites (RBS) and detecting the system capacity for producing chiral amino acid. Results demonstrated that the dual-enzyme system had good enantioselectivity, low cost, high stability, high conversion rate and approximately 100% substrate conversion. This study has paved a new way of exploring metabolic mechanism of functional genes and engineering whole cell-catalysts for synthesis of chiral α-hydroxy acids or chiral amino acids. PMID:27456301

  13. ''A Time-Resolved Infrared Spectroscopic Study of Reactive Acyl Intermediates Relevant to Cobalt-Catalyzed Hydroformylation''

    SciTech Connect

    Ford, Peter C.; S.M. Massick; J.Rabor; S.Elbers; J.Marhanke; S.Bernhard; J.R. Schoonover

    2000-12-31

    OAK-B135 Mechanisms of the homogeneous catalytic and photocatalytic activation of small molecules such as carbon monoxide, dihydrogen and various hydrocarbons. Time-resolved spectroscopic investigations of rhodium and iridium haol cabonyl complex intermediates proposed in methanol carbonylation catalysts.

  14. [Medium chain acyl-CoA dehydrogenase deficiency. Apropos of a case with demonstration of this enzyme deficiency].

    PubMed

    Collet, J P; Divry, P; Blanc, J F; Guibaud, P; David, M; Macabeo, V; Vibert, J; Hermier, M

    1984-12-01

    The medium chain acyl-CoA deshydrogenase defect: a new inherited metabolic disorder. This enzymatic defect blocks the catabolism of non esterified fatty acids during fasting. Thus, this disease is revealed by a coma due to hypoglycemia in a young child; the presence of dicarboxylic aciduria in such a situation is the main evidence for this diagnosis. Finally, the enzymatic studies performed on skin fibroblasts show a defect in medium chain acyl-CoA deshydrogenase. When a child is investigated away from a coma episode, the ketotic diet induces dicarboxylic aciduria but must be performed in an intensive care unit for its dangers. PMID:6535973

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

    PubMed

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

    2016-08-18

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

  16. Structure of a low-population intermediate state in the release of an enzyme product

    PubMed Central

    De Simone, Alfonso; Aprile, Francesco A; Dhulesia, Anne; Dobson, Christopher M; Vendruscolo, Michele

    2015-01-01

    Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product. DOI: http://dx.doi.org/10.7554/eLife.02777.001 PMID:25575179

  17. Reactive intermediates produced from the metabolism of the vanilloid ring of capsaicinoids by p450 enzymes.

    PubMed

    Reilly, Christopher A; Henion, Fred; Bugni, Tim S; Ethirajan, Manivannan; Stockmann, Chris; Pramanik, Kartick C; Srivastava, Sanjay K; Yost, Garold S

    2013-01-18

    This study characterized electrophilic and radical products derived from the metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from the trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5'-Dicapsaicin, presumably arising from the bimolecular coupling of free radical intermediates was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated quinone methide and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems. PMID:23088752

  18. Reactive Intermediates Produced from Metabolism of the Vanilloid Ring of Capsaicinoids by P450 Enzymes

    PubMed Central

    Reilly, Christopher A.; Henion, Fred; Bugni, Tim S.; Ethirajan, Manivannan; Stockmann, Chris; Pramanik, Kartick C.; Srivastava, Sanjay K.; Yost, Garold S.

    2012-01-01

    This study characterized electrophilic and radical products derived from metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin, were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5’-Dicapsaicin, presumably arising from bi-molecular coupling of free radical intermediates, was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated, quinone methide, and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems. PMID:23088752

  19. Acylated Carrageenan Changes the Physicochemical Properties of Mixed Enzyme-Lipid Ultrathin Films and Enhances the Catalytic Properties of Sucrose Phosphorylase Nanostructured as Smart Surfaces.

    PubMed

    Rocha, Jefferson M; Pavinatto, Adriana; Nobre, Thatyane M; Caseli, Luciano

    2016-06-23

    Control over the catalytic activity of enzymes is important to construct biosensors with a wide range of detectability and higher stability. For this, immobilization of enzymes on solid supports as nanostructured films is a current approach that permits easy control of the molecular architecture as well as tuning of the properties. In this article, we employed acylated carrageenan (AC) mixed with phospholipids at the air-water interface to facilitate the adsorption of the enzyme sucrose phosphorylase (SP). AC stabilized the adsorption of SP at the phospholipid monolayer, as detected by tensiometry, by which thermodynamic parameters could be inferred from the surface pressure-area isotherm. Also, infrared spectroscopy applied in situ over the monolayer showed that the AC-phospholipid system not only permitted the enzyme to be adsorbed but also helped conserve its secondary structure. The mixed monolayers were then transferred onto solid supports as Langmuir-Blodgett (LB) films and investigated with transfer ratio, quartz crystal microbalance, fluorescence spectroscopy, and atomic force microscopy. The enzyme activity of the LB film was then determined, revealing that although there was an expected reduction in activity in relation to the homogeneous environment the activity could be better preserved after 1 month, revealing enhanced stability. PMID:27249064

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

    SciTech Connect

    Chen, H.H.

    1989-01-01

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

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

    PubMed

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

    2012-08-01

    The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids. PMID:22353623

  2. SIRT3 and SIRT5 Regulate the Enzyme Activity and Cardiolipin Binding of Very Long-Chain Acyl-CoA Dehydrogenase

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  6. Copper(II)-catalyzed room temperature aerobic oxidation of hydroxamic acids and hydrazides to acyl-nitroso and azo intermediates, and their Diels-Alder trapping.

    PubMed

    Chaiyaveij, Duangduan; Cleary, Leah; Batsanov, Andrei S; Marder, Todd B; Shea, Kenneth J; Whiting, Andrew

    2011-07-01

    CuCl(2), in the presence of a 2-ethyl-2-oxazoline ligand, is an effective catalyst for the room temperature, aerobic oxidation of hydroxamic acids and hydrazides, to acyl-nitroso and azo dienophiles respectively, which are efficiently trapped in situ via both inter- and intramolecular hetero-Diels-Alder reactions with dienes. Both inter- and intramolecular variants of the Diels-Alder reaction are suitable under the reaction conditions using a variety of solvents. Under the same conditions, an acyl hydrazide was also oxidized to give an acyl-azo dienophile which was trapped intramolecularly by a diene. PMID:21644530

  7. Enzymatic acylation of starch.

    PubMed

    Alissandratos, Apostolos; Halling, Peter J

    2012-07-01

    Starch a cheap, abundant and renewable natural material has been chemically modified for many years. The popular modification acylation has been used to adjust rheological properties as well as deliver polymers with internal plasticizers and other potential uses. However the harsh reaction conditions required to produce these esters may limit their use, especially in sensitive applications (foods, pharmaceuticals, etc.). The use of enzymes to catalyse acylation may provide a suitable alternative due to high selectivities and mild reaction conditions. Traditional hydrolase-catalysed synthesis in non-aqueous apolar media is hard due to lack of polysaccharide solubility. However, acylated starch derivatives have recently been successfully produced in other non-conventional systems: (a) surfactant-solubilised subtilisin and suspended amylose in organic media; (b) starch nanoparticles dispersed in organic medium with immobilised lipase; (c) aqueous starch gels with lipase and dispersed fatty acids. We attempt a systematic review that draws parallels between the seemingly unrelated approaches described. PMID:22138593

  8. Structures of the Michaelis Complex (1.2A) and the Covalent Acyl Intermediate (2.0A ) of Cefamandole Bound in the Active Sites of the Mycobacterium tuberculosis beta-Lactamase K72A and E166A Mutants

    SciTech Connect

    L Tremblay; h Xu; J Blanchard

    2011-12-31

    The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active {beta}-lactamase, BlaC, that imparts TB with resistance to {beta}-lactam chemotherapy. The structure of covalent BlaC-{beta}-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 {angstrom}, respectively. These structures provide insight into the details of the catalytic mechanism.

  9. Thermal fragmentation of acyl thiolato complexes to reactive metal sulfido intermediates. Structure of Ru([eta][sup 6]-SC[sub 3]Me[sub 3]COMe)(PPh[sub 3])[sub 2

    SciTech Connect

    Feng, Q.; Krautscheid, H.; Rauchfuss, T.B.; Skaugset, A.E.; Venturelli, A. )

    1995-01-01

    In this study we have examined the thermal fragmentation of acyl thiolate complexes of the type (ring)M(SC[sub 3]Me[sub 3]COMe). While the (cymene)Ru derivative only gave insoluble materials, the thermolysis of the corresponding osmium compound, (cymene)Os(SC[sub 3]Me[sub 3]COMe) (2), proved very informative. Here it was established that the tetramethylfuran is indeed released prior to formation of the free cymene. Furthermore, two intermediates were observed, the second of which was identified as (cymene)[sub 3]Os[sub 3]S[sub 2]. This species is more stable with respect to loss of cymene than the corresponding (cymene)[sub 3]Ru[sub 3]S[sub 2]. Attempts were made to intercept intermediates in these fragmentation processes by employing PPh[sub 3] as a trapping agent. For the Ru case, PPh[sub 3] completely changed the course of the reaction and no tetramethylfuran was generated. Instead we obtained the structurally unusual [eta][sup 6]-acyl thiolate complex (5). We propose that this unimolecular step involves the formation of ([eta][sup 4]-cymene)Ru([eta][sup 6]-SC[sub 3]Me[sub 3]COMe). The forward rate for this equilibration is far slower than the rate of isomerization of the acyl thiolate and far faster than that for the unassisted thermal decomposition of (cymene)Ru(SC[sub 3]MesCOMe). This result leads to a more global assessment of the reactivity of the acyl thiolates: the orientation of the carbonyl center determines the decomposition pathway. 21 refs., 7 figs., 1 tab.

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

    PubMed

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

    2015-10-30

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

  11. DHHC Protein S-Acyltransferases Use Similar Ping-Pong Kinetic Mechanisms but Display Different Acyl-CoA Specificities*

    PubMed Central

    Jennings, Benjamin C.; Linder, Maurine E.

    2012-01-01

    DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of Vmax and Km values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells. PMID:22247542

  12. Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.

    PubMed

    Machutta, Carl A; Bommineni, Gopal R; Luckner, Sylvia R; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2010-02-26

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton. PMID:20018879

  13. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaene variabilis

    SciTech Connect

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

    1987-04-01

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

  14. Intermediate divergence levels maximize the strength of structure-sequence correlations in enzymes and viral proteins.

    PubMed

    Jackson, Eleisha L; Shahmoradi, Amir; Spielman, Stephanie J; Jack, Benjamin R; Wilke, Claus O

    2016-07-01

    Structural properties such as solvent accessibility and contact number predict site-specific sequence variability in many proteins. However, the strength and significance of these structure-sequence relationships vary widely among different proteins, with absolute correlation strengths ranging from 0 to 0.8. In particular, two recent works have made contradictory observations. Yeh et al. (Mol. Biol. Evol. 31:135-139, 2014) found that both relative solvent accessibility (RSA) and weighted contact number (WCN) are good predictors of sitewise evolutionary rate in enzymes, with WCN clearly out-performing RSA. Shahmoradi et al. (J. Mol. Evol. 79:130-142, 2014) considered these same predictors (as well as others) in viral proteins and found much weaker correlations and no clear advantage of WCN over RSA. Because these two studies had substantial methodological differences, however, a direct comparison of their results is not possible. Here, we reanalyze the datasets of the two studies with one uniform analysis pipeline, and we find that many apparent discrepancies between the two analyses can be attributed to the extent of sequence divergence in individual alignments. Specifically, the alignments of the enzyme dataset are much more diverged than those of the virus dataset, and proteins with higher divergence exhibit, on average, stronger structure-sequence correlations. However, the highest structure-sequence correlations are observed at intermediate divergence levels, where both highly conserved and highly variable sites are present in the same alignment. PMID:26971720

  15. Biological channeling of a reactive intermediate in the bifunctional enzyme DmpFG.

    PubMed

    Smith, Natalie E; Vrielink, Alice; Attwood, Paul V; Corry, Ben

    2012-02-22

    It has been hypothesized that the bifunctional enzyme DmpFG channels its intermediate, acetaldehyde, from one active site to the next using a buried intermolecular channel identified in the crystal structure. This channel appears to switch between an open and a closed conformation depending on whether the coenzyme NAD(+) is present or absent. Here, we applied molecular dynamics and metadynamics to investigate channeling within DmpFG in both the presence and absence of NAD(+). We found that substrate channeling within this enzyme is energetically feasible in the presence of NAD(+) but was less likely in its absence. Tyr-291, a proposed control point at the channel's entry, does not appear to function as a molecular gate. Instead, it is thought to orientate the substrate 4-hydroxy-2-ketovalerate in DmpG before reaction occurs, and may function as a proton shuttle for the DmpG reaction. Three hydrophobic residues at the channel's exit appear to have an important role in controlling the entry of acetaldehyde into the DmpF active site. PMID:22385858

  16. Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes.

    PubMed

    Jiang, Yongying; He, Xiang; Ortiz de Montellano, Paul R

    2006-01-17

    Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent. PMID:16401082

  17. Acyl-lipid metabolism.

    PubMed

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

    2013-01-01

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

  18. Acyl-lipid metabolism.

    PubMed

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

    2010-01-01

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

  19. Acyl-Lipid Metabolism

    PubMed Central

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

    2010-01-01

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

  20. Acyl-Lipid Metabolism

    PubMed Central

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

    2013-01-01

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

  1. Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase.

    PubMed

    Wu, Jiaquan; Kinoshita, Kenji; Khosla, Chaitan; Cane, David E

    2004-12-28

    The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead

  2. Human α/β hydrolase domain containing 10 (ABHD10) is responsible enzyme for deglucuronidation of mycophenolic acid acyl-glucuronide in liver.

    PubMed

    Iwamura, Atsushi; Fukami, Tatsuki; Higuchi, Ryota; Nakajima, Miki; Yokoi, Tsuyoshi

    2012-03-16

    Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is primarily metabolized by glucuronidation to a phenolic glucuronide (MPAG) and an acyl glucuronide (AcMPAG). It is known that AcMPAG, which may be an immunotoxic metabolite, is deglucuronidated in human liver. However, it has been reported that recombinant β-glucuronidase does not catalyze this reaction. AcMPAG deglucuronidation activity was detected in both human liver cytosol (HLC) and microsomes (HLM). In this study, the enzyme responsible for AcMPAG deglucuronidation was identified by purification from HLC with column chromatographic purification steps. The purified enzyme was identified as α/β hydrolase domain containing 10 (ABHD10) by amino acid sequence analysis. Recombinant ABHD10 expressed in Sf9 cells efficiently deglucuronidated AcMPAG with a K(m) value of 100.7 ± 10.2 μM, which was similar to those in HLM, HLC, and human liver homogenates (HLH). Immunoblot analysis revealed ABHD10 protein expression in both HLC and HLM. The AcMPAG deglucuronidation by recombinant ABHD10, HLC, and HLH were potently inhibited by AgNO(3), CdCl(2), CuCl(2), PMSF, bis-p-nitrophenylphosphate, and DTNB. The CL(int) value of AcMPAG formation from MPA, which was catalyzed by human UGT2B7, in HLH was increased by 1.8-fold in the presence of PMSF. Thus, human ABHD10 would affect the formation of AcMPAG, the immunotoxic metabolite. PMID:22294686

  3. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean.

    PubMed

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants' pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  4. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean

    PubMed Central

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants’ pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  5. Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase.

    PubMed

    Fitzpatrick, Paul F; Orville, Allen M; Nagpal, Akanksha; Valley, Michael P

    2005-01-01

    While several flavoproteins will oxidize nitroalkanes in addition to their physiological substrates, nitroalkane oxidase (NAO) is the only one which does not require the anionic nitroalkane. This, in addition to the induction of NAO by nitroethane seen in Fusarium oxysporum, suggests that oxidation of a nitroaliphatic species is the physiological role of the enzyme. Mechanistic studies of the reaction with nitroethane as substrate have established many of the details of the enzymatic reaction. The enzyme is unique in being the only flavoprotein to date for which a carbanion is definitively established as an intermediate in catalysis. Recent structural analyses show that NAO is homologous to the acyl-CoA dehydrogenase and acyl-CoA oxidase families of enzymes. In NAO, the glutamate which acts as the active site base in the latter enzymes is replaced by an aspartate. PMID:15581574

  6. The Physiology of Protein S-acylation

    PubMed Central

    Chamberlain, Luke H.; Shipston, Michael J.

    2015-01-01

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

  7. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

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

  8. LuxR homolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa.

    PubMed

    Chugani, Sudha; Greenberg, Everett Peter

    2010-06-01

    Pseudomonas aeruginosa quorum control of gene expression involves three LuxR-type signal receptors LasR, RhlR, and QscR that respond to the LasI- and RhlI-generated acyl-homoserine lactone (acyl-HSL) signals 3OC12-HSL and C4-HSL. We found that a LasR-RhlR-QscR triple mutant responds to acyl-HSLs by regulating at least 37 genes. LuxR homolog-independent activation of the representative genes antA and catB also occurs in the wild type. Expression of antA was influenced the most by C10-HSL and to a lesser extent by other acyl-HSLs, including the P. aeruginosa 3OC12-HSL and C4-HSL signals. The ant and cat operons encode enzymes for the degradation of anthranilate to tricarboxylic acid cycle intermediates. Our results indicate that LuxR homolog-independent acyl-HSL control of the ant and cat operons occurs via regulation of antR, which codes for the transcriptional activator of the ant operon. Although P. aeruginosa has multiple pathways for anthranilate synthesis, one pathway-the kynurenine pathway for tryptophan degradation-is required for acyl-HSL activation of the ant operon. The kynurenine pathway is also the critical source of anthranilate for energy metabolism via the antABC gene products, as well as the source of anthranilate for synthesis of the P. aeruginosa quinolone signal. Our discovery of LuxR homolog-independent responses to acyl-HSLs provides insight into acyl-HSL signaling. PMID:20498077

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

    PubMed

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

    2015-11-01

    Acyl-CoA mutases are a growing class of adenosylcobalamin-dependent radical enzymes that perform challenging carbon skeleton rearrangements in primary and secondary metabolism. Members of this class of enzymes must precisely control substrate positioning to prevent oxidative interception of radical intermediates during catalysis. Our understanding of substrate specificity and catalysis in acyl-CoA mutases, however, is incomplete. Here, we present crystal structures of IcmF, a natural fusion protein variant of isobutyryl-CoA mutase, in complex with the adenosylcobalamin cofactor and four different acyl-CoA substrates. These structures demonstrate how the active site is designed to accommodate the aliphatic acyl chains of each substrate. The structures suggest that a conformational change of the 5'-deoxyadenosyl group from C2'-endo to C3'-endo could contribute to initiation of catalysis. Furthermore, detailed bioinformatic analyses guided by our structural findings identify critical determinants of acyl-CoA mutase substrate specificity and predict new acyl-CoA mutase-catalyzed reactions. These results expand our understanding of the substrate specificity and the catalytic scope of acyl-CoA mutases and could benefit engineering efforts for biotechnological applications ranging from production of biofuels and commercial products to hydrocarbon remediation. PMID:26318610

  10. Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.

    PubMed

    Shen, Z; Fice, D; Byers, D M

    1992-07-01

    A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis. PMID:1514693

  11. Lipid Acyl Chain Remodeling in Yeast

    PubMed Central

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

    2015-01-01

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

  12. QM/MM studies of monozinc β-lactamase CphA suggest that the crystal structure of an enzyme-intermediate complex represents a minor pathway.

    PubMed

    Wu, Shanshan; Xu, Dingguo; Guo, Hua

    2010-12-29

    QM/MM studies of the hydrolysis of a β-lactam antibiotic molecule (biapenem) catalyzed by a monozinc β-lactamase (CphA) have revealed the complete reaction mechanism and shown that an experimentally determined enzyme-intermediate complex is a stable intermediate or product in a minor pathway. PMID:21138257

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    ERIC Educational Resources Information Center

    Thomas, Jacob

    2009-01-01

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

  16. Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase.

    PubMed

    Ingram-Smith, Cheryl; Woods, Barrett I; Smith, Kerry S

    2006-09-26

    AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site. PMID:16981708

  17. 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

    PubMed Central

    Jiang, Yanfang; Morgan-Kiss, Rachael M.; Campbell, John W.; Chan, Chi Ho; Cronan, John E.

    2010-01-01

    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. PMID:20028080

  18. Formation of a square-planar Co(I) B12 intermediate. Implications for enzyme catalysis.

    PubMed Central

    Wirt, M D; Sagi, I; Chance, M R

    1992-01-01

    X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond. PMID:1420887

  19. Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate.

    PubMed Central

    Connelly, G. P.; Withers, S. G.; McIntosh, L. P.

    2000-01-01

    NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements. PMID:10752613

  20. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  1. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

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

    2009-01-01

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

  2. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate

    PubMed Central

    Baumann, Anna-Maria T.; Bakkers, Mark J. G.; Buettner, Falk F. R.; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A.; de Groot, Raoul J.; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  3. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

    PubMed

    Baumann, Anna-Maria T; Bakkers, Mark J G; Buettner, Falk F R; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A; de Groot, Raoul J; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  4. LPS impairs phospholipid synthesis by triggering beta-transducin repeat-containing protein (beta-TrCP)-mediated polyubiquitination and degradation of the surfactant enzyme acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1).

    PubMed

    Zou, Chunbin; Butler, Phillip L; Coon, Tiffany A; Smith, Rebecca M; Hammen, Gary; Zhao, Yutong; Chen, Bill B; Mallampalli, Rama K

    2011-01-28

    Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβ(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis. PMID:21068446

  5. Involvement of Two Cytosolic Enzymes and a Novel Intermediate, 5′-Oxoaverantin, in the Pathway from 5′-Hydroxyaverantin to Averufin in Aflatoxin Biosynthesis

    PubMed Central

    Sakuno, Emi; Yabe, Kimiko; Nakajima, Hiromitsu

    2003-01-01

    During aflatoxin biosynthesis, 5′-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5′-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed. PMID:14602595

  6. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

    PubMed Central

    Radisky, Evette S.; Lee, Justin M.; Lu, Chia-Jung Karen; Koshland, Daniel E.

    2006-01-01

    Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction. PMID:16636277

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

    PubMed

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

    2015-05-19

    Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long chain deacylation, in addition to the well-known NAD(+)-dependent deacetylation activity [Feldman, J. L., Baeza, J., and Denu, J. M. (2013) J. Biol. Chem. 288, 31350-31356]. Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD(+)-binding and acyl-group selectivity for a diverse series of human Sirtuins, SIRT1-SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD(+) saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl substrate chain length. Though the rate of nucleophilic attack of the 2'-hydroxyl on the C1'-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however, for SIRT6, this step is no longer rate-limiting for long chain substrates. Cocrystallization of SIRT2 with myristoylated peptide and NAD(+) yielded a co-complex structure with reaction product 2'-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately determined co-complex structures containing either a myristoylated peptide or 2'-O-myristoyl-ADP-ribose indicates there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl substrate preferences and how different reaction kinetics influence NAD(+) dependence. The biological implications are discussed. PMID:25897714

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2014-03-01

    Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification. PMID:24467666

  10. A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

    PubMed

    Hayashi-Iwasaki, Y; Numata, K; Yamagishi, A; Yutani, K; Sakurai, M; Tanaka, N; Oshima, T

    1996-03-01

    The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface. PMID:8868488

  11. 3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

    PubMed Central

    Tai, H; Jaworski, J G

    1993-01-01

    A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals. PMID:8290632

  12. LIGNIN ACYLATION IN GRASSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acylation of lignin during growth and development is a commonly found among some plant species. Grasses form unique acylated lignins involving p-coumarate (pCA). In corn rind tissue, it is exclusively attached to the gamma-carbon of lignin monomers, with a strong preference (over 90%) for attachment...

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

    PubMed

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

    2012-02-01

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

  14. Oxidative acylation using thioacids

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.

    1997-01-01

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

  15. Comparison of pH-dependent allostery and dissociation for phosphofructokinases from Artemia embryos and rabbit muscle: nature of the enzymes acylated with diethylpyrocarbonate.

    PubMed

    Carpenter, J F; Hand, S C

    1986-07-01

    Purified Artemia phosphofructokinase (PFK), unlike the rabbit skeletal muscle enzyme, displays allosteric kinetics at pH 8, a feature that is functionally significant since the intracellular pH of the developing brine shrimp embryo is greater than or equal to 7.9. Catalytic activity of the Artemia enzyme is severely suppressed by acidic pH even when assayed at the adenylate nucleotide concentrations existing in anaerobic embryos, which is consistent with the lack of a Pasteur effect in these organisms. For both PFK homologs, carbethoxylation reduces the sensitivity to ATP and citrate inhibition, the cooperativity as a function of fructose 6-phosphate concentration and the degree of activation in the presence ADP, AMP, and fructose 2,6-bisphosphate. Considering the role of histidine protonation in PFK allosteric control, the capacity for regulatory kinetics seen at pH 8 in the Artemia enzyme could be explained in part by upward shifts in pKa values of ionizable residues. pH-induced dissociation of tetrameric Artemia PFK into inactive subunits does not occur during catalytic inhibition at acidic pH (pH 6.5, 6 degrees C), as judged by 90 degree light scattering. This observation contrasts markedly with the dimerization and inactivation of rabbit PFK, but is shown not to be unique when compared to other selected PFK homologs. Neither the acute pH sensitivity of Artemia PFK nor the pH-induced hysteretic inactivation displayed by the rabbit enzyme are altered by carbethoxylation, suggesting that ionizable residues involved in these two processes are not the same ones involved in allosteric kinetics. PMID:2942107

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

    PubMed

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

    2006-03-01

    Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation. PMID:16466685

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

    SciTech Connect

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

    2011-12-31

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

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

    SciTech Connect

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

    2011-02-18

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

  19. Direct Acylation of Carrier Proteins with Functionalized β-Lactones

    PubMed Central

    Amoroso, Jon W.; Borketey, Lawrence S.; Prasad, Gitanjeli

    2014-01-01

    As the key component of many biosynthetic assemblies, acyl-carrier proteins offer a robust entry point for introduction of small molecule probes and pathway intermediates. Current labeling strategies primarily rely on modifications to the phosphopantetheine cofactor or its biosynthetic precursors followed by attachment to the apo form of a given carrier protein. As a greatly simplified alternative, direct and selective acylation of holo-acyl-carrier proteins using readily accessible β-lactones as electrophilic partners for the phosphopantetheine-thiol has been demonstrated. PMID:20433156

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

    SciTech Connect

    Not Available

    1990-01-01

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

  1. Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates*

    PubMed Central

    Egger, Sigrid; Chaikuad, Apirat; Klimacek, Mario; Kavanagh, Kathryn L.; Oppermann, Udo; Nidetzky, Bernd

    2012-01-01

    Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD+-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu161) by an incompetent analog (Gln161) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD+, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys276 was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD+. The proposed catalytic mechanism of human UGDH involves Lys220 as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp280 deprotonates Cys276 to function as an aldehyde trap and also provides oxyanion stabilization. Glu161 is the Brønsted base catalytically promoting the thioester hydrolysis. PMID:22123821

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

    PubMed Central

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-01-01

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

  3. "One-Pot" Approach to 8-Acylated 2-Quinolinones via Palladium-Catalyzed Regioselective Acylation of Quinoline N-Oxides.

    PubMed

    Chen, Xiaopei; Cui, Xiuling; Wu, Yangjie

    2016-05-20

    A "one-pot" facile and efficient protocol for 8-acylated 2-quinolinones has been developed through palladium-catalyzed acylation of quinoline N-oxides, which proceeds with high selectivity at the C8-position. The desired products were isolated in up to 95% yield and good functional group tolerance. A palladacycle was isolated from the catalytic process and proposed as a key intermediate. PMID:27153298

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

    PubMed Central

    2011-01-01

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

  5. A method for the determination of the hepatic enzyme activity catalyzing bile acid acyl glucuronide formation by high-performance liquid chromatography with pulsed amperometric detection.

    PubMed

    Ikegawa, S; Oohashi, J; Murao, N; Goto, J

    2000-05-01

    A method for the determination of the activity of hepatic glucuronyltransferase catalyzing formation of bile acid 24-glucuronides using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) has been developed. Bile acid 24-glucuronides were simultaneously separated on a semimicrobore column, Capcell Pak C18UG120, using 20 mM ammonium phosphate (pH 6.0)-acetonitrile (27:10 and 16:10) as the mobile phase in the stepwise gradient elution mode. A 1 M potassium hydroxide solution for the hydrolysis of the 24-glucuronides, which liberates the corresponding bile acids and glucuronic acid, was mixed with the mobile phase in a post-column mode, and the resulting eluant was heated at 90 degrees C, the 24-glucuronides being monitored using a pulsed amperometric detector; the limit of detection was 10 ng. The proposed method was applied to the determination of the hepatic enzyme activity catalyzing bile acid 24-glucuronide formation and the result exhibited the efficient 24-glucuronide formation of the monohydroxylated bile acid, lithocholic acid. PMID:10850616

  6. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  7. Structural Insight into How Streptomyces coelicolor Maltosyl Transferase GlgE Binds α-Maltose 1-Phosphate and Forms a Maltosyl-enzyme Intermediate

    PubMed Central

    2014-01-01

    GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure–function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme. PMID:24689960

  8. Progress in the Experimental Observation of Thiamin Diphosphate-bound Intermediates on Enzymes and Mechanistic Information Derived from these Observations

    PubMed Central

    Jordan, Frank; Nemeria, Natalia S.

    2014-01-01

    Thiamin diphosphate (ThDP), the vitamin B1 coenzyme is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/carbene/C2-carbanion of the thiazolium ring and the C2α-carbanion/enamine, once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, solid-state NMR and circular dichroism detection of the states of ionization and tautomerization of the 4′-aminopyrimidine moiety of ThDP in some of the intermediates. These methods also enabled in some cases determination of the rate-limiting step in the complex series of steps. This review is an update of a review with the same title published by the authors in 2005 in this Journal. Much progress has been made in the intervening decade in the identification of the intermediates and their application to gain additional mechanistic insight. PMID:15888311

  9. Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues.

    PubMed

    Manaf, A M; Harwood, J L

    2000-01-01

    Glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.15) catalyses the first step of the Kennedy pathway for acyl lipid formation. This enzyme was studied using high-speed particulate fractions from oil palm (Elaeis guineensis Jacq.) tissue cultures and mesocarp acetone powders. The fractions were incubated with [(14)C]glycerol 3-phosphate and incorporation of radioactivity into Kennedy pathway intermediates studied. Optimal conditions were broadly similar between the two preparations but those from fruit mesocarp clearly contained more active enzymes for the subsequent stages of the Kennedy pathway - as exemplified by the appreciable accumulation of radioactivity in triacylglycerol. Experiments with different acyl-CoA substrates showed that the GPAT in both high-speed particulate preparations had a significant preference for palmitate. Glycerol 3-phosphate acyltransferase was solubilised from both preparations with optimal solubilisation being achieved at 0.5% (w/v) CHAPS concentrations. Solubilised GPATs were purified further using DE52 ion-exchange chromatography and Sephadex G-100 molecular exclusion chromatography. Purifications of up to about 70-fold were achieved. The purified GPATs showed a strong preference for palmitoyl-CoA compared to other acyl-CoA donors, in keeping with the importance of palmitate in palm oil. PMID:10664139

  10. A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

    PubMed Central

    Doherty, F J; Wassell, J A; Mayer, R J

    1987-01-01

    Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy. Images Fig. 1. Fig. 5. PMID:3593223

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

    PubMed Central

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

    2002-01-01

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

  12. Spectroscopic and Kinetic Characterization of Peroxidase-Like π-Cation Radical Pinch-Porphyrin-Iron(III) Reaction Intermediate Models of Peroxidase Enzymes.

    PubMed

    Hernández Anzaldo, Samuel; Arroyo Abad, Uriel; León García, Armando; Ramírez Rosales, Daniel; Zamorano Ulloa, Rafael; Reyes Ortega, Yasmi

    2016-01-01

    The spectroscopic and kinetic characterization of two intermediates from the H₂O₂ oxidation of three dimethyl ester [(proto), (meso), (deuteroporphyrinato) (picdien)]Fe(III) complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively) pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III) quantum mixed spin (qms) ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1-3 + guaiacol + H₂O₂ → oxidation guaiacol products). The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III) and H₂O₂, resulting in only two types of kinetics that were developed during the first 0-4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III) family with the ligand picdien [N,N'-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, ¹H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity. PMID:27355940

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

    SciTech Connect

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

    2010-03-05

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

  14. The structure of putative N-acetyl glutamate kinase from Thermus thermophilus reveals an intermediate active site conformation of the enzyme.

    PubMed

    Sundaresan, Ramya; Ragunathan, Preethi; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2012-04-13

    The de novo biosynthesis of arginine in microorganisms and plants is accomplished via several enzymatic steps. The enzyme N-acetyl glutamate kinase (NAGK) catalyzes the phosphorylation of the γ-COO(-) group of N-acetyl-L-glutamate (NAG) by adenosine triphosphate (ATP) which is the second rate limiting step in arginine biosynthesis pathway. Here we report the crystal structure of putative N-acetyl glutamate kinase (NAGK) from Thermus thermophilus HB8 (TtNAGK) determined at 1.92Å resolution. The structural analysis of TtNAGK suggests that the dimeric quaternary state of the enzyme and arginine insensitive nature are similar to mesophilic Escherichia coli NAGK. These features are significantly different from its thermophilic homolog Thermatoga maritima NAGK which is hexameric and arginine-sensitive. TtNAGK is devoid of its substrates but contains two sulfates at the active site. Very interestingly the active site of the enzyme adopts a conformation which is not completely open or closed and likely represents an intermediate stage in the catalytic cycle unlike its structural homologs, which all exist either in the open or closed conformation. Engineering arginine biosynthesis pathway enzymes for the production of l-arginine is an important industrial application. The structural comparison of TtNAGK with EcNAGK revealed the structural basis of thermostability of TtNAGK and this information could be very useful to generate mutants of NAGK with increased overall stability. PMID:22452987

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

    PubMed Central

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

    2006-01-01

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

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

    SciTech Connect

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

    1985-09-01

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

  17. Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

    PubMed

    Bui, Soi; von Stetten, David; Jambrina, Pablo G; Prangé, Thierry; Colloc'h, Nathalie; de Sanctis, Daniele; Royant, Antoine; Rosta, Edina; Steiner, Roberto A

    2014-12-01

    Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5-OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site. PMID:25314114

  18. Anaerobic Toluene Catabolism of Thauera aromatica: the bbs Operon Codes for Enzymes of β Oxidation of the Intermediate Benzylsuccinate

    PubMed Central

    Leuthner, Birgitta; Heider, Johann

    2000-01-01

    The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent β-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in β oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the β-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp. PMID:10629170

  19. Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation.

    PubMed

    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. PMID:20382402

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

    PubMed Central

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

    2013-01-01

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

  1. Intermediate Energy Metabolism of Leptospira

    PubMed Central

    Baseman, J. B.; Cox, C. D.

    1969-01-01

    Metabolic studies were performed on three representative serotypes of Leptospira: a water isolate designated B16 and two pathogenic serotypes, pomona and schueffneri. Examination of whole cells of B16 for their ability to oxidize various substrates revealed that oleate significantly stimulated oxygen uptake. The respiratory quotient of 0.7 implied that oleate was degraded to carbon dioxide and water. Other substrates, such as carbohydrates, alcohols, intermediates of the citric acid cycle, and short-chain acids, including selected amino acids, did not stimulate endogenous respiration of whole cells. No oxygen uptake could be measured when cell-free extracts were tested with the substrates used with whole cells. Enzymatic analyses of cell-free extracts of the three strains demonstrated enzymes of the citric acid cycle, enzymes of the glycolytic and pentose pathways, and the general acyl coenzyme A dehydrogenase required for β-oxidation of fatty acids. Strain B16 and the two pathogenic serotypes appeared to possess similar metabolic capabilities. Enzymatic data might also explain the apparent inability of B16 to oxidize other substrates; kinases necessary for activation of common nonphosphorylated compounds were not detected in leptospiral extracts. These findings emphasized the dependence of leptospiral growth upon long-chain fatty acids. PMID:5776541

  2. Characterization of C₃--C₄ intermediate species in the genus Heliotropium L. (Boraginaceae): anatomy, ultrastructure and enzyme activity.

    PubMed

    Muhaidat, Riyadh; Sage, Tammy L; Frohlich, Michael W; Dengler, Nancy G; Sage, Rowan F

    2011-10-01

    Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C₄ acid decarboxylases, and immunolocalization of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and the P-subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C₃ plants, while Heliotropium texanum and Heliotropium polyphyllum are C₄ species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C₃, but exhibit 'proto-Kranz' anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C₃--C₄ intermediates, with Kranz-like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C₃--C₄ species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C₄ evolution. Where existing models such as Flaveria emphasize diversification of C₃--C₄ intermediates, Heliotropium has numerous C₃ species expressing proto-Kranz traits that could represent a critical initial phase in the evolutionary origin of C₄ photosynthesis. PMID:21631534

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2011-01-01

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

  5. The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡

    PubMed Central

    Shah, Manish B.; Ingram-Smith, Cheryl; Cooper, Leroy L.; Qu, Jun; Meng, Yu; Smith, Kerry S.; Gulick, Andrew M.

    2009-01-01

    The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket. PMID:19544569

  6. Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

    DOE PAGESBeta

    Poust, Sean; Yoon, Isu; Adams, Paul D.; Katz, Leonard; Petzold, Christopher J.; Keasling, Jay D.

    2014-10-06

    Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-likemore » subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.« less

  7. Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

    SciTech Connect

    Poust, Sean; Yoon, Isu; Adams, Paul D.; Katz, Leonard; Petzold, Christopher J.; Keasling, Jay D.

    2014-10-06

    Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.

  8. Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

    PubMed

    Peshenko, Igor V; Olshevskaya, Elena V; Lim, Sunghyuk; Ames, James B; Dizhoor, Alexander M

    2012-04-20

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity. PMID:22383530

  9. Interactions of the acyl chain with the Saccharomyces cerevisiae acyl carrier protein.

    PubMed

    Perez, Daniel R; Leibundgut, Marc; Wider, Gerhard

    2015-04-01

    Acyl carrier protein (ACP) domains are critical integral components of multifunctional type I fatty acid synthases (FAS I) and polyketide synthases (PKSs), where they shuttle the growing adducts of the synthesis between the catalytic domains. In contrast to ACP of mammalian FAS I, PKSs, and the dissociated fatty acid synthase type II systems (FAS II) of bacteria, fungal FAS I ACP consists of two subdomains, one comprising the canonical ACP fold observed in all FAS systems and the other representing an extra structural subdomain. While ACPs of dissociated FAS II are able to sequester the reaction intermediates during substrate shuttling, such a transport mechanism has not been observed in ACP domains of multifunctional FAS I and PKS systems. For a better understanding of the interaction between the canonical subdomain of fungal ACP with the growing acyl chain and the role of the structural subdomain, we determined the structure of the isolated Saccharomyces cerevisiae acyl carrier protein (ScACP) domain by NMR spectroscopy and investigated the interactions between ScACP and covalently attached substrate acyl chains of varying length by monitoring chemical shift perturbations. The interactions were mapped to the hydrophobic core of the canonical subdomain, while no perturbations were detected in the structural subdomain. A population analysis revealed that only approximately 15% of covalently attached decanoyl chains are sequestered by the ACP core, comparable to the mammalian FAS I and multifunctional PKS systems, which do not sequester their substrates. Finally, denaturation experiments show that both ScACP subdomains unfold cooperatively and that the weak interaction of the acyl chain with the hydrophobic core does not significantly affect the ACP stability. PMID:25774789

  10. INTERMEDIATE CLEAVAGE PEPTIDASE55 Modifies Enzyme Amino Termini and Alters Protein Stability in Arabidopsis Mitochondria1[OPEN

    PubMed Central

    Huang, Shaobai; Nelson, Clark J.; Li, Lei; Taylor, Nicolas L.; Ströher, Elke; Petereit, Jakob; Millar, A. Harvey

    2015-01-01

    Precursor proteins containing mitochondrial peptide signals are cleaved after import by a mitochondrial processing peptidase. In yeast (Saccharomyces cerevisiae) and human (Homo sapiens), INTERMEDIATE CLEAVAGE PEPTIDASE55 (ICP55) plays a role in stabilizing mitochondrial proteins by the removal of single amino acids from mitochondrial processing peptidase-processed proteins. We have investigated the role of a metallopeptidase (At1g09300) from Arabidopsis (Arabidopsis thaliana) that has sequence similarity to yeast ICP55. We identified this protein in mitochondria by mass spectrometry and have studied its function in a transfer DNA insertion line (icp55). Monitoring of amino-terminal peptides showed that Arabidopsis ICP55 was responsible for the removal of single amino acids, and its action explained the −3 arginine processing motif of a number of mitochondrial proteins. ICP55 also removed single amino acids from mitochondrial proteins known to be cleaved at nonconserved arginine sites, a subset of mitochondrial proteins specific to plants. Faster mitochondrial protein degradation rates not only for ICP55 cleaved protein but also for some non-ICP55 cleaved proteins were observed in Arabidopsis mitochondrial samples isolated from icp55 than from the wild type, indicating that a complicated protease degradation network has been affected. The lower protein stability of isolated mitochondria and the lack of processing of target proteins in icp55 were complemented by transformation with the full-length ICP55. Analysis of in vitro degradation rates and protein turnover rates in vivo of specific proteins indicated that serine hydroxymethyltransferase was affected in icp55. The maturation of serine hydroxymethyltransferase by ICP55 is unusual, as it involves breaking an amino-terminal diserine that is not known as an ICP55 substrate in other organisms and that is typically considered a sequence that stabilizes rather than destabilizes a protein. PMID:25862457

  11. Role of Nγ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine

    PubMed Central

    Cánovas, David; Borges, Nuno; Vargas, Carmen; Ventosa, Antonio; Nieto, Joaquín J.; Santos, Helena

    1999-01-01

    Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Nγ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A 1H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine. PMID:10473374

  12. The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

    PubMed Central

    Gumaa, K. A.; McLean, Patricia

    1969-01-01

    1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The Km values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and Pi found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [14C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with

  13. Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme-pRNA intermediate formation.

    PubMed

    Neubauer, Julie; Ogino, Minako; Green, Todd J; Ogino, Tomoaki

    2016-01-01

    The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3-8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop-start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs. PMID:26602696

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

    PubMed

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

    2015-11-01

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

  15. beta-Lactamase-catalyzed hydrolysis of acyclic depsipeptides and acyl transfer to specific amino acid acceptors.

    PubMed Central

    Pratt, R F; Govardhan, C P

    1984-01-01

    beta-Lactamases from all three classes, A, B, and C, catalyze the hydrolysis of specific acyclic depsipeptide (PhCH2CONHCR1R2CO2CHR3CO2H) analogs of acyl-D-alanyl-D-alanine peptides. The depsipeptides investigated, which are chemically as reactive toward nucleophiles as penicillins, are in general poor substrates, although differences between the classes of beta-lactamases have been observed: the order of effectiveness seems to be C greater than B greater than A. Certain class A and C beta-lactamases also catalyze phenylacetylglycyl transfer between phenylacetylglycyl depsipeptides and specific amino acid acceptors, a type of reaction hitherto identified more closely with D-alanyl-D-alanine transpeptidases than with beta-lactamases. Preliminary indications of an acyl-enzyme intermediate in these reactions have been obtained. These results support the suggestion [Tipper, D.J. and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. USA 54, 1133-1141] that beta-lactamases are evolutionary descendants of bacterial cell wall D-alanyl-D-alanine transpeptidases. PMID:6424114

  16. Effects of ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme activities in rats. Relation between hepatic carnitine concentration and carnitine acetyltransferase activity.

    PubMed Central

    Bhuiyan, A K; Bartlett, K; Sherratt, H S; Agius, L

    1988-01-01

    The effects of feeding the peroxisome proliferators ciprofibrate (a hypolipidaemic analogue of clofibrate) or POCA (2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate) (an inhibitor of CPT I) to rats for 5 days on the distribution of carnitine and acylcarnitine esters between liver, plasma and muscle and on hepatic CoA concentrations (free and acylated) and activities of carnitine acetyltransferase and acyl-CoA hydrolases were determined. Ciprofibrate and POCA increased hepatic [total CoA] by 2 and 2.5 times respectively, and [total carnitine] by 4.4 and 1.9 times respectively, but decreased plasma [carnitine] by 36-46%. POCA had no effect on either urinary excretion of acylcarnitine esters or [acylcarnitine] in skeletal muscle. By contrast, ciprofibrate decreased [acylcarnitine] and [total carnitine] in muscle. In liver, ciprofibrate increased the [carnitine]/[CoA] ratio and caused a larger increase in [acylcarnitine] (7-fold) than in [carnitine] (4-fold), thereby increasing the [short-chain acylcarnitine]/[carnitine] ratio. POCA did not affect the [carnitine]/[CoA] and the [short-chain acylcarnitine]/[carnitine] ratios, but it decreased the [long-chain acylcarnitine]/[carnitine] ratio. Ciprofibrate and POCA increased the activities of acyl-CoA hydrolases, and carnitine acetyltransferase activity was increased 28-fold and 6-fold by ciprofibrate and POCA respectively. In cultures of hepatocytes, ciprofibrate caused similar changes in enzyme activity to those observed in vivo, although [carnitine] decreased with time. The results suggest that: (1) the reactions catalysed by the short-chain carnitine acyltransferases, but not by the carnitine palmitoyltransferases, are near equilibrium in liver both before and after modification of metabolism by administration of ciprofibrate or POCA; (2) the increase in hepatic [carnitine] after ciprofibrate or POCA feeding can be explained by redistribution of carnitine between tissues; (3) the activity of carnitine

  17. New players in the fatty acyl ethanolamide metabolism.

    PubMed

    Rahman, Iffat Ara Sonia; Tsuboi, Kazuhito; Uyama, Toru; Ueda, Natsuo

    2014-08-01

    Fatty acyl ethanolamides represent a class of endogenous bioactive lipid molecules and are generally referred to as N-acylethanolamines (NAEs). NAEs include palmitoylethanolamide (anti-inflammatory and analgesic substance), oleoylethanolamide (anorexic substance), and anandamide (endocannabinoid). The endogenous levels of NAEs are mainly regulated by enzymes responsible for their biosynthesis and degradation. In mammalian tissues, the major biosynthetic pathway starts from glycerophospholipids and is composed of two enzyme reactions. The first step is N-acylation of ethanolamine phospholipids catalyzed by Ca(2+)-dependent N-acyltransferase and the second step is the release of NAEs from N-acylated ethanolamine phospholipids by N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD). As for the degradation of NAEs, fatty acid amide hydrolase plays the central role. However, recent studies strongly suggest the involvement of other enzymes in the NAE metabolism. These enzymes include members of the HRAS-like suppressor family (also called phospholipase A/acyltransferase family), which were originally discovered as tumor suppressors but can function as Ca(2+)-independent NAPE-forming N-acyltransferases; multiple enzymes involved in the NAPE-PLD-independent multi-step pathways to generate NAE from NAPE, which came to light by the analysis of NAPE-PLD-deficient mice; and a lysosomal NAE-hydrolyzing acid amidase as a second NAE hydrolase. These newly recognized enzymes may become the targets for the development of new therapeutic drugs. Here, we focus on recent enzymological findings in this area. PMID:24747663

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

    SciTech Connect

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

    2012-10-01

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

  19. Acylation of Ferrocene: A Greener Approach

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  20. Evaluation of antioxidant enzymes activities and identification of intermediate products during phytoremediation of an anionic dye (C.I. Acid Blue 92) by pennywort (Hydrocotyle vulgaris).

    PubMed

    Vafaei, Fatemeh; Movafeghi, Ali; Khataee, Alireza

    2013-11-01

    The potential of pennywort (Hydrocotyle vulgaris) for phytoremediation of C.I. Acid Blue 92 (AB92) was evaluated. The effects of various experimental parameters including pH, temperature, dye concentration and plant weight on dye removal efficiency were investigated. The results showed that the optimal condition for dye removal were pH 3.5 and temperature 25 degree C. Moreover, the absolute dye removal enhanced with increase in the initial dye concentration and plant weight. Pennywort showed the same removal efficiency in repeated experiments (four runs) as that obtained from the first run (a 6-day period). Therefore, the ability of the plant in consecutive removal of AB92 confirmed the biodegradation process. Accordingly, a number of produced intermediate compounds were identified. The effect of treatment on photosynthesis and antioxidant defense system including superoxide dismutase, peroxidase and catalase in plant roots and leaves were evaluated. The results revealed a reduction in photosynthetic pigments content under dye treatments. Antioxidant enzyme responses showed marked variations with respect to the plant organ and dye concentration in the liquid medium. Overall, the increase in antioxidant enzyme activity under AB92 stress in the roots was much higher than that in the leaves. Nevertheless, no significant increase in malondialdehyde content was detected in roots or leaves, implying that the high efficiency of antioxidant system in the elimination of reactive oxygen species. Based on these results, pennywort was founded to be a capable species for phytoremediation of AB92-contaminated water, may be effective for phytoremediation dye-contaminated polluted aquatic ecosystems. PMID:24552049

  1. The mechanism of acyl specific phospholipid remodeling by tafazzin

    PubMed Central

    Schlame, Michael; Acehan, Devrim; Berno, Bob; Xu, Yang; Valvo, Salvatore; Ren, Mindong; Stokes, David L.; Epand, Richard M.

    2013-01-01

    Cardiolipin is a mitochondrial phospholipid with a characteristic acyl chain composition that depends on the function of tafazzin, a phospholipid-lysophospholipid transacylase, although the enzyme itself lacks acyl specificity. We incubated isolated tafazzin with various mixtures of phospholipids and lysophospholipids, characterized the lipid phase by 31P-NMR, and measured newly formed molecular species by mass spectrometry. Significant transacylation was observed only in non-bilayer lipid aggregates and the substrate specificity was highly sensitive to the lipid phase. In particular, tetralinoleoyl-cardiolipin, a prototype molecular species, formed only under conditions that favor the inverted hexagonal phase. In isolated mitochondria, <1 percent of lipids participated in transacylations, suggesting that the action of tafazzin is limited to privileged lipid domains. We propose that tafazzin reacts with non-bilayer type lipid domains that occur in curved or hemifused membrane zones, and that acyl specificity is driven by the packing properties of these domains. PMID:22941046

  2. Regioselective self-acylating cyclodextrins in organic solvent

    PubMed Central

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

    2016-01-01

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

  3. Regioselective self-acylating cyclodextrins in organic solvent.

    PubMed

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

    2016-01-01

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

  4. Regioselective self-acylating cyclodextrins in organic solvent

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  5. Evaluating Nonpolar Surface Area and LC/MS Response: An Application for Site Occupancy Measurements for Enzyme Intermediates in Polyketide Biosynthesis

    PubMed Central

    Randall, Shan M.; Koryakina, Irina; Williams, Gavin J.; Muddiman, David C.

    2014-01-01

    RATIONALE Site occupancy measurements using LC/MS are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases. METHODS Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of nonpolar surface area. RESULTS A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied. CONCLUSIONS Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements. PMID:25366398

  6. Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles inacyl 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 se...

  7. A Conserved Glutamate Controls the Commitment to Acyl-adenylate Formation in Asparagine Synthetase†

    PubMed Central

    Meyer, Megan E.; Gutierrez, Jemy A.; Raushel, Frank M.; Richards, Nigel G. J.

    2010-01-01

    Inhibitor docking studies have implicated a conserved glutamate residue (Glu-348) as a general base in the synthetase active site of the enzyme asparagine synthetase B from Escherichia coli (AS-B). We now report steady-state kinetic, isotope transfer and positional isotope exchange experiments for a series of site-directed AS-B mutants in which Glu-348 is substituted by conservative amino acid replacements. We find that formation of the β-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, Glu-348 may also play a role in mediating the conformational changes needed to (i) coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme, and (ii) establish the structural integrity of the intramolecular tunnel along which ammonia is translocated. The importance of Glu-348 in mediating acyl-adenylate formation contrasts with the functional role of the cognate residues in β-lactam synthetase (BLS) and carbapenam synthetase (CPS) (Tyr-348 and Tyr-345, respectively), which both likely evolved from asparagine synthetase. Given the similarity of the chemistry catalyzed by AS-B, BLS and CPS, our work highlights the difficulty of predicting the functional outcome of single site mutations on enzymes that catalyze almost identical chemical transformations. PMID:20853825

  8. Cardiolipin molecular species with shorter acyl chains accumulate in Saccharomyces cerevisiae mutants lacking the acyl coenzyme A-binding protein Acb1p: new insights into acyl chain remodeling of cardiolipin.

    PubMed

    Rijken, Pieter J; Houtkooper, Riekelt H; Akbari, Hana; Brouwers, Jos F; Koorengevel, Martijn C; de Kruijff, Ben; Frentzen, Margrit; Vaz, Frédéric M; de Kroon, Anton I P M

    2009-10-01

    The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates. PMID:19656950

  9. Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases.

    PubMed

    Zhang, Xiujun; Li, Mai; Agrawal, Arpita; San, Ka-Yiu

    2011-11-01

    Microbial biosynthesis of fatty acid-like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Free fatty acids can be produced by introducing an acyl-acyl carrier protein thioesterase gene into Escherichia coli. The presence of the acyl-ACP thioesterase will break the fatty acid elongation cycle and release free fatty acid. Depending on their sequence similarity and substrate specificity, class FatA thioesterase is active on unsaturated acyl-ACPs and class FatB prefers saturated acyl group. Different acyl-ACP thioesterases have different degrees of chain length specificity. Although some of these enzymes have been characterized from a number of sources, information on their ability to produce free fatty acid in microbial cells has not been extensively examined until recently. In this study, we examined the effect of the overexpression of acyl-ACP thioesterase genes from Diploknema butyracea, Gossypium hirsutum, Ricinus communis and Jatropha curcas on free fatty acid production. In particular, we are interested in studying the effect of different acyl-ACP thioesterase on the quantities and compositions of free fatty acid produced by an E. coli strain ML103 carrying these constructs. It is shown that the accumulation of free fatty acid depends on the acyl-ACP thioesterase used. The strain carrying the acyl-ACP thioesterase gene from D. butyracea produced approximately 0.2g/L of free fatty acid while the strains carrying the acyl-ACP thioesterase genes from R. communis and J. curcas produced the most free fatty acid at a high level of more than 2.0 g/L at 48 h. These two strains accumulated three major straight chain free fatty acids, C14, C16:1 and C16 at levels about 40%, 35% and 20%, respectively. PMID:22001432

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

    PubMed

    Resh, Marilyn D

    2016-07-01

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

  11. Cardiolipin Molecular Species with Shorter Acyl Chains Accumulate in Saccharomyces cerevisiae Mutants Lacking the Acyl Coenzyme A-binding Protein Acb1p

    PubMed Central

    Rijken, Pieter J.; Houtkooper, Riekelt H.; Akbari, Hana; Brouwers, Jos F.; Koorengevel, Martijn C.; de Kruijff, Ben; Frentzen, Margrit; Vaz, Frédéric M.; de Kroon, Anton I. P. M.

    2009-01-01

    The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates. PMID:19656950

  12. Effect of carbon chain length in acyl coenzyme A on the efficiency of enzymatic transformation of okadaic acid to 7-O-acyl okadaic acid.

    PubMed

    Furumochi, Sachie; Onoda, Tatsuya; Cho, Yuko; Fuwa, Haruhiko; Sasaki, Makoto; Yotsu-Yamashita, Mari; Konoki, Keiichi

    2016-07-01

    Okadaic acid (OA), a product of dinoflagellate Prorocentrum spp., is transformed into 7-O-acyl OA in various bivalve species. The structural transformation proceeds enzymatically in vitro in the presence of the microsomal fraction from the digestive gland of bivalves. We have been using LC-MS/MS to identify OA-transforming enzymes by detecting 7-O-acyl OA, also known as dinophysistoxin 3 (DTX3). However, an alternative assay for DTX3 is required because the OA-transforming enzyme is a membrane protein, and surfactants for solubilizing membrane proteins decrease the sensitivity of LC-MS/MS. The present study examined saturated fatty acyl CoAs with a carbon chain length of 10 (decanoyl), 12 (dodecanoyl), 14 (tetradecanoyl), 16 (hexadecanoyl) and 18 (octadecanoyl) as the substrate for the in vitro acylation reaction. Saturated fatty acyl CoAs with a carbon chain length of 14, 16 and 18 exhibited higher yields than those with a carbon chain length of 10 or 12. Acyl CoAs with carbon chain lengths from 14 to 18 and containing either a diene unit, an alkyne unit, or an azide unit in the carbon chain were synthesized and shown to provide the corresponding DTX3 with a yield comparable to that of hexadecanoyl CoA. The three functional units can be conjugated with fluorescent reagents and are applicable to the development of a novel assay for DTX3. PMID:27231127

  13. Specificity of acyl-homoserine lactone synthases examined by mass spectrometry.

    PubMed

    Gould, Ty A; Herman, Jake; Krank, Jessica; Murphy, Robert C; Churchill, Mair E A

    2006-01-01

    Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources. PMID:16385066

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

    PubMed

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

    2016-08-01

    In metabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides. After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates. The feasibility of MGE was first discovered for sialylated glycans, by using N-acyl-modified mannosamines as precursor molecules for unnatural sialic acids. Prerequisite is the promiscuity of the enzymes of the Roseman-Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N-acyl side chain of mannosamine analogues, for example, elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications). Unnatural sialic acids are incorporated into glycoconjugates of cells and organs. MGE has intriguing biological consequences for treated cells (aliphatic MGE) and offers the opportunity to visualize the topography and dynamics of sialylated glycans in vitro, ex vivo, and in vivo (bioorthogonal MGE). PMID:27435524

  15. Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity.

    PubMed

    Füllekrug, Joachim; Poppelreuther, Margarete

    2016-01-01

    Long-chain fatty acyl-CoA synthetases (ACS) are a family of essential enzymes of lipid metabolism, activating fatty acids by thioesterification with coenzyme A. Fatty acyl-CoA molecules are then readily utilized for the biosynthesis of storage and membrane lipids, or for the generation of energy by ß-oxidation. Acyl-CoAs also function as transcriptional activators, allosteric inhibitors, or precursors for inflammatory mediators. Recent work suggests that ACS enzymes may drive cellular fatty acid uptake by metabolic trapping, and may also regulate the channeling of fatty acids towards specific metabolic pathways. The implication of ACS enzymes in widespread lipid associated diseases like type 2 diabetes has rekindled interest in this protein family. Here, we describe in detail how to measure long-chain fatty acyl-CoA synthetase activity by a straightforward radiometric assay. Cell lysates are incubated with ATP, coenzyme A, Mg(2+), and radiolabeled fatty acid bound to BSA. Differential phase partitioning of fatty acids and acyl-CoAs is exploited to quantify the amount of generated acyl-CoA by scintillation counting. The high sensitivity of this assay also allows the analysis of small samples like patient biopsies. PMID:26552674

  16. Metabolic and Tissue-Specific Regulation of Acyl-CoA Metabolism

    PubMed Central

    Ellis, Jessica M.; Bowman, Caitlyn E.; Wolfgang, Michael J.

    2015-01-01

    Acyl-CoA formation initiates cellular fatty acid metabolism. Acyl-CoAs are generated by the ligation of a fatty acid to Coenzyme A mediated by a large family of acyl-CoA synthetases (ACS). Conversely, acyl-CoAs can be hydrolyzed by a family of acyl-CoA thioesterases (ACOT). Here, we have determined the transcriptional regulation of all ACS and ACOT enzymes across tissues and in response to metabolic perturbations. We find patterns of coordinated regulation within and between these gene families as well as distinct regulation occurring in a tissue- and physiologically-dependent manner. Due to observed changes in long-chain ACOT mRNA and protein abundance in liver and adipose tissue, we determined the consequence of increasing cytosolic long-chain thioesterase activity on fatty acid metabolism in these tissues by generating transgenic mice overexpressing a hyperactive mutant of Acot7 in the liver or adipose tissue. Doubling cytosolic acyl-CoA thioesterase activity failed to protect mice from diet-induced obesity, fatty liver or insulin resistance, however, overexpression of Acot7 in adipocytes rendered mice cold intolerant. Together, these data suggest distinct modes of regulation of the ACS and ACOT enzymes and that these enzymes act in a coordinated fashion to control fatty acid metabolism in a tissue-dependent manner. PMID:25760036

  17. Fatty acyl-CoA reductases of birds

    PubMed Central

    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

  18. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

    PubMed

    Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

    2006-06-01

    The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important. PMID:16531404

  19. N-Acylation During Glidobactin Biosynthesis by the Tridomain Nonribosomal Peptide Synthetase Module GlbF

    PubMed Central

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

    2011-01-01

    Summary Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on co-expression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr1 amino group and generate the fatty acyl-Thr1-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis. PMID:21035730

  20. Enhanced Activity of Nanocrystalline Beta Zeolite for Acylation of Veratrole with Acetic Anhydride.

    PubMed

    Aisha Mahmood Abdulkareem, Al-Turkustani; Selvin, Rosilda

    2016-04-01

    Friedel-Craft acylation of veratrole using homogeneous acid catalysts such as AlCl3, FeCl3, ZnCl2, and HF etc. produces acetoveratrone, (3',4'-dimethoxyacetophenone), which is the intermediate for synthesis of papavarine alkaloids. The problems associated with these homogeneous catalysts can be overcome by using heterogeneous solid catalysts. Since acetoveratrone is a larger molecule, large pore Beta zeolites with smaller particle sizes are beneficial for the liquid-phase acylation of veratrole, for easy diffusion of reactants and products. The present study aims in the acylation of veratrole with acetic anhydride using nanocrystalline Beta Zeolite catalyst. A systematic investigation of the effects of various reaction parameters was done. The catalysts were characterized for their structural features by using XRD, TEM and DLS analyses. The catalytic activity of nanocrystalline Beta zeolite was compared with commercial Beta zeolite for the acylation and was found that nanocrystalline Beta zeolite possessed superior activity. PMID:27451793

  1. Hybrid Potential Simulation of the Acylation of Enterococcus faecium l,d-Transpeptidase by Carbapenems.

    PubMed

    Bhattacharjee, Nicholus; Field, Martin J; Simorre, Jean-Pierre; Arthur, Michel; Bougault, Catherine M

    2016-06-01

    The l,d-transpeptidases, Ldts, catalyze peptidoglycan cross-linking in β-lactam-resistant mutant strains of several bacteria, including Enterococcus faecium and Mycobacterium tuberculosis. Although unrelated to the essential d,d-transpeptidases, which are inactivated by the β-lactam antibiotics, they are nevertheless inhibited by the carbapenem antibiotics, making them potentially useful targets in the treatment of some important diseases. In this work, we have investigated the acylation mechanism of the Ldt from E. faecium by the carbapenem, ertapenem, using computational techniques. We have employed molecular dynamics simulations in conjunction with QC/MM hybrid potential calculations to map out possible reaction paths. We have focused on determining the following: (i) the protonation state of the nucleophilic cysteine of the enzyme when it attacks; (ii) whether nucleophilic attack and β-lactam ring-opening are concerted or stepwise, the latter occurring via an oxyanion intermediate; and (iii) the identities of the proton acceptors at the beginning and end of the reaction. Overall, we note that there is considerable plasticity in the mechanisms, owing to the significant flexibility of the enzyme, but find that the preferred pathways are ones in which nucleophilic attack of cysteine thiolate is concerted with β-lactam ring-opening. PMID:27196382

  2. Membrane Topology and Transient Acylation of Toxoplasma gondii Glycosylphosphatidylinositols

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    1995-01-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

  4. Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition.

    PubMed

    Moreno-Pérez, Antonio J; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2011-01-01

    The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results. PMID:21071236

  5. Free Energy Contribution Analysis Using Response Kernel Approximation: Insights into the Acylation Reaction of a Beta-Lactamase.

    PubMed

    Asada, Toshio; Ando, Kanta; Bandyopadhyay, Pradipta; Koseki, Shiro

    2016-09-01

    A widely applicable free energy contribution analysis (FECA) method based on the quantum mechanical/molecular mechanical (QM/MM) approximation using response kernel approaches has been proposed to investigate the influences of environmental residues and/or atoms in the QM region on the free energy profile. This method can evaluate atomic contributions to the free energy along the reaction path including polarization effects on the QM region within a dramatically reduced computational time. The rate-limiting step in the deactivation of the β-lactam antibiotic cefalotin (CLS) by β-lactamase was studied using this method. The experimentally observed activation barrier was successfully reproduced by free energy perturbation calculations along the optimized reaction path that involved activation by the carboxylate moiety in CLS. It was found that the free energy profile in the QM region was slightly higher than the isolated energy and that two residues, Lys67 and Lys315, as well as water molecules deeply influenced the QM atoms associated with the bond alternation reaction in the acyl-enzyme intermediate. These facts suggested that the surrounding residues are favorable for the reactant complex and prevent the intermediate from being too stabilized to proceed to the following deacylation reaction. We have demonstrated that the free energy contribution analysis should be a useful method to investigate enzyme catalysis and to facilitate intelligent molecular design. PMID:27501066

  6. RNA SHAPE chemistry with aromatic acylating reagents.

    PubMed

    Nodin, Laura; Noël, Olivier; Chaminade, Françoise; Maskri, Ouerdia; Barbier, Vincent; David, Olivier; Fossé, Philippe; Xie, Juan

    2015-02-01

    As chemical methods for RNA secondary structure determination, SHAPE chemistry (selective 2'-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature. In order to improve the specificity of acylating reagents towards unpaired nucleotides, we have explored the reactivity of symmetric anhydrides, acyl fluorides, active esters like succinimidyl ester and cyanomethyl esters for 2'-O-acylation reaction. Among the tested compounds, only the acyl fluoride 4 showed a low reactivity (compared to NMIA). However, this study is the first to show that nucleophilic catalysts like DMAP greatly improved the selective 2'-hydroxyl acylation by symmetric anhydrides, acyl fluorides and succinimidyl ester, with the 2-fluorobenzoic anhydride 5 being the most reactive. PMID:25557357

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

    PubMed Central

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

    2012-01-01

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

  8. Crystal structures of intermediates in the nitroalkane oxidase reaction.

    PubMed

    Héroux, Annie; Bozinovski, Dragana M; Valley, Michael P; Fitzpatrick, Paul F; Orville, Allen M

    2009-04-21

    The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 A resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes [Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 125, 8738-8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped [Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066]. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle. PMID:19265437

  9. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

    SciTech Connect

    Heroux, A.; Bozinovski, D; Valley, M; Fitzpatrick, P; Orville, A

    2009-01-01

    The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-10-01

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

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

    PubMed Central

    Burton, Mark; Rose, Timothy M.; Færgeman, Nils J.; Knudsen, Jens

    2005-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein that binds C12–C22 acyl-CoA esters with high affinity. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-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 in all four eukaryotic kingdoms, Animalia, Plantae, Fungi and Protista, and eleven eubacterial species. ACBP homologues were not detected in any other known bacterial species, or in archaea. Nearly all of the ACBP-containing bacteria are pathogenic to plants or animals, suggesting that an ACBP gene could have been acquired from a eukaryotic host by horizontal gene transfer. Many bacterial, fungal and higher eukaryotic species only harbour a single ACBP homologue. However, a number of species, ranging from protozoa to vertebrates, have evolved two to six lineage-specific paralogues through gene 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-specific paralogues have evolved altered functions. The appearance of ACBP very early on in evolution points towards a fundamental role of ACBP in acyl-CoA metabolism, including ceramide synthesis and in signalling. PMID:16018771

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

    PubMed Central

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

    2013-01-01

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

  15. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  16. Accumulation of glucosaminyl(acyl)phosphatidylinositol in an S3 HeLa subline expressing normal dolicholphosphomannose synthase activity.

    PubMed Central

    Sevlever, D; Schiemann, D; Guidubaldi, J; Medof, M E; Rosenberry, T L

    1997-01-01

    Glucosaminyl(acyl)phosphatidylinositol [GlcN(acyl)PI], the third intermediate in the mammalian glycosylphosphatidylinositol (GPI) anchor pathway, is undetectable in most cells. This intermediate was previously shown to accumulate, however, in murine lymphoma mutant E and in yeast mutant dpm1, both of which lack dolicholphosphomannose synthase activity. Here we report that a mammalian HeLa S3 subline, denoted D, produces large amounts of GlcN(acyl)PI. The level of GlcN(acyl)PI in this subline is twice that in the murine lymphoma mutant E and 4 times that in the parental S3 line. This HeLa D subline differs from the previously reported mutants that accumulate GlcN(acyl)PI because no defects in the synthesis or utilization of dolicholphosphomannose were found. Kinetic analysis indicated that in this HeLa subline there is an increased rate of synthesis of GlcN(acyl)PI, whereas the rate of metabolism for this GPI is comparable to that in wild-type cells. Furthermore, HeLa D cells accumulate GlcN(acyl)PI without a block in the synthesis of the downstream mannosylated GPI anchor precursors and GPI-anchored proteins. These findings might be relevant for understanding the regulation of the GPI pathway. PMID:9032473

  17. Modified acyl-ACP desaturase

    DOEpatents

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

    1998-01-06

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

  18. Modified Acyl-ACP desaturase

    DOEpatents

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

    1999-03-30

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme.

    PubMed

    Tan, S L; Kopczynski, M G; Bachovchin, W W; Orme-Johnson, W H; Babior, B M

    1986-03-15

    During the deamination of S-2-aminopropanol by the AdoCbl-dependent ethanolamine ammonia-lyase of Clostridia sp., a catalytic intermediate accumulates whose active site contains two paramagnetic species: cob(II)alamin and a free radical derived from the substrate molecule. Spin-echo spectroscopy has revealed that the unpaired electron on the substrate-derived radical is delocalized over a nitrogen atom that from its quadrupole splittings is probably a component of a secondary amide group. Experiments with 15N- and deuterium-labeled propanolamine gave no evidence of an interaction between this unpaired electron and the nitrogen originally attached to the substrate molecule. These results strongly suggest that the substrate-derived radical in this intermediate has already lost its nitrogen, and that this radical is stabilized by delocalization of the unpaired electron onto a nitrogen most likely situated in one of the peptide bonds of the enzyme backbone. PMID:3949775

  1. Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

    PubMed Central

    Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

    2016-01-01

    An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

  2. Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2015-10-01

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

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

    PubMed Central

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

    2012-01-01

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

  4. Nickel-Catalyzed Decarbonylative Borylation of Amides: Evidence for Acyl C-N Bond Activation.

    PubMed

    Hu, Jiefeng; Zhao, Yue; Liu, Jingjing; Zhang, Yemin; Shi, Zhuangzhi

    2016-07-18

    A nickel/N-heterocyclic carbene catalytic system has been established for decarbonylative borylation of amides with B2 nep2 by C-N bond activation. This transformation shows good functional-group compatibility and can serve as a powerful synthetic tool for late-stage borylation of amide groups in complex compounds. More importantly, as a key intermediate, the structure of an acyl nickel complex was first confirmed by X-ray analysis. Furthermore, the decarbonylative process was also observed. These findings confirm the key mechanistic features of the acyl C-N bond activation process. PMID:27258597

  5. Metabolism of acyl-lipids in Chlamydomonas reinhardtii.

    PubMed

    Li-Beisson, Yonghua; Beisson, Fred; Riekhof, Wayne

    2015-05-01

    Microalgae are emerging platforms for production of a suite of compounds targeting several markets, including food, nutraceuticals, green chemicals, and biofuels. Many of these products, such as biodiesel or polyunsaturated fatty acids (PUFAs), derive from lipid metabolism. A general picture of lipid metabolism in microalgae has been deduced from well characterized pathways of fungi and land plants, but recent advances in molecular and genetic analyses of microalgae have uncovered unique features, pointing out the necessity to study lipid metabolism in microalgae themselves. In the past 10 years, in addition to its traditional role as a model for photosynthetic and flagellar motility processes, Chlamydomonas reinhardtii has emerged as a model organism to study lipid metabolism in green microalgae. Here, after summarizing data on total fatty acid composition, distribution of acyl-lipid classes, and major acyl-lipid molecular species found in C. reinhardtii, we review the current knowledge on the known or putative steps for fatty acid synthesis, glycerolipid desaturation and assembly, membrane lipid turnover, and oil remobilization. A list of characterized or putative enzymes for the major steps of acyl-lipid metabolism in C. reinhardtii is included, and subcellular localizations and phenotypes of associated mutants are discussed. Biogenesis and composition of Chlamydomonas lipid droplets and the potential importance of lipolytic processes in increasing cellular oil content are also highlighted. PMID:25660108

  6. Modified Acyl-ACP desaturase

    DOEpatents

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

    1999-03-30

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

  7. Modified acyl-ACP desaturase

    DOEpatents

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

    1998-01-06

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

  8. Theoretical study of enzymatic catalysis explains why the trapped covalent intermediate in the E303C mutant of glycosyltransferase GTB was not detected in the wild-type enzyme.

    PubMed

    Bobovská, Adela; Tvaroška, Igor; Kóňa, Juraj

    2015-01-01

    Hybrid quantum mechanics/molecular mechanics calculations were used to study the catalytic mechanism of the retaining human α-(1,3)-galactosyltransferase (GTBWT) and its E303C mutant (GTBE303C). Both backside (via covalent glycosyl-enzyme intermediate, CGEI) and frontside SNi-like mechanisms (via oxocarbenium-ion intermediate, OCII) were investigated. The calculations suggest that both mechanisms are feasible in the enzymatic catalysis. The nucleophilic attack of the acceptor substrate to the anomeric carbon of OCII is the rate-determining step with an overall reaction barrier (ΔE(‡) = 19.5 kcal mol(-1)) in agreement with an experimental rate constant (kcat = 5.1 s(-1)). A calculated α-secondary kinetic isotope effect (α-KIE) of 1.27 (GTBWT) and 1.26 (GTBE303C) predicts dissociative character of the transition state in agreement with experimentally measured α-KIE of other retaining glycosyltransferases. Remarkably, stable CGEI in GTBE303C compared with its counterpart in GTBWT may explain why the CGEI has been detected by mass spectrometry only in GTBE303C ( Soya N, Fang Y, Palcic MM, Klassen JS. 2011. Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases. Glycobiology, 21: 547-552). PMID:25138306

  9. Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane.

    PubMed Central

    Mannaerts, G P; Van Veldhoven, P; Van Broekhoven, A; Vandebroek, G; Debeer, L J

    1982-01-01

    1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes. PMID:7115321

  10. Glycosyltransferases from Oat (Avena) Implicated in the Acylation of Avenacins*

    PubMed Central

    Owatworakit, Amorn; Townsend, Belinda; Louveau, Thomas; Jenner, Helen; Rejzek, Martin; Hughes, Richard K.; Saalbach, Gerhard; Qi, Xiaoquan; Bakht, Saleha; Roy, Abhijeet Deb; Mugford, Sam T.; Goss, Rebecca J. M.; Field, Robert A.; Osbourn, Anne

    2013-01-01

    Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid. PMID:23258535

  11. Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins.

    PubMed

    Owatworakit, Amorn; Townsend, Belinda; Louveau, Thomas; Jenner, Helen; Rejzek, Martin; Hughes, Richard K; Saalbach, Gerhard; Qi, Xiaoquan; Bakht, Saleha; Roy, Abhijeet Deb; Mugford, Sam T; Goss, Rebecca J M; Field, Robert A; Osbourn, Anne

    2013-02-01

    Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid. PMID:23258535

  12. Acylation Type Determines Ghrelin's Effects on Energy Homeostasis in Rodents

    PubMed Central

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

    2012-01-01

    Ghrelin is a gastrointestinal polypeptide that acts through the ghrelin receptor (GHSR) to promote food intake and increase adiposity. Activation of GHSR requires the presence of a fatty-acid (FA) side chain on amino acid residue serine 3 of the ghrelin molecule. However, little is known about the role that the type of FA used for acylation plays in the biological action of ghrelin. We therefore evaluated a series of differentially acylated peptides to determine whether alterations in length or stability of the FA side chain have an impact on the ability of ghrelin to activate GHSR in vitro or to differentially alter food intake, body weight, and body composition in vivo. Fatty acids principally available in the diet (such as palmitate C16) and therefore representing potential substrates for the ghrelin-activating enzyme ghrelin O-acyltransferase (GOAT) were used for dose-, time-, and administration/route-dependent effects of ghrelin on food intake, body weight, and body composition in rats and mice. Our data demonstrate that altering the length of the FA side chain of ghrelin results in the differential activation of GHSR. Additionally, we found that acylation of ghrelin with a long-chain FA (C16) delays the acute central stimulation of food intake. Lastly, we found that, depending on acylation length, systemic and central chronic actions of ghrelin on adiposity can be enhanced or reduced. Together our data suggest that modification of the FA side-chain length can be a novel approach to modulate the efficacy of pharmacologically administered ghrelin. PMID:22865372

  13. Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin.

    PubMed

    Xiong, Jian; Bhaskar, Ujjwal; Li, Guoyun; Fu, Li; Li, Lingyun; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J

    2013-09-10

    Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C₅-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin. PMID:23835156

  14. Biogenesis of ER subdomains containing DGAT2, an enzyme involved in industrial oil biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) are enzymes that catalyze the committed step in triacylglycerol (TAG) biosynthesis by transferring a fatty acyl group from the acyl-CoA pool to the sn-3 position of diacylglycerol. The substrate specificity and overall activity of these enzymes play a key role...

  15. Stability-increasing effects of anthocyanin glycosyl acylation.

    PubMed

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

    2017-01-01

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

  16. Macrocyclic prolinyl acyl guanidines as inhibitors of β-secretase (BACE).

    PubMed

    Boy, Kenneth M; Guernon, Jason M; Wu, Yong-Jin; Zhang, Yunhui; Shi, Joe; Zhai, Weixu; Zhu, Shirong; Gerritz, Samuel W; Toyn, Jeremy H; Meredith, Jere E; Barten, Donna M; Burton, Catherine R; Albright, Charles F; Good, Andrew C; Grace, James E; Lentz, Kimberley A; Olson, Richard E; Macor, John E; Thompson, Lorin A

    2015-11-15

    The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented. PMID:26497283

  17. Alkylation and acylation of cyclotriphosphazenes.

    PubMed

    Benson, Mark A; Zacchini, Stefano; Boomishankar, Ramamoorthy; Chan, Yuri; Steiner, Alexander

    2007-08-20

    Phosphazenes (RNH)6P3N3 (R = n-propyl, isobutyl, isopropyl, cyclohexyl, tert-butyl, benzyl) are readily alkylated at ring N sites by alkyl halides forming N-alkyl phosphazenium cations. Alkylation of two ring N sites occurred after prolonged heating in the presence of methyl iodide or immediately at room temperature with methyl triflate yielding N,N'-dimethyl phosphazenium dications. Geminal dichloro derivatives Cl2(RNH)4P3N3 are methylated by methyl iodide at the ring N site adjacent to both P centers carrying four RNH groups. X-ray crystal structures showed that the alkylation of ring N sites leads to substantial elongation of the associated P-N bonds. Both N-alkyl and N,N'-dialkyl phosphazenium salts form complex supramolecular networks in the solid state via NH...X interactions. Systems carrying less-bulky RNH groups show additional NH...N bonds between N-alkyl phosphazenium ions. N-Alkyl phosphazenium halides form complexes with silver ions upon treatment with silver nitrate. Depending on the steric demand of RNH substituents, either one or both of the vacant ring N sites engage in coordination to silver ions. Treatment of (RNH)6P3N3 (R = isopropyl) with acetyl chloride and benzoyl chloride, respectively, yielded N-acyl phosphazenium ions. X-ray crystal structures revealed that elongation of P-N bonds adjacent to the acylated ring N site is more pronounced than it is in the case of N-alkylated species. Salts containing N-alkyl phosphazenium ions are stable toward water and other mild nucleophiles, while N,N'-dialkyl and N-acyl phosphazenium salts are readily hydrolyzed. The reaction of (RNH)6P3N3 with bromoacetic acid led to N-alkylation at one ring N site in addition to formation of an amide via condensation of an adjacent RNH substituent with the carboxylic acid group. The resulting bromide salt contains mono cations of composition (RNH)5P3N3CH2CONR in which a CH2-C(O) unit is embedded between a ring N and an exocyclic N site of the phosphazene. PMID

  18. Long chain acyl-CoA synthetases and other acyl activating enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proper synthesis and breakdown of molecules containing carboxylic acids is a vital part of metabolism in all living organisms. Given the relatively inert chemical nature of many carboxylic acids, activation is a necessary step prior to use in the various anabolic and catabolic pathways that utilize...

  19. In-Silico docking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate

    PubMed Central

    Savarino, Andrea

    2007-01-01

    Background HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain. Methods Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD). Results Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC50s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N-Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those

  20. Friedel-Crafts Acylation with Amides

    PubMed Central

    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

  1. Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine.

    PubMed

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman; Ottenwälder, Birgit; Schnölzer, Martina; Kartenbeck, Jürgen; Lyer, Stefan; Autschbach, Frank; Poustka, Annemarie; Otto, Herwart F; Mollenhauer, Jan

    2004-02-01

    Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease or coeliac disease, respectively. Acyl-CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl-CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine. PMID:14743501

  2. Impaired expression of acyl-CoA-synthetase 5 in epithelial tumors of the small intestine.

    PubMed

    Gassler, Nikolaus; Schneider, Armin; Kopitz, Jürgen; Schnölzer, Martina; Obermüller, Nicholas; Kartenbeck, Jürgen; Otto, Herwart F; Autschbach, Frank

    2003-10-01

    Fatty acids are implicated in tumorigenesis, but data are limited concerning endogenous fatty acid metabolism of tumor cells in adenomas and adenocarcinomas of the small intestine. The recently cloned human acyl-CoA-synthetase 5 (ACS5) is predominantly found in the small intestine and represents a key enzyme in providing cytosolic acyl-CoA thioesters. Protein synthesis and mRNA expression of ACS5 were studied in human intestinal tissues using different methods, including a newly established monoclonal antibody. In the healthy small intestine, expression of ACS5 was restricted to the villus surface epithelium but was not detectable in enterocytes lining crypts. ACS5 protein and mRNA were progressively diminished in epithelial cells of adenomas and adenocarcinomas of the small intestine. In conclusion, altered expression of ACS5 is probably related to the adenoma-carcinoma sequence of small intestinal epithelial tumors due to an impaired acyl-CoA thioester synthesis. PMID:14608540

  3. An orphan esterase ABHD10 modulates probenecid acyl glucuronidation in human liver.

    PubMed

    Ito, Yusuke; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki

    2014-12-01

    Probenecid, a widely used uricosuric agent, is mainly metabolized to probenecid acyl glucuronide (PRAG), which is considered a causal substance of severe allergic or anaphylactoid reactions. PRAG can be hydrolyzed (deglucuronidated) to probenecid. The purpose of this study was to identify enzymes responsible for probenecid acyl glucuronidation and PRAG deglucuronidation in human livers and to examine the effect of deglucuronidation in PRAG formation. In human liver homogenates (HLHs), the intrinsic clearance (CLint) of PRAG deglucuronidation was much greater (497-fold) than that of probenecid acyl glucuronidation. Evaluation of PRAG formation by recombinant UDP-glucuronosyltransferase (UGT) isoforms and an inhibition study using HLHs as an enzyme source demonstrated that multiple UGT isoforms, including UGT1A1, UGT1A9, and UGT2B7, catalyzed probenecid acyl glucuronidation. We found that recombinant α/β hydrolase domain containing 10 (ABHD10) substantially catalyzed PRAG deglucuronidation activity, whereas carboxylesterases did not. Similar inhibitory patterns by chemicals between HLHs and recombinant ABHD10 supported the major contribution of ABHD10 to PRAG deglucuronidation in human liver. Interestingly, it was demonstrated that the CLint value of probenecid acyl glucuronidation in HLHs was increased by 1.7-fold in the presence of phenylmethylsulfonyl fluoride, which potently inhibited ABHD10 activity. In conclusion, we found that PRAG deglucuronidation catalyzed by ABHD10 suppressively regulates PRAG formation via multiple UGT enzymes in human liver. The balance of activities by these enzymes is important for the formation of PRAG, which may be associated with the adverse reactions observed after probenecid administration. PMID:25217485

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  6. Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme–pRNA intermediate formation

    PubMed Central

    Neubauer, Julie; Ogino, Minako; Green, Todd J.; Ogino, Tomoaki

    2016-01-01

    The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3–8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop–start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs. PMID:26602696

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

    PubMed

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

    1991-06-01

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

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

    PubMed

    Martinelle, M; Hult, K

    1995-09-01

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid. PMID:7669809

  9. Biosynthesis of lipid a precursors: cloning, expression and partial purification of E. coli UDP-GlcNAc acyl transferase; identification of UDP-3-O-acyl-GlcNAc as its enzymatic product

    SciTech Connect

    Anderson, M.S.; Crowell, D.N.; Schlafmann, S.E.; Raetz, C.R.H.

    1986-05-01

    The initial steps in the biosynthesis of E. coli lipid A disaccharides have been shown to proceed by two acylations of UDP-GlcNAc to form UDP-2,3-diacyl-GlcN (UDP-DAG). In vitro synthesis of UDP-DAG appears to involve a previously undescribed, monoacylated form of UDP-GlcNAc. The authors now show (1) definitive evidence that this metabolite has the structure UDP-3-O-acyl-GlcNAc, and (2) that a gene encoding or controlling this acylating enzyme is located on a 2.4 Kb fragment mapping between dnaE and cds on the E. coli chromosome. Insertion of this fragment into an expression vector directs overproduction of an enzyme catalyzing monoacylation of UDP-GlcNAc by acyl-acyl carrier protein. The activity was partially purified from an overproducing strain by chromatography of the soluble fraction of a cell extract on DEAE cellulose in the presence of 1% octyl glucoside. Peak fractions were 2200x purified relative to wild type with an 80% yield. The product generated by this enzyme was isolated from a preparative scale incubation and structurally identified by /sup 1/H-NMR.

  10. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    PubMed

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  11. Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme.

    PubMed

    Lukoyanov, Dmitriy; Khadka, Nimesh; Yang, Zhi-Yong; Dean, Dennis R; Seefeldt, Lance C; Hoffman, Brian M

    2016-08-24

    We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology. PMID:27529724

  12. Gamma-glutamyltranspeptidase-mediated degradation of diclofenac-S-acyl-glutathione in vitro and in vivo in rat.

    PubMed

    Grillo, Mark P; Hua, Fengmei; March, Kristi L; Benet, Leslie Z; Knutson, Charles G; Ware, Joseph A

    2008-10-01

    Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac-S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic gamma-glutamyltranspeptidase (gamma-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic gamma-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for gamma-GT in vivo in rat and in vitro with commercially available gamma-GT enzyme. We also examined the ability of D-SG to inhibit gamma-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the gamma-GT-mediated D-SG degradation product diclofenac-N-acyl-cysteinylglycine (D- N-CG), where a total of approximately 8 microg was excreted 6 h postadministration. When D-SG (100 microM) was incubated with gamma-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where approximately 94 microM D- N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of gamma-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the gamma-GT enzyme. Results from theses studies indicate that D-SG is a substrate for gamma-GT; however, the conjugate may not contribute significantly to the decrease in gamma-GT activity reported to occur in vivo in rat. PMID:18693772

  13. An Adenosine Triphosphate-Phosphate Exchange Catalyzed by a Soluble Enzyme Couple Inhibited by Uncouplers of Oxidative Phosphorylation

    PubMed Central

    Allison, William S.; Benitez, Lita V.

    1972-01-01

    The sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), which is an acyl phosphatase, will catalyze an acetyl phosphate-Pi exchange reaction. This exchange reaction is reversibly inhibited by the uncouplers of oxidative phosphorylation, 2,4-dinitrophenol, m-Cl carbonylcyanide-phenylhydrazone, pentachlorophenol, and 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanalide, and is irreversibly inhibited by cyanide and dicumarol. An ATP-Pi exchange reaction similar to that catalyzed by mitochondria can be simulated by a system composed of oxidized glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (EC 2.7.1.28), 3-phosphoglycerate, ATP, 32Pi, and appropriate cofactors. The ATP-Pi exchange is inhibited by uncouplers of oxidative phosphorylation. Higher concentrations of uncouplers will also inhibit the ATPase reaction catalyzed by the coupled enzyme system. The exchange reactions catalyzed by the sulfenic acid form of glyceraldehyde-3-phosphate are consistent with a sulfenyl carboxylate intermediate. On the basis of these observations, a reaction scheme has been postulated for covalent coupling in oxidative phosphorylation that includes a sulfenyl carboxylate as a nonphosphorylated, high energy intermediate and an acyl phosphate as a phosphorylated, high energy intermediate. PMID:4507619

  14. Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets[S

    PubMed Central

    Snyder, Nathaniel W.; Basu, Sankha S.; Worth, Andrew J.; Mesaros, Clementina; Blair, Ian A.

    2015-01-01

    Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [13C1]propionate and [13C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[2H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings. PMID:25424005

  15. Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.

    PubMed

    Snyder, Nathaniel W; Basu, Sankha S; Worth, Andrew J; Mesaros, Clementina; Blair, Ian A

    2015-01-01

    Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [(13)C1]propionate and [(13)C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[(2)H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings. PMID:25424005

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

    PubMed

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

    2016-07-01

    Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 μM), but different Km for arachidonic acid (0.5 and 6 μM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid β-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for β-oxidation in the fat body, and is also required for normal reproduction. PMID:27091636

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

    PubMed

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

    2014-09-01

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

  18. Microbial Tailoring of Acyl Peptidic Siderophores

    PubMed Central

    2015-01-01

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

  19. Microbial tailoring of acyl peptidic siderophores.

    PubMed

    Gauglitz, Julia M; Iinishi, Akira; Ito, Yusai; Butler, Alison

    2014-04-29

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

  20. The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.

    PubMed

    Wille, Georg; Meyer, Danilo; Steinmetz, Andrea; Hinze, Erik; Golbik, Ralph; Tittmann, Kai

    2006-06-01

    Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase. PMID:16680160

  1. Remodeling of host phosphatidylcholine by Chlamydia acyltransferase is regulated by acyl-CoA binding protein ACBD6 associated with lipid droplets

    PubMed Central

    Soupene, Eric; Wang, Derek; Kuypers, Frans A

    2015-01-01

    The bacterial human pathogen Chlamydia trachomatis invades cells as an infectious elementary body (EB). The EB is internalized into a vacuole that is hidden from the host defense mechanism, and is modified to sustain the development of the replicative reticulate body (RB). Inside this parasitophorous compartment, called the inclusion, the pathogen survives supported by an active exchange of nutrients and proteins with the host cell. We show that host lipids are scavenged and modified into bacterial-specific lipids by the action of a shared human-bacterial acylation mechanism. The bacterial acylating enzymes for the essential lipids 1-acyl-sn-glycerol 3-phosphate and 1-acyl-sn-phosphatidylcholine were identified as CT453 and CT775, respectively. Bacterial CT775 was found to be associated with lipid droplets (LDs). During the development of C. trachomatis, the human acyl-CoA carrier hACBD6 was recruited to cytosolic LDs and translocated into the inclusion. hACBD6 protein modulated the activity of CT775 in an acyl-CoA dependent fashion and sustained the activity of the bacterial acyltransferase by buffering the concentration of acyl-CoAs. We propose that disruption of the binding activity of the acyl-CoA carrier might represent a new drug-target to prevent growth of C. trachomatis. PMID:25604091

  2. Acylation of lysolecithin in the intestinal mucosa of rats

    PubMed Central

    Subbaiah, P. V.; Sastry, P. S.; Ganguly, J.

    1970-01-01

    1. The presence of an active acyl-CoA–lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-14C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-14C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the β-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-14C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order. PMID:5484668

  3. Acylation and deacylation mechanism of Helicobacter pylori AmiF formamidase: A computational DFT study

    NASA Astrophysics Data System (ADS)

    He, Rongxing; Yang, Qinlei; Li, Ming

    2014-04-01

    The acylation and deacylation mechanisms of Helicobacter pylori AmiF formamidase were investigated using DFT method. In the constructed active site, residues Glu60, Glu141 and His167 were taken into account besides Lys133 and Cys166. Calculations provided insight on the details of mechanism and explained crucial roles played by Glu60, Glu141 and His167. For acetylation, we proposed a new stepwise mechanism in which the thiol group first attacks the carbon atom of formamide and produces tetrahedral intermediate. In deacylation, Glu60 activates a water molecule to perform nucleophilic attack and then forms an intermediate, which is different from the usually suggested mechanism.

  4. Cloning, characterization, and expression analysis of acyl-acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis).

    PubMed

    Dong, Shubin; Huang, Jiacong; Li, Yannan; Zhang, Jing; Lin, Shanzhi; Zhang, Zhixiang

    2014-05-25

    Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)△FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches. PMID:24631366

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

    PubMed

    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

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

    PubMed Central

    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

  7. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain...

  8. Characterization of soluble acyl-ACP desaturases from Camelina sativa, Macadamia tetraphylla and Dolichandra unguis-cati.

    PubMed

    Rodríguez, Manuel Fernando Rodríguez; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-04-15

    Acyl-acyl carrier protein (ACP) desaturases (EC 1.14.19.2) are soluble enzymes that catalyse the insertion of a double bond into saturated fatty acid bound in saturated acyl chains bound to ACP in higher plants, producing cis-monounsaturated fatty acids. Three types of soluble acyl-ACP desaturases have been described: Δ(9)-acyl-ACP, Δ(6)-acyl-ACP and Δ(4)-acyl-ACP desaturases, which differ in the substrate specificity and the position in which the double bond is introduced. In the present work, Camelina sativa (CsSAD), Macadamia tetraphylla (MtSAD) and Dolichandra unguis-cati (DuSAD) desaturases were cloned, sequenced and characterized. Single copies of CsSAD, MtSAD and DuSAD with three, one and two different alleles, respectively, were found. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization in protein extracts. The recombinant CsSAD enzyme showed 300-fold higher specificity towards 18:0-ACP than 16:0-ACP. Similar profile exhibited MtSAD although the differences in the specificity were lower, around 170-fold higher for 18:0-ACP than 16:0-ACP. Furthermore, DuSAD presented a profile showing preference towards 16:0-ACP against 18:0-ACP, around twice more, being so a Δ(9) palmitoyl-ACP desaturase. Also, we reported the expression profile of CsSAD, which showed the highest levels of expression in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm. Moreover, the possibility to express a new desaturase in C. sativa (oilseed crop that store high levels of oil and is easy to transform) to create a new line rich in short monounsaturated fatty acid is discussed. PMID:25765361

  9. Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

    PubMed Central

    Coleman, R A; Haynes, E B

    1984-01-01

    The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta. PMID:6696738

  10. Isolation of Acyl-CoA:cholesterol acyltransferase inhibitor from Persicaria vulgaris.

    PubMed

    Song, Hye Young; Rho, Mun-Chual; Lee, Seung Woong; Kwon, Oh Eok; Chang, Young-Duck; Lee, Hyun Sun; Kim, Young-Kook

    2002-09-01

    In the course of our search for Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors from natural sources, a new type of ACAT inhibitor was isolated from the methanol extract of Persicaria vulgaris. On the basis of spectral evidence, the structure of the active compound was identified as pheophorbide A. Pheophorbide A inhibited ACAT activity with an IC 50 value of 1.1 microg/ml in an enzyme assay using rat liver microsomes with a dose dependent fashion. PMID:12357403

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

    PubMed Central

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

    2016-01-01

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

  12. Discovery of acyl guanidine tryptophan hydroxylase-1 inhibitors.

    PubMed

    Goldberg, Daniel R; De Lombaert, Stéphane; Aiello, Robert; Bourassa, Patricia; Barucci, Nicole; Zhang, Qing; Paralkar, Vishwas; Stein, Adam J; Valentine, Jim; Zavadoski, William

    2016-06-15

    An increasing number of diseases have been linked to a dysfunctional peripheral serotonin system. Given that tryptophan hydroxylase 1 (TPH1) is the rate limiting enzyme in the biosynthesis off serotonin, it represents an attractive target to regulate peripheral serotonin. Following up to our first disclosure, we report a new chemotype of TPH1 inhibitors where-by the more common central planar heterocycle has been replaced with an open-chain, acyl guanidine surrogate. Through our work, we found that compounds of this nature provide highly potent TPH1 inhibitors with favorable physicochemical properties that were effective in reducing murine intestinal 5-HT in vivo. Furthermore, we obtained a high resolution (1.90Å) X-ray structure crystal structure of one of these inhibitors (compound 51) that elucidated the active conformation along with revealing a dimeric form of TPH1 for the first time. PMID:27146606

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

    PubMed Central

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

    2008-01-01

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

  14. Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

    PubMed Central

    Roviello, G. N.; Gröschel, S.; Pedone, C.

    2009-01-01

    Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy. PMID:19629638

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

    SciTech Connect

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

    2008-04-02

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

  16. Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

    PubMed Central

    Garland, P. B.; Shepherd, D.; Yates, D. W.

    1965-01-01

    1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50μμmoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by β-oxidation takes place without detectable accumulation of acyl-CoA intermediates. PMID:16749169

  17. Synthesis of Cobalt-Acyl-Complexes of the Type MeC(O)Co(CO)3PR3; Characterization of Reactive Intermediates Using TR IR Techniques, Torsten Buetner, Diplom thesis submitted to the Univ. Jena, Germany based on DOE supported studies at UCSB, 2000

    SciTech Connect

    Ford, Peter C.

    2000-12-31

    OAK-B135 Mechanisms of the homogeneous catalytic and photocatalytic activation of small molecules such as carbon monoxide, dihydrogen and various hydrocarbons. Time-resolved spectroscopic investigations of rhodium and iridium halo carbonyl complex intermediates proposed in methanol carbonylation catalysts.

  18. Crystal Structures of Protein Glutaminase and Its Pro Forms Converted into Enzyme-Substrate Complex*

    PubMed Central

    Hashizume, Ryota; Maki, Yukiko; Mizutani, Kimihiko; Takahashi, Nobuyuki; Matsubara, Hiroyuki; Sugita, Akiko; Sato, Kimihiko; Yamaguchi, Shotaro; Mikami, Bunzo

    2011-01-01

    Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed. PMID:21926168

  19. High acyl gellan as an emulsion stabilizer.

    PubMed

    Vilela, Joice Aline Pires; da Cunha, Rosiane Lopes

    2016-03-30

    High acyl gellan (0.01-0.2% w/w) was used as stabilizer in oil in water emulsions containing 30% (w/w) of sunflower oil and prepared under different process conditions. Stable emulsions to phase separation could be obtained using high acyl gellan (HA) content above 0.05% (w/w), while low acyl gellan (LA) prepared at the same conditions could not stabilize emulsions. Emulsions properties depended on the process used to mix the oil and gellan dispersion since high pressure homogenization favored stabilization while very high energy density applied by ultrasound led to systems destabilization. Emulsions prepared using high pressure homogenization showed zeta potential values ranging from -50 up to -59 mV, suggesting that electrostatic repulsion could be contributing to the systems stability. Rheological properties of continuous phase were also responsible for emulsions stabilization, since HA gellan dispersions showed high viscosity and gel-like behavior. The high viscosity of the continuous phase could be associated to the presence of high acyl gellan microgels/aggregates. Disentanglement of these aggregates performed by ultrasound strongly decreased the viscosity and consequently affected the emulsions behavior, reducing the stability to phase separation. PMID:26794954

  20. Tight binding inhibitors of N-acyl amino sugar and N-acyl amino acid deacetylases.

    PubMed

    Xu, Chengfu; Hall, Richard; Cummings, Jennifer; Raushel, Frank M

    2006-04-01

    Very potent inhibitors were synthesized for the enzymatic deacetylation of N-acetyl-d-glucosamine-6-phosphate (NagA) and N-acetyl-d-glutamate (DGD). The methyl phosphonamidate derivative of d-glucosamine-6-phosphate bound to N-acetyl-d-glucosamine-6-phosphate deacetylase with an equilibrium dissociation constant of 34 +/- 5 nM at pH 7.5 and an association rate constant of 6.1 x 103 M-1 s-1. The inhibition constant is 4000-fold lower than the Michaelis constant for the substrate N-acetyl-d-glucosamine-6-phosphate. N-Acetyl-d-glutamate deacetylase was inhibited by the methyl phosphonamidate derivative of d-glutamate with an inhibition constant of 460 +/- 70 pM at pH 7.6. The inhibitor bound to the enzyme 500 000-fold tighter than the Michaelis constant for N-formyl-d-glutamate. These compounds mimic the putative tetrahedral intermediate formed upon nucleophilic attack of an activated water molecule on the amide bond of the target substrate. These inhibitors should prove useful in the elucidation of the enzyme-substrate interactions for enzymes within the amidohydrolase superfamily. PMID:16568996

  1. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    PubMed Central

    Rodríguez-Durán, Luis V.; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C.; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N.

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

  2. Dimerization and activation of porcine pancreatic phospholipase A2 via substrate level acylation of lysine 56.

    PubMed

    Tomasselli, A G; Hui, J; Fisher, J; Zürcher-Neely, H; Reardon, I M; Oriaku, E; Kézdy, F J; Heinrikson, R L

    1989-06-15

    The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration. Octanoylation of Lys-56 activates the enzyme some 180-fold toward 4-nitro-3-octanoyloxybenzoate and more than 100-fold toward monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Acylation also attends the enzymatic hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with the incorporation of 1 eq of palmitate. Kinetic analysis of the early phase of reaction with 4-nitro-3-octanoyloxybenzoate shows that in this initial step the rate of activation is first order with respect to enzyme and substrate. A much more rapid, autocatalytic activation occurs in the later phases of the reaction where the activation of the enzyme is catalyzed by the activated enzyme itself. These findings with porcine pancreatic phospholipase A2, together with those relative to a snake venom enzyme monomer (Cho, W., Tomasselli, A. G., Heinrikson, R. L., and Kézdy, F. J. (1988) J. Biol. Chem. 263, 11237-11241), strongly support the proposal that interfacial activation of monomeric phospholipases is due to substrate-level autoacylation resulting in fully potentiated dimeric enzymes. PMID:2498336

  3. Acyl-coenzyme A:cholesterol O-acyltransferase is not identical to liver microsomal carboxylesterase.

    PubMed

    Diczfalusy, M A; Björkhem, I; Einarsson, K; Alexson, S E

    1996-04-01

    Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, DL-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein. PMID:8624784

  4. Enzymatic Acylation of Anthocyanin Isolated from Black Rice with Methyl Aromatic Acid Ester as Donor: Stability of the Acylated Derivatives.

    PubMed

    Yan, Zheng; Li, Chunyang; Zhang, Lixia; Liu, Qin; Ou, Shiyi; Zeng, Xiaoxiong

    2016-02-10

    The enzymatic acylation of anthocyanin from black rice with aromatic acid methyl esters as acyl donors and Candida antarctica lipase B was carried out under reduced pressure. The highest conversion of 91% was obtained with benzoic acid methyl ester as acyl donor; cyanidin 3-(6″-benzoyl)-glucoside, cyanidin 3-(6″-salicyloyl)-glucoside, and cyanidin 3-(6″-cinnamoyl)-glucoside were successfully synthesized. This is the first report on the enzymatic acylation of anthocyanin from black rice with methyl aromatic esters as acyl donors and lipase as biocatalyst. Furthermore, the acylation with aromatic carboxylic acids enhanced both the thermostability and light resistivity of anthocyanin. In particular, cyanidin 3-(6″-cinnamoyl)-glucoside was the most stable among the three acylated anthocyanins synthesized. PMID:26766135

  5. Two Clades of Type-1 Brassica napus Diacylglycerol Acyltransferase Exhibit Differences in Acyl-CoA Preference.

    PubMed

    Greer, Michael S; Pan, Xue; Weselake, Randall J

    2016-06-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to produce triacylglycerol, which is the main component of the seed oil of Brassica oilseed species. Phylogenetic analysis of the amino acid sequences encoded by four transcriptionally active DGAT1 genes from Brassica napus suggests that the gene forms diverged over time into two clades (I and II), with representative members in each genome (A and C). The majority of the amino acid sequence differences in these forms of DGAT1, however, reside outside of motifs suggested to be involved in catalysis. Despite this, the clade II enzymes displayed a significantly enhanced preference for linoleoyl-CoA when assessed using in-vitro enzyme assays with yeast microsomes containing recombinant enzyme forms. These findings contribute to our understanding of triacylglycerol biosynthesis in B. napus, and may advance our ability to engineer DGAT1s with desired substrate selectivity properties. PMID:27138895

  6. Identification of a Long-Chain Polyunsaturated Fatty Acid Acyl-Coenzyme A Synthetase from the Diatom Thalassiosira pseudonana1

    PubMed Central

    Tonon, Thierry; Qing, Renwei; Harvey, David; Li, Yi; Larson, Tony Robert; Graham, Ian Alexander

    2005-01-01

    The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast. PMID:15821149

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    1995-11-01

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

  9. Efficient regioselective acylation of quercetin using Rhizopus oryzae lipase and its potential as antioxidant.

    PubMed

    Kumar, Vinod; Jahan, Firdaus; Mahajan, Richi V; Saxena, Rajendra Kumar

    2016-10-01

    The present investigation describes the regioselective enzymatic acylation of quercetin with ferulic acid using Rhizopus oryzae lipase. Optimization of reaction parameters resulted in 93.2% yield of the ester synthesized using 750IU of lipase in cyclo-octane at a temperature of 45°C. The reaction was successfully carried out upto 25g scale. The ester synthesized was analyzed by (1)H Nuclear magnetic resonance spectroscopy. The ester synthesized (quercetin ferulate) showed higher antiradical activity as compared to ascorbic acid using the 2,2-diphenyl-1-picrylhydrazyl radical method. These results on enzyme-catalyzed acylation of quercetin might be used to prepare and scale-up other flavonoids derivatives. PMID:27372535

  10. Ab initio study on the transition state of acylation step of trypsin catalysis.

    PubMed

    Kubodera, H; Nakagawa, S; Umeyama, H

    1990-03-01

    The transition state of acylation step of trypsin catalysis was determined by molecular orbital calculations. The calculations were carried out at the RHF-LCAO-SCF approximation level with double zeta basis set (plus polarization functions). The role of His57 residue in the acylation step of the catalytic reaction of trypsin was analysed from a quantum mechanical point of view. The influences of surrounding residues, such as oxyanion hole and Asp102-, and the electrostatic effect of the other regions of the enzyme were also studied. His57 was proved to capture the proton from Ser195 side chain terminus with its lone pair and to transfer it to substrate with electrostatic assistance of Asp102- and oxyanion hole. PMID:2165153

  11. A Thiolate Anion Buried within the Hydrocarbon Ruler Perturbs PagP Lipid Acyl Chain Selection†

    PubMed Central

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

    2016-01-01

    The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP exhibits remarkable selectivity because its binding pocket for lipid acyl chains excludes those differing in length from palmitate by a solitary methylene unit. This narrow detergent-binding hydrophobic pocket buried within the eight-strand antiparallel β-barrel is known as the hydrocarbon ruler. Gly88 lines the acyl chain binding pocket floor, and its substitution can raise the floor to correspondingly shorten the selected acyl chain. An aromatic exciton interaction between Tyr26 and Trp66 provides an intrinsic spectroscopic probe located immediately adjacent to Gly88. The Gly88Cys PagP enzyme was engineered to function as a dedicated myristoyltransferase, but the mutant enzyme instead selected both myristoyl and pentadecanoyl groups, was devoid of the exciton, and displayed a 21 °C reduction in thermal stability. We now demonstrate that the structural perturbation results from a buried thiolate anion attributed to suppression of the Cys sulfhydryl group pKa from 9.4 in aqueous solvent to 7.5 in the hydrocarbon ruler microenvironment. The Cys thiol is sandwiched at the interface between a nonpolar and a polar β-barrel interior milieu, suggesting that local electrostatics near the otherwise hydrophobic hydrocarbon ruler pocket serve to perturb the thiol pKa. Neutralization of the Cys thiolate anion by protonation restores wild-type exciton and thermal stability signatures to Gly88Cys PagP, which then functions as a dedicated myristoyltransferase at pH 7. Gly88Cys PagP assembled in bacterial membranes recapitulates lipid A myristoylation in vivo. Hydrocarbon ruler–exciton coupling in PagP thus reveals a thiol–thiolate ionization mechanism for modulating lipid acyl chain selection. PMID:20175558

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

    PubMed

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

    2013-11-22

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

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

    PubMed Central

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

    2013-01-01

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

  14. Protein engineering of a bacterial N-acyl-d-glucosamine 2-epimerase for improved stability under process conditions.

    PubMed

    Klermund, Ludwig; Riederer, Amelie; Hunger, Annique; Castiglione, Kathrin

    2016-06-01

    Enzymatic cascade reactions, i.e. the combination of several enzyme reactions in one pot without isolation of intermediates, have great potential for the establishment of sustainable chemical processes. However, many cascade reactions suffer from cross-inhibitions and enzyme inactivation by components of the reaction system. This study focuses on the two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-d-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE) in combination with an N-acetylneuraminate lyase (NAL) from Escherichia coli. AvaAGE epimerizes N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), which then reacts with pyruvate in a NAL-catalyzed aldol condensation to form Neu5Ac. However, AvaAGE is inactivated by high pyruvate concentrations, which are used to push the NAL reaction toward the product side. A biphasic inactivation was observed in the presence of 50-800mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis revealed that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE. Because ATP is an allosteric activator of the epimerase and the binding of the nucleotide is crucial for its catalytic properties, saturation mutagenesis at position K160 was performed to identify the most compatible amino acid exchanges. The best variants, K160I, K160N and K160L, showed no inactivation by pyruvate, but significantly impaired kinetic parameters. For example, depending on the mutant, the turnover number kcat was reduced by 51-68% compared with the wild-type enzyme. A mechanistic model of the Neu5Ac synthesis was established, which can be used to select the AvaAGE variant that is most favorable for a given process condition. The results show that mechanistic models can greatly facilitate the choice of the right enzyme for an enzymatic cascade reaction with multiple cross-inhibitions and inactivation phenomena

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

    PubMed Central

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

    2014-01-01

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

  16. Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606.

    PubMed

    Richie, Daryl L; Takeoka, Kenneth T; Bojkovic, Jade; Metzger, Louis E; Rath, Christopher M; Sawyer, William S; Wei, Jun-Rong; Dean, Charles R

    2016-01-01

    The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3-O-[(R)-3-OH-C14]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A. baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth. PMID:27526195

  17. Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606

    PubMed Central

    Richie, Daryl L.; Takeoka, Kenneth T.; Bojkovic, Jade; Metzger, Louis E.; Rath, Christopher M.; Sawyer, William S.; Wei, Jun-Rong; Dean, Charles R.

    2016-01-01

    The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3-O-[(R)-3-OH-C14]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A. baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth. PMID:27526195

  18. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

    DOE PAGESBeta

    Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.; Erskine, Peter T.; Tomanicek, Stephen J.; Ostermann, Andreas; Schrader, Tobias E.; Ginell, Stephan L.; Coates, Leighton

    2015-12-02

    The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacksmore » a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less

  19. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

    SciTech Connect

    Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.; Erskine, Peter T.; Tomanicek, Stephen J.; Ostermann, Andreas; Schrader, Tobias E.; Ginell, Stephan L.; Coates, Leighton

    2015-12-02

    The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.

  20. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography.

    PubMed

    Vandavasi, Venu Gopal; Weiss, Kevin L; Cooper, Jonathan B; Erskine, Peter T; Tomanicek, Stephen J; Ostermann, Andreas; Schrader, Tobias E; Ginell, Stephan L; Coates, Leighton

    2016-01-14

    The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery. PMID:26630115

  1. Radical migration of substituents of aryl groups on quinazolinones derived from N-acyl cyanamides.

    PubMed

    Larraufie, Marie-Hélène; Courillon, Christine; Ollivier, Cyril; Lacôte, Emmanuel; Malacria, Max; Fensterbank, Louis

    2010-03-31

    A newly designed radical cascade involving N-acyl cyanamides is reported. It builds on aromatic homolytic substitutions as intermediate events and leads to complex heteroaromatic structures via an unprecedented radical migration of a substituent on aryl groups of quinazolinones (hydrogen or alkyl). Mechanistic considerations are detailed, which allowed us to devise fine control over the domino processes. The latter could be predictably stopped at several stages, depending on the reaction conditions. Finally, a surgical introduction of a trifluoromethyl substituent on a quinazolinone was achieved via the reported migration. PMID:20205425

  2. Enantioselective acyl transfer catalysis by a combination of common catalytic motifs and electrostatic interactions

    PubMed Central

    Mandai, Hiroki; Fujii, Kazuki; Yasuhara, Hiroshi; Abe, Kenko; Mitsudo, Koichi; Korenaga, Toshinobu; Suga, Seiji

    2016-01-01

    Catalysts that can promote acyl transfer processes are important to enantioselective synthesis and their development has received significant attention in recent years. Despite noteworthy advances, discovery of small-molecule catalysts that are robust, efficient, recyclable and promote reactions with high enantioselectivity can be easily and cost-effectively prepared in significant quantities (that is, >10 g) has remained elusive. Here, we demonstrate that by attaching a binaphthyl moiety, appropriately modified to establish H-bonding interactions within the key intermediates in the catalytic cycle, and a 4-aminopyridyl unit, exceptionally efficient organic molecules can be prepared that facilitate enantioselective acyl transfer reactions. As little as 0.5 mol% of a member of the new catalyst class is sufficient to generate acyl-substituted all-carbon quaternary stereogenic centres in quantitative yield and in up to 98:2 enantiomeric ratio (er) in 5 h. Kinetic resolution or desymmetrization of 1,2-diol can be performed with high efficiency and enantioselectivity as well. PMID:27079273

  3. Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

    PubMed

    Rodríguez-Rodríguez, Manuel Fernando; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2014-11-01

    Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results. PMID:25212866

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

    PubMed

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

    2013-04-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae

    PubMed Central

    Mori, Natsumi; Moriyama, Takashi; Toyoshima, Masakazu; Sato, Naoki

    2016-01-01

    Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid

  7. Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae.

    PubMed

    Mori, Natsumi; Moriyama, Takashi; Toyoshima, Masakazu; Sato, Naoki

    2016-01-01

    Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid

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

    PubMed Central

    Carter, Michael S.

    2015-01-01

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

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

    PubMed Central

    Kawelke, Steffen; Feussner, Ivo

    2015-01-01

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

  10. Ion channel regulation by protein S-acylation

    PubMed Central

    2014-01-01

    Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease. PMID:24821965

  11. Secondary Acylation of Klebsiella pneumoniae Lipopolysaccharide Contributes to Sensitivity to Antibacterial Peptides*

    PubMed Central

    Clements, Abigail; Tull, Dedreia; Jenney, Adam W.; Farn, Jacinta L.; Kim, Sang-Hyun; Bishop, Russell E.; McPhee, Joseph B.; Hancock, Robert E. W.; Hartland, Elizabeth L.; Pearse, Martin J.; Wijburg, Odilia L. C.; Jackson, David C.; McConville, Malcolm J.; Strugnell, Richard A.

    2016-01-01

    Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055ΔlpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055ΔlpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins. PMID:17371870

  12. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    SciTech Connect

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

  13. Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats.

    PubMed

    Ohtomo, Takayuki; Hoshino, Atsuko; Yajima, Masako; Tsuchiya, Akiharu; Momose, Atsushi; Tanonaka, Kouichi; Toyoda, Hiroo; Kato, Tetsuta; Yamada, Junji

    2013-08-01

    Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage. PMID:23385637

  14. Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases.

    PubMed

    Koryakina, Irina; McArthur, John; Randall, Shan; Draelos, Matthew M; Musiol, Ewa M; Muddiman, David C; Weber, Tilmann; Williams, Gavin J

    2013-01-18

    Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides. PMID:23083014

  15. Cloning of a palmitoyl-acyl carrier protein thioesterase from oil palm.

    PubMed

    Othman, A; Lazarus, C; Fraser, T; Stobart, K

    2000-12-01

    A palmitoyl-acyl carrier protein (ACP) thioesterase cDNA clone was isolated from an oil palm cDNA library. The cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and a crude bacterial extract was assayed for acyl-CoA-hydrolysing activity. The recombinant enzyme was able to hydrolyse medium- and long-chain acyl-CoAs. Northern-blot analysis showed a high level of gene expression in leaf, flower and 15-, 17- and 18-week mesocarp tissues. Low-level gene expression was detected in germinated seedlings and 8- and 12-week mesocarp tissues, but no transcript was detected in any kernel tissues. Southern-blot analysis indicated the presence of a single gene and we have also isolated a genomic clone using the cDNA as a probe. Two genomic fragments were subcloned and a 7 kb contiguous stretch of the oil palm genome was sequenced. Comparison of this sequence with the cDNA sequence identified a putative 93 amino acid transit peptide, most of which is missing from the cDNA. The coding region of the gene consisted of seven exons and six introns. PMID:11171146

  16. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

    SciTech Connect

    Van De Loo, F.J.; Broun, P.; Turner, S.; Somerville, C.

    1995-07-18

    Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.

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

    PubMed

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

    2001-12-14

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

  18. Crystal structure of human mitochondrial acyl-CoA thioesterase (ACOT2)

    SciTech Connect

    Mandel, Corey R.; Tweel, Benjamin; Tong, Liang

    2009-08-13

    Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of CoA esters to free CoA and carboxylic acids and have important functions in lipid metabolism and other cellular processes. Type I ACOTs are found only in animals and contain an {alpha}/{beta} hydrolase domain, through currently no structural information is available on any of these enzymes. We report here the crystal structure at 2.1 {angstrom} resolution of human mitochondrial ACOT2, a type I enzyme. The structure contains two domains, N and C domains. The C domain has the {alpha}/{beta} hydrolase fold, with the catalytic triad Ser294-His422-Asp388. The N domain contains a seven-stranded {beta}-sandwich, which has some distant structural homologs in other proteins. The active site is located in a large pocket at the interface between the two domains. The structural information has significant relevance for other type I ACOTs and related enzymes.

  19. Cyclic AMP-dependent Protein Lysine Acylation in Mycobacteria Regulates Fatty Acid and Propionate Metabolism*

    PubMed Central

    Nambi, Subhalaxmi; Gupta, Kallol; Bhattacharyya, Moitrayee; Ramakrishnan, Parvathy; Ravikumar, Vaishnavi; Siddiqui, Nida; Thomas, Ann Terene; Visweswariah, Sandhya S.

    2013-01-01

    Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host. PMID:23553634

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

    PubMed

    Kandra, L; Wagner, G J

    1990-11-01

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

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

    PubMed Central

    Kandra, Lili; Wagner, George J.

    1990-01-01

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

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

    SciTech Connect

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

    2010-11-30

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

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

    PubMed

    Miklaszewska, Magdalena; Banaś, Antoni

    2016-08-01

    Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols. PMID:27297992

  4. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    SciTech Connect

    Ohlrogge, John B.

    2013-11-05

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

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

    PubMed

    Kem, Michelle P; Butler, Alison

    2015-06-01

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

  6. Vertebrate fatty acyl desaturase with Δ4 activity

    PubMed Central

    Li, Yuanyou; Monroig, Oscar; Zhang, Liang; Wang, Shuqi; Zheng, Xiaozhong; Dick, James R.; You, Cuihong; Tocher, Douglas R.

    2010-01-01

    Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 18∶2n - 6 to produce 18∶3n - 6 that is elongated to 20∶3n - 6 followed by Δ5 desaturation. Synthesis of EPA from 18∶3n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22∶5n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22∶5n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above. PMID:20826444

  7. 2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage.

    PubMed

    Agrawal, H C; Sprinkle, T J; Agrawal, D

    1990-07-15

    2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP1 and CNP2 with Mr of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNP1 and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32% by cycloheximide. These results provide conclusive evidence that CNP1 and CNP2 are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis. PMID:2164018

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

    SciTech Connect

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

    2006-02-01

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

  9. The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis.

    PubMed

    Cumplido-Laso, Guadalupe; Medina-Puche, Laura; Moyano, Enriqueta; Hoffmann, Thomas; Sinz, Quirin; Ring, Ludwig; Studart-Wittkowski, Claudia; Caballero, José Luis; Schwab, Wilfried; Muñoz-Blanco, Juan; Blanco-Portales, Rosario

    2012-06-01

    Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour. PMID:22563120

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

    PubMed

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

    2011-01-01

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

  11. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

  12. Structure of enzyme-bound substrates: resonance Raman and kinetic evidence for differential enzyme-substrate contacts in N-(Pentafluoro-benzoyl)glycine dithioacyl and thioacyl papain

    NASA Astrophysics Data System (ADS)

    Lee, H.; Angus, R. H.; Storer, A. C.; Carey, P. R.

    1989-12-01

    Resonance Raman (RR) spectroscopy is used to probe the structure of the substrate in the substrate-enzyme complex N-pentafluorobenzoyl) glycine (dithioacyl) papain (C 6F 5C(=O) NHCH 2C(=S)S-papain). This system was chosen since the high electron withdrawing capacity of the C 6F 5 group markedly affects electron density of the -NH- moiety which, in turn, is known to change catalytic activity. The RR spectrum of the enzyme-substrate complex is interpreted by reference to the model compound N-(pentafluorobenzoyl) glycine ethyl dithioester (C 6F 5(CO))NHCH 2C(=S)SC 2H 5. The RR spectra of this compound in aqueous or organic solvents can be understood in terms of the known conformational states of N-acylglycine dithioesters. Comparison of model with enzyme-substrate RR spectra shows that the substrate is binding in the active site in a conformer known as conformer B characterized by a small-NHCH 2CS(thiol) torsional angle and close N-to-S (thiol) contact. Kinetic rate-structure correlations are developed involving k3, the rate constant for deacylation, and the strength of the N-to-S (thiol) interaction. N-(Pentafluorobenzoyl) glycine dithioacyl papain fits the rate-structure correlation whereas the corresponding pentafluorobenzoyl glycine thiol intermediate does not. It is proposed that the difference in the size of the CS compared to the CO group brings about a small change in the dithioacyl papain compared to the thiolacyl papain conformation such that enzyme-substrate contacts involving ortho and meta F atoms in the thiol acyl enzyme case are weakened or removed in the case of the dithioacyl papain.

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

    PubMed Central

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

    2015-01-01

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

  14. Probing Intra- versus Interchain Kinetic Preferences of L-Thr Acylation on Dimeric VibF with Mass Spectrometry

    PubMed Central

    Hicks, Leslie M.; Balibar, Carl J.; Walsh, Christopher T.; Kelleher, Neil L.; Hillson, Nathan J.

    2006-01-01

    We present a method to probe intra- and interchain activities within dimeric nonribosomal peptide synthetases. Utilizing domain inactivation and analytical mass mutants in conjunction with rapid-quench, mass spectrometry, and a probabilistic kinetic model, we have elucidated the pre-steady-state intra- and interchain rates and the corresponding flux of the acylation of L-Thr onto VibF. Although the intra rate is significantly faster than the inter rate, the data are most consistent with an even flux of covalent substrate loading where neither pathway dominates. These pre-steady-state results confirm previous steady-state in vitro mutant complementation studies of VibF. Extension of this methodology to other dimeric nonribosomal peptide synthetases, and to the related fatty acid and polyketide synthases, will further our biophysical understanding of their acyl-intermediate-processing pathways. PMID:16815901

  15. Palladium-Catalyzed Environmentally Benign Acylation.

    PubMed

    Suchand, Basuli; Satyanarayana, Gedu

    2016-08-01

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

  16. Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

    PubMed Central

    O'Brien, W J; Frerman, F E

    1977-01-01

    The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates. PMID:334745

  17. Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase.

    PubMed Central

    Knight, Kirsty; Scrutton, Nigel S

    2002-01-01

    The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+). The second and third steps represent further reduction of the enzyme flavins and NADP(+) release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP(+) release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+) is modestly slower in calmodulin-depleted enzyme. Our studies indicate the

  18. Identification of a Mycoloyl Transferase Selectively Involved in O-Acylation of Polypeptides in Corynebacteriales

    PubMed Central

    Huc, Emilie; de Sousa-D'Auria, Célia; de la Sierra-Gallay, Inès Li; Salmeron, Christophe; van Tilbeurgh, Herman; Bayan, Nicolas; Houssin, Christine

    2013-01-01

    We have previously described the posttranslational modification of pore-forming small proteins of Corynebacterium by mycolic acid, a very-long-chain α-alkyl and β-hydroxy fatty acid. Using a combination of chemical analyses and mass spectrometry, we identified the mycoloyl transferase (Myt) that catalyzes the transfer of the fatty acid residue to yield O-acylated polypeptides. Inactivation of corynomycoloyl transferase C (cg0413 [Corynebacterium glutamicum mytC {CgmytC}]), one of the six Cgmyt genes of C. glutamicum, specifically abolished the O-modification of the pore-forming proteins PorA and PorH, which is critical for their biological activity. Expectedly, complementation of the cg0413 mutant with either the wild-type gene or its orthologues from Corynebacterium diphtheriae and Rhodococcus, but not Nocardia, fully restored the O-acylation of the porins. Consistently, the three-dimensional structure of CgMytC showed the presence of a unique loop that is absent from enzymes that transfer mycoloyl residues onto both trehalose and the cell wall arabinogalactan. These data suggest the implication of this structure in the enzyme specificity for protein instead of carbohydrate. PMID:23852866

  19. Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae).

    PubMed Central

    Dormann, P.; Frentzen, M.; Ohlrogge, J. B.

    1994-01-01

    This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide. PMID:12232130

  20. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    SciTech Connect

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2005-01-01

    The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria. PMID:15687201

  3. Anti-Tumor Effects of Novel 5-O-Acyl Plumbagins Based on the Inhibition of Mammalian DNA Replicative Polymerase Activity

    PubMed Central

    Kawamura, Moe; Kuriyama, Isoko; Maruo, Sayako; Kuramochi, Kouji; Tsubaki, Kazunori; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2014-01-01

    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. PMID:24520419

  4. Site-specific analysis of protein S-acylation by resin-assisted capture[S

    PubMed Central

    Forrester, Michael T.; Hess, Douglas T.; Thompson, J. Will; Hultman, Rainbo; Moseley, M. Arthur; Stamler, Jonathan S.; Casey, Patrick J.

    2011-01-01

    Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells. PMID:21044946

  5. Pseudo-enzymatic S-acylation of a myristoylated yes protein tyrosine kinase peptide in vitro may reflect non-enzymatic S-acylation in vivo.

    PubMed Central

    Bañó, M C; Jackson, C S; Magee, A I

    1998-01-01

    Covalent attachment of a variety of lipid groups to proteins is now recognized as a major group of post-translational modifications. S-acylation of proteins at cysteine residues is the only modification considered dynamic and thus has the potential for regulating protein function and/or localization. The activities that catalyse reversible S-acylation have not been well characterized and it is not clear whether both the acylation and the deacylation steps are regulated, since in principle it would be sufficient to control only one of them. Both apparently enzymatic and non-enzymatic S-acylation of proteins have previously been reported. Here we show that a synthetic myristoylated c-Yes protein tyrosine kinase undecapeptide undergoes spontaneous S-acylation in vitro when using a long chain acyl-CoA as acyl donor in the absence of any protein. The S-acylation was dependent on myristoylation of the substrate, the length of the incubation period, temperature and substrate concentration. When COS cell fractions were added to the S-acylation reaction no additional peptide:S-acyltransferase activity was detected. These results are consistent with the possibility that membrane-associated proteins may undergo S-acylation in vivo by non-enzymatic transfer of acyl groups from acyl-CoA. In this case, the S-acylation-deacylation process could be controlled by a regulated depalmitoylation mechanism. PMID:9480882

  6. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making

    PubMed Central

    Tuladhar, Rubina; Lum, Lawrence

    2015-01-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  7. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making.

    PubMed

    Tuladhar, Rubina; Lum, Lawrence

    2015-04-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  8. Quorum quenching enzymes.

    PubMed

    Fetzner, Susanne

    2015-05-10

    Bacteria use cell-to-cell communication systems based on chemical signal molecules to coordinate their behavior within the population. These quorum sensing systems are potential targets for antivirulence therapies, because many bacterial pathogens control the expression of virulence factors via quorum sensing networks. Since biofilm maturation is also usually influenced by quorum sensing, quenching these systems may contribute to combat biofouling. One possibility to interfere with quorum sensing is signal inactivation by enzymatic degradation or modification. Such quorum quenching enzymes are wide-spread in the bacterial world and have also been found in eukaryotes. Lactonases and acylases that hydrolyze N-acyl homoserine lactone (AHL) signaling molecules have been investigated most intensively, however, different oxidoreductases active toward AHLs or 2-alkyl-4(1H)-quinolone signals as well as other signal-converting enzymes have been described. Several approaches have been assessed which aim at alleviating virulence, or biofilm formation, by reducing the signal concentration in the bacterial environment. These involve the application or stimulation of signal-degrading bacteria as biocontrol agents in the protection of crop plants against soft-rot disease, the use of signal-degrading bacteria as probiotics in aquaculture, and the immobilization or entrapment of quorum quenching enzymes or bacteria to control biofouling in membrane bioreactors. While most approaches to use quorum quenching as antivirulence strategy are still in the research phase, the growing number of organisms and enzymes known to interfere with quorum sensing opens up new perspectives for the development of innovative antibacterial strategies. PMID:25220028

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

    PubMed

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

    2016-09-01

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

  10. Comparison of metabolic fluxes of cis-5-enoyl-CoA and saturated acyl-CoA through the beta-oxidation pathway.

    PubMed Central

    Tserng, K Y; Chen, L S; Jin, S J

    1995-01-01

    The metabolic fluxes of cis-5-enoyl-CoAs through the beta-oxidation cycle were studied in solubilized rat liver mitochondrial samples and compared with saturated acyl-CoAs of equal chain length. These studies were accomplished using either spectrophotometric assay of enzyme activities and/or the analysis of metabolites and precursors using a gas chromatographic method after conversion of CoA esters into their free acids. Cis-5-enoyl-CoAs were dehydrogenated by acyl-CoA oxidase or acyl-CoA dehydrogenases at significantly lower rates (10-44%) than saturated acyl-CoAs. However, enoyl-CoA hydratase hydrated trans-2-cis-5-enoyl-CoA at a faster rate (at least 1.5-fold) than trans-2-enoyl-CoA. The combined activities of 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase for 3-hydroxy-cis-5-enoyl-CoAs derived from cis-5-enoyl-CoAs were less than 40% of the activity for the corresponding 3-hydroxyacyl-CoAs prepared from saturated acyl-CoAs. Rat liver mitochondrial beta-oxidation enzymes were capable of metabolizing cis-5-enoyl-CoA via one cycle of beta-oxidation to cis-3-enoyl-CoA with two less carbons. However, the overall rates of one cycle of beta-oxidation, as determined with stable-isotope-labelled tracer, was only 15-25%, for cis-5-enoyl-CoA, of that for saturated acyl-CoA. In the presence of NADPH, the metabolism of cis-5-enoyl-CoAs was switched to the reduction pathway.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7717980

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-03-01

    The substrate specificity of the acyl-acyl carrier protein (ACP) thioesterases significantly determines the type of fatty acids that are exported from plastids. Thus, designing acyl-ACP thioesterases with different substrate specificities or kinetic properties would be of interest for plant lipid biotechnology to produce oils enriched in specialty fatty acids. In the present work, the FatA thioesterase from Helianthus annuus was used to test the impact of changes in the amino acids present in the binding pocket on substrate specificity and catalytic efficiency. Amongst all the mutated enzymes studied, Q215W was especially interesting as it had higher specificity towards saturated acyl-ACP substrates and higher catalytic efficiency compared to wild-type H. annuus FatA. Null, wild type and high-efficiency alleles were transiently expressed in tobacco leaves to check their effect on lipid biosynthesis. Expression of active FatA thioesterases altered the composition of leaf triacylglycerols but did not alter total lipid content. However, the expression of the wild type and the high-efficiency alleles in Arabidopsis thaliana transgenic seeds resulted in a strong reduction in oil content and an increase in total saturated fatty acid content. The role and influence of acyl-ACP thioesterases in plant metabolism and their possible applications in lipid biotechnology are discussed. PMID:24327259

  13. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    PubMed

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. PMID:24163338

  14. Protonation of a Peroxodiiron(III) Complex and Conversion to a Diiron(III/IV) Intermediate: Implications for Proton-assisted O-O Bond Cleavage in Nonheme Diiron Enzymes

    PubMed Central

    Cranswick, Matthew A.; Meier, Katlyn K.; Shan, Xiaopeng; Stubna, Audria; Kaizer, Jószef; Mehn, Mark P.; Münck, Eckard; Que, Lawrence

    2012-01-01

    Oxygenation of a diiron(II) complex,[FeII2(μ-OH)2(BnBQA)2(NCMe)2]2+ (2) (where BnBQA is N-benzyl-N,N-bis(2-quinolinylmethyl)amine) results in the formation of a metastable peroxodiferric intermediate (3). Treatment of 3 with strong acid affords its conjugate acid 4 in which the (μ-oxo)(μ-1,2-peroxo)diiron(III) core of 3 is protonated at the oxo bridge. The core structures of 3 and 4 are characterized in detail by UV-vis, Mössbauer, resonance Raman, and X-ray absorption spectroscopies. Complex 4 is shorter lived than 3 and decays to generate in 20–25% yield a diiron(III/IV) species (5) that can be identified by EPR and Mössbauer spectroscopy. This reaction sequence demonstrates for the first time that protonation of the oxo bridge of a (μ-oxo)(μ-1,2-peroxo)diiron(III) complex leads to the cleavage of the peroxo O–O bond and formation of a high-valent diiron complex, thereby mimicking the steps involved in the formation of intermediate X in the activation cycle of ribonucleotide reductase. PMID:22971084

  15. SMc01553 is the sixth acyl carrier protein in Sinorhizobium meliloti 1021.

    PubMed

    Dávila-Martínez, Yadira; Ramos-Vega, Ana Laura; Contreras-Martínez, Sandra; Encarnación, Sergio; Geiger, Otto; López-Lara, Isabel M

    2010-01-01

    Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. In Sinorhizobium meliloti 1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence of S. meliloti 1021 also reveals the ORF SMc01553, annotated as a putative ACP. smc01553 is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed in Escherichia coli was labelled in vivo with [(3)H]beta-alanine, a biosynthetic building block of the 4'-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase from S. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures of S. meliloti. However, a mutant in which the two duplicated regions were deleted did not show any different phenotype with respect to the wild-type in the free-living or symbiotic lifestyle. PMID:19797355

  16. Deciphering the roles of acyl-CoA-binding proteins in plant cells.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-09-01

    Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed. PMID:26340904

  17. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    SciTech Connect

    Bagautdinov, Bagautdin Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The crystal structure of 3-oxoacyl-(acyl-carrier protein) synthase II from T. thermophilus HB8 has been determined at 2.0 Å resolution and compared with the structures of β-keto-ACP synthases from other sources. The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain.

  18. Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin

    PubMed Central

    Kudryashova, Elena; Kalda, Caitlin; Kudryashov, Dmitri S.

    2012-01-01

    Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K0.5 = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg2+-GTP to support crosslinking, but the kinetic parameters (KM = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0–9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions. PMID:23029200

  19. Synthesis of 1,2-dihydro-2-oxo-4-quinolinyl phosphates from 2-acyl-benzoic acids

    PubMed Central

    He, Xinhua; Aglio, Tharcilla; Deschamps, Jeffrey R.; Rai, Rachita; Xue, Fengtian

    2015-01-01

    We report a facile synthesis of 1,2-dihydro-2-oxo-4-quinolinyl phosphates (1a-l) starting from 2-acyl-benzoic acids (2a-l) in the presence of phosphoryl azides via a one-pot cascade reaction involving a Curtius rearrangement, an intramolecular nucleophilic addition of the enol carbon to the isocyanate intermediate, and an addition-elimination of the enol oxygen to the phosphoryl azide. During the reaction three new bonds are formed under mild conditions to yield 1,2-dihydro-2-oxo-4-quinolinyl phosphates in modest yields. PMID:25937677

  20. Chemical and enzymic studies on the characterization of intermediates during the removal of the 14alpha-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives.

    PubMed Central

    Akhtar, M; Alexander, K; Boar, R B; McGhie, J F; Barton, D H

    1978-01-01

    By using cell-free preparations of rat liver it was shown that the removal of the 14alpha-methyl group (C-32) of steroids containing either a delta7(8) or a delta8(9) double bond is attended exclusively by the formation of the corresponding 7,14- and 8,14-dienes respectively (structures of types III and VIII). Cumulative evidence from a variety of experimental approaches had led to the deduction that delta8(14)-steroids are not involved as intermediates on the major pathway of cholesterol biosynthesis. The metabolism of [32-3H]lanost-7-ene-3beta,32-diol (structure of type I) results in the formation of radioactive formic acid, no labelled formaldehyde being formed. By using appropriately labelled species of the compound (I) it was found that the release of formic acid and the formation of 4,4-dimethylcholesta-7,14-dien-3beta-ol (strurcture of type III) were closely linked processes, and that in the conversion of compound (I) into compound (III), 3-beta-hydroxylanost-7-en-32-al (II) is an obligatory intermediate. Both the conversion of lanost-7-ene-3beta,32-diol (I) into 3beta-hydroxylanost-7-en-32-al (II) and the further metabolism of the latter (II) to 4,4-dimethylcholesta-7,14-dien-3beta-ol (III) exhibited a requirement for NADPH and O2. This suggests that the oxidation of the 32-hydroxy group of compound (I) to the aldehyde group of compound (II) does not occur by the conventional alcohol dehydrogenase type of reaction, but may proceed by a novel mechanism involving the intermediacy of a gem-diol. A detailed overall pathway for the 14alpha-demethylation in cholesterol biosynthesis is considered, and proposals about the mechanism of individual steps in the pathway are made. PMID:25646

  1. Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH. PMID:24015914

  2. Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry.

    PubMed Central

    Ding, A; Ojingwa, J C; McDonagh, A F; Burlingame, A L; Benet, L Z

    1993-01-01

    Acyl glucuronide metabolites of bilirubin and many drugs can react with serum albumin in vivo to form covalent adducts. Such adducts may be responsible for some toxic effects of carboxylic nonsteroidal antiinflammatory agents. The mechanism of formation of the adducts and their chemical structures are unknown. In this paper we describe the use of tandem mass spectrometry to locate binding sites and elucidate the binding mechanism involved in the formation of covalent adducts from tolmetin glucuronide and albumin in vitro. Human serum albumin and excess tolmetin glucuronide were coincubated in the presence of sodium cyanoborohydride to trap imine intermediates. The total protein product was reduced, carboxymethylated, and digested with trypsin. Six tolmetin-containing peptides (indicated by absorbance at 313 nm) were isolated by high-pressure liquid chromatography and analyzed by liquid secondary-ion mass spectrometry and collision-induced dissociation, using a four-sector tandem mass spectrometer. All six peptides contained tolmetin linked covalently via a glucuronic acid to protein lysine groups. Major attachment sites on the protein were Lys-195, -199, and -525; minor sites were identified as Lys-137, -351, and -541. Our results show unambiguously that the glucuronic acid moiety of acyl glucuronides can be retained within the structure when these reactive metabolites bind covalently to proteins, and they suggest that acyl migration followed by Schiff base (imine) formation is a credible mechanism for the generation of covalent adducts in vivo. PMID:8483897

  3. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

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

    PubMed

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

    2016-05-11

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

  5. Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes.

    PubMed

    Datta, S C; Ghosh, M K; Hajra, A K

    1990-05-15

    The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75

  6. X-Ray Crystal Structure of Mycobacterium Tuberculosis β-Ketoacyl Acyl Carrier Protein Synthase II (mtKasB)

    PubMed Central

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

    2007-01-01

    Summary Mycolic acids are long chain α-alkyl branched, β-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C56) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis β-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis β-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 Å resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds which inhibit

  7. A Mechanochemical Switch to Control Radical Intermediates

    PubMed Central

    2015-01-01

    B12-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side reactions. Using hybrid quantum mechanical/molecular mechanical simulations, we follow the full catalytic cycle of an AdoB12-dependent enzyme and present the details of a mechanism that utilizes a highly effective mechanochemical switch. When the switch is “off”, the 5′-deoxyadenosyl radical moiety is stabilized by releasing the internal strain of an enzyme-imposed conformation. Turning the switch “on,” the enzyme environment becomes the driving force to impose a distinct conformation of the 5′-deoxyadenosyl radical to avoid deleterious radical transfer. This mechanochemical switch illustrates the elaborate way in which enzymes attain selectivity of extremely chemically challenging reactions. PMID:24846280

  8. A mechanochemical switch to control radical intermediates.

    PubMed

    Brunk, Elizabeth; Kellett, Whitney F; Richards, Nigel G J; Rothlisberger, Ursula

    2014-06-17

    B₁₂-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side reactions. Using hybrid quantum mechanical/molecular mechanical simulations, we follow the full catalytic cycle of an AdoB₁₂-dependent enzyme and present the details of a mechanism that utilizes a highly effective mechanochemical switch. When the switch is "off", the 5'-deoxyadenosyl radical moiety is stabilized by releasing the internal strain of an enzyme-imposed conformation. Turning the switch "on," the enzyme environment becomes the driving force to impose a distinct conformation of the 5'-deoxyadenosyl radical to avoid deleterious radical transfer. This mechanochemical switch illustrates the elaborate way in which enzymes attain selectivity of extremely chemically challenging reactions. PMID:24846280

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases.

    PubMed

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

    2016-07-01

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

  11. Protein S-ACYL Transferase10 is critical for development and salt tolerance in Arabidopsis.

    PubMed

    Zhou, Liang-Zi; Li, Sha; Feng, Qiang-Nan; Zhang, Yu-Ling; Zhao, Xinying; Zeng, Yong-lun; Wang, Hao; Jiang, Liwen; Zhang, Yan

    2013-03-01

    Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins. PMID:23482856

  12. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    PubMed Central

    Bagautdinov, Bagautdin; Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-01-01

    The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P21212, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain. PMID:18453702

  13. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8.

    PubMed

    Bagautdinov, Bagautdin; Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The beta-ketoacyl-(acyl carrier protein) synthases (beta-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 A resolution. The crystal is orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 A, and contains one homodimer in the asymmetric unit. The subunits adopt the well known alpha-beta-alpha-beta-alpha thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ;open' conformation of the Phe396 side chain. PMID:18453702

  14. Structure and mechanism of ORF36, an Aminosugar Oxidizing Enzyme in Everninomicin Biosynthesis†

    PubMed Central

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T. M.

    2010-01-01

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitrosugar, l-evernitrose, analogs of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically-generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of 18O2 establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products, and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 Å resolution x-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-coA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36. PMID:20866105

  15. Structure and Mechanism of ORF36, an Amino Sugar Oxidizing Enzyme in Everninomicin Biosynthesis

    SciTech Connect

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T.M.

    2010-12-07

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, L-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-L-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-L-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of {sup 18}O{sub 2} establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 {angstrom} resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

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

    PubMed

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

    2016-01-01

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

  17. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

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

    2016-01-01

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

  18. Acylated flavonol glycosides from the forage legume, Onobrychis viciifolia (sainfoin).

    PubMed

    Veitch, Nigel C; Regos, Ionela; Kite, Geoffrey C; Treutter, Dieter

    2011-04-01

    Ten acylated flavonol glycosides were isolated from aqueous acetone extracts of the aerial parts of the forage legume, Onobrychis viciifolia, and their structures determined using spectroscopic methods. Among these were eight previously unreported examples which comprised either feruloylated or sinapoylated derivatives of 3-O-di- and 3-O-triglycosides of kaempferol (3,5,7,4'-tetrahydroxyflavone) or quercetin (3,5,7,3',4'-pentahydroxyflavone). The diglycosides were acylated at the primary Glc residue of O-α-Rhap(1→6)-β-Glcp (rutinose), whereas the triglycosides were acylated at the terminal Rha residues of the branched trisaccharides, O-α-Rhap(1→2)[α-Rhap(1→6)]-β-Galp or O-α-Rhap(1→2)[α-Rhap(1→6)]-β-Glcp. Identification of the primary 3-O-linked hexose residues as either Gal or Glc was carried out by negative ion electrospray and serial MS, and cryoprobe NMR spectroscopy. Analysis of UV and MS spectra of the acylated flavonol glycosides provided additional diagnostic features relevant to direct characterisation of these compounds in hyphenated analyses. Quantitative analysis of the acylated flavonol glycosides present in different aerial parts of sainfoin revealed that the highest concentrations were in mature leaflets. PMID:21292287

  19. An Artificial Enzyme Made by Covalent Grafting of an Fe(II) Complex into β-Lactoglobulin: Molecular Chemistry, Oxidation Catalysis, and Reaction-Intermediate Monitoring in a Protein.

    PubMed

    Buron, Charlotte; Sénéchal-David, Katell; Ricoux, Rémy; Le Caër, Jean-Pierre; Guérineau, Vincent; Méjanelle, Philippe; Guillot, Régis; Herrero, Christian; Mahy, Jean-Pierre; Banse, Frédéric

    2015-08-17

    An artificial metalloenzyme based on the covalent grafting of a nonheme Fe(II) polyazadentate complex into bovine β-lactoglobulin has been prepared and characterized by using various spectroscopic techniques. Attachment of the Fe(II) catalyst to the protein scaffold is shown to occur specifically at Cys121. In addition, spectrophotometric titration with cyanide ions based on the spin-state conversion of the initial high spin (S=2) Fe(II) complex into a low spin (S=0) one allows qualitative and quantitative characterization of the metal center's first coordination sphere. This biohybrid catalyst activates hydrogen peroxide to oxidize thioanisole into phenylmethylsulfoxide as the sole product with an enantiomeric excess of up to 20 %. Investigation of the reaction between the biohybrid system and H2 O2 reveals the generation of a high spin (S=5/2) Fe(III) (η(2) -O2 ) intermediate, which is proposed to be responsible for the catalytic sulfoxidation of the substrate. PMID:26178593

  20. A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis

    PubMed Central

    Jenkins, Ronald J.; Dotson, Garry D.

    2012-01-01

    UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of Lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among Gram-negative bacteria which synthesize lipopolysaccharide, and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP) dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Herein, we exploit the single free-thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol specific labeling reagent, ThioGlo. The assay is continuously monitored as a change in fluorescence at λex = 379 nm and λem = 513 nm using a microtiter plate reader. This assay marks the first continuous and non-radioactive assay for either acyltransferase. PMID:22381368

  1. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  2. A shotgun lipidomics study of a putative lysophosphatidic acid acyl transferase (PlsC) in Sinorhizobium meliloti.

    PubMed

    Basconcillo, Libia Saborido; Zaheer, Rahat; Finan, Turlough M; McCarry, Brian E

    2009-09-15

    A shotgun lipidomics approach was used to study the knockout mutant of a putative lysophosphatidic acyl acid transferase (PlsC) in order to delineate the function of this enzyme in Sinorhizobium meliloti. In plsC knockout mutant lipids that contained 16:0 and 16:1 fatty acids and their biosynthetically related cyclopropane fatty acid (cis-9,10-methylene hexadecanoic acid) decreased up to 93%. Tandem mass spectrometry experiments in the presence of added Li(+) showed that the putative PlsC (SMc00714) functioned as a lysophosphatidic acid acyl transferase specific for the transfer of C16 fatty acids to the sn-2 position of lipids. The levels of lipids containing C18 fatty acids were unaffected in plsC mutant, suggesting the presence of one or more fatty acyl transferases in the genome of S. meliloti with selectivity towards C18 fatty acids. Two non-phosphorus containing lipid classes, sulfoquinovosyldiacylglycerol and 1,2-diacylglyceryl-trimethylhomoserine lipids, showed similar decreases in C16 fatty acid content as phospholipids in plsC knockout mutant; these non-phosphorus containing lipids share a common biosynthetic origin with phospholipids, most likely involving phosphatidic acid. Ornithine lipids containing C16 fatty acids also showed decreased levels in PlsC knockout mutant, suggesting that PlsC is also involved in their biosynthesis. PMID:19525157

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

    PubMed

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

    2015-10-20

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

  4. Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress.

    PubMed

    Hentchel, Kristy L; Escalante-Semerena, Jorge C

    2015-09-01

    Acylation of biomolecules (e.g., proteins and small molecules) is a process that occurs in cells of all domains of life and has emerged as a critical mechanism for the control of many aspects of cellular physiology, including chromatin maintenance, transcriptional regulation, primary metabolism, cell structure, and likely other cellular processes. Although this review focuses on the use of acetyl moieties to modify a protein or small molecule, it is clear that cells can use many weak organic acids (e.g., short-, medium-, and long-chain mono- and dicarboxylic aliphatics and aromatics) to modify a large suite of targets. Acetylation of biomolecules has been studied for decades within the context of histone-dependent regulation of gene expression and antibiotic resistance. It was not until the early 2000s that the connection between metabolism, physiology, and protein acetylation was reported. This was the first instance of a metabolic enzyme (acetyl coenzyme A [acetyl-CoA] synthetase) whose activity was controlled by acetylation via a regulatory system responsive to physiological cues. The above-mentioned system was comprised of an acyltransferase and a partner deacylase. Given the reversibility of the acylation process, this system is also referred to as reversible lysine acylation (RLA). A wealth of information has been obtained since the discovery of RLA in prokaryotes, and we are just beginning to visualize the extent of the impact that this regulatory system has on cell function. PMID:26179745

  5. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    SciTech Connect

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  6. Acylated sucroses and acylated quinic acids analogs from the flower buds of Prunus mume and their inhibitory effect on melanogenesis.

    PubMed

    Nakamura, Seikou; Fujimoto, Katsuyoshi; Matsumoto, Takahiro; Nakashima, Souichi; Ohta, Tomoe; Ogawa, Keiko; Matsuda, Hisashi; Yoshikawa, Masayuki

    2013-08-01

    The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang Province, China, showed an inhibitory effect on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the methanolic extract, five acylated sucroses, mumeoses A-E, and three acylated quinic acid analogs, 5-O-(E)-p-coumaroylquinic acid ethyl ester, and mumeic acid-A and its methyl ester, were isolated together with 13 known compounds. The chemical structures of the compounds were elucidated on the basis of chemical and physicochemical evidence. Inhibitory effects of the isolated compounds on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were also investigated. Acylated quinic acid analogs substantially inhibited melanogenesis. In particular, 5-O-(E)-feruloylquinic acid methyl ester exhibited a potent inhibitory effect [inhibition (%): 21.5±1.0 (P<0.01) at 0.1 μM]. Moreover, its biological effect was much stronger than that of the reference compound, arbutin [inhibition (%): 10.6±0.6 (P<0.01) at 10 μM]. Interestingly, the obtained acylated quinic acid analogs displaying melanogenesis inhibitory activity showed no cytotoxicity [cell viability >97% at 10 μM]. It is concluded that acylated quinic acid analogs are promising therapeutic agents for the treatment of skin disorders. PMID:23693120

  7. Asymmetric Allylboration of Acyl Imines Catalyzed by Chiral Diols

    PubMed Central

    Lou, Sha; Moquist, Philip N.; Schaus, Scott E.

    2008-01-01

    Chiral BINOL-derived diols catalyze the enantioselective asymmetric allylboration of acyl imines. The reaction requires 15 mol% of (S)-3,3′-Ph2-BINOL as the catalyst and allyldiisopropoxyborane as the nucleophile. The reaction products are obtained in good yields (75 – 94%) and high enantiomeric ratios (95:5 – 99.5:0.5) for aromatic and aliphatic imines. High diastereoselectivities (dr > 98:2) and enantioselectivities (er > 98:2) are obtained in the reactions of acyl imines with crotyldiisopropoxyboranes. This asymmetric transformation is directly applied to the synthesis of maraviroc, the selective CCR5 antagonist with potent activity against HIV-1 infection. Mechanistic investigations of the allylboration reaction including IR, NMR, and mass spectrometry study indicate that acyclic boronates are activated by chiral diols via exchange of one of the boronate alkoxy groups with activation of the acyl imine via hydrogen bonding. PMID:18020334

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

    SciTech Connect

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

    2005-11-11

    Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC{sub 50} values in the range 5.1-8.2 {mu}M), whereas the two compounds with a single unsaturated bond were less potent (IC{sub 50} values 19 and 21 {mu}M). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC{sub 50} values of 12 and 32 {mu}M, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC{sub 50} value 4.5 {mu}M). Introduction of an {alpha}-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.

  9. Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. I. Production, isolation, and biological properties.

    PubMed

    Tomoda, H; Kim, Y K; Nishida, H; Masuma, R; Omura, S

    1994-02-01

    Aspergillus fumigatus FO-1289, a soil isolate, was found to produce a series of novel inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Four active compounds, named pyripyropenes A, B, C and D, were isolated from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography, ODS column chromatography and preparative HPLC. Pyripyropenes A, B, C and D show very potent ACAT inhibitory activity in an enzyme assay system using rat liver microsomes with IC50 values of 58, 117, 53 and 268 nM, respectively. PMID:8150709

  10. Differential acylation in vitro with tetradecanoyl coenzyme A and tetradecanoic acid (+ATP) of three polypeptides shown to have induced synthesis in Photobacterium phosphoreum

    SciTech Connect

    Wall, L.; Rodriguez, A.; Meighen, E.

    1984-02-01

    Acylation of extracts of Photobacterium phosphoreum at different stages of growth with (/sup 3/H)tetradecanoic acid (+ATP) has shown that two polypeptides found in the fatty acid reductase complex, the fatty acid activating enzyme (50K) and the 34K polypeptide, were specifically labeled during induction of the luminescent system. An alternate method for in vitro acylation of polypeptides in the luminescence system was developed using tetradecanoyl-CoA. Both the 34K polypeptide and, to a lesser extent, the acyl-CoA reductase component (58K) in the complex, were acylated with (/sup 3/H)tetradecanoyl-CoA. In contrast, the fatty acid activating enzyme (50K) was not labeled. Labeling of both the 34K and 58K polypeptides with (/sup 3/H)tetradecanoyl-CoA as well as the acyl-CoA reductase activity in extracts paralleled the induction of luciferase during growth. Differential labeling of P. phosphoreum cells with (/sup 35/S)methionine before luminescence induction and with (/sup 3/H)methionine after the onset of luminescence followed by purification of luciferase and the polypeptides in the fatty acid reductase complex demonstrated that the ..cap alpha.. and ..beta.. subunits of luciferase and the 34K, 50K and 58K polypeptides of the complex had /sup 3/H//sup 35/S ratios at least 7-fold higher than the constitutive proteins. These results give evidence that the synthesis of the component polypeptides of the fatty acid reductase are induced during the development of bioluminescence and may be under the same control as luciferase. The experiments also showed that P. phosphoreum may have the highest content of luciferase of any luminescent bacterium, constituting approximately 20% of the total soluble protein in extracts.

  11. Chemical and Biochemical Transfer of Acyl Groups: A New Look at an Old Mechanism

    ERIC Educational Resources Information Center

    Douglas, Kenneth T.; Williams, Andrew

    1976-01-01

    Examines recent studies of the elimination-addition mechanism of acyl group transfer, in which an acid function moves from one acceptor to another. Presents diagnostic evidence for this mechanism and discusses acyl group transfers in metabolism. (MLH)

  12. Direct N-acylation of lactams, oxazolidinones, and imidazolidinones with aldehydes by Shvo's catalyst.

    PubMed

    Zhang, Jian; Hong, Soon Hyeok

    2012-09-01

    Direct N-acylation of lactams, oxazolidinones, and imidazolidinones was achieved with aldehydes by Shvo's catalyst without using any other stoichiometric reagent. The N-acylations with α,β-unsaturated aldehydes were achieved with excellent yields. PMID:22913512

  13. Synthesis, crystal structure, and in vitro and in silico molecular docking of novel acyl thiourea derivatives

    NASA Astrophysics Data System (ADS)

    Haribabu, Jebiti; Subhashree, Govindarajulu Rangabashyam; Saranya, Sivaraj; Gomathi, Kannayiram; Karvembu, Ramasamy; Gayathri, Dasararaju

    2015-08-01

    In the present study, a series of six biologically active substituted acyl thiourea compounds (1-6) has been synthesized from cyclohexanecarbonyl isothiocyanate and various primary amines (2-methyl aniline, aniline, 4-methoxy aniline, 4-ethoxy aniline, benzyl amine and 2-methoxy aniline). The synthesized compounds were characterized by elemental analyses, UV-Visible, FT-IR, 1H & 13C NMR and mass spectroscopic techniques. Three dimensional molecular structure of two compounds (1 and 5) was determined by single crystal X-ray crystallography. All the synthesized compounds show good anti-oxidant and anti-haemolytic activities. In silico molecular docking studies were performed to screen against DprE1 and HSP90 enzymes targeting tuberculosis and cancer respectively.

  14. Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

    PubMed Central

    Jiang, D W; Englund, P T

    2001-01-01

    As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

  15. Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides

    PubMed Central

    He, Xin; Alian, Akram; Ortiz de Montellano, Paul R.

    2007-01-01

    InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screening led to the discovery of a potent InhA inhibitor with in vitro IC50 = 90 nM, representing a 34-fold potency improvement over the lead compound. PMID:17723305

  16. N-Acyl Dehydrotyrosines, Tyrosinase Inhibitors from the Marine Bacterium Thalassotalea sp. PP2-459.

    PubMed

    Deering, Robert W; Chen, Jianwei; Sun, Jiadong; Ma, Hang; Dubert, Javier; Barja, Juan L; Seeram, Navindra P; Wang, Hong; Rowley, David C

    2016-02-26

    Thalassotalic acids A-C and thalassotalamides A and B are new N-acyl dehydrotyrosine derivatives produced by Thalassotalea sp. PP2-459, a Gram-negative bacterium isolated from a marine bivalve aquaculture facility. The structures were elucidated via a combination of spectroscopic analyses emphasizing two-dimensional NMR and high-resolution mass spectrometric data. Thalassotalic acid A (1) displays in vitro inhibition of the enzyme tyrosinase with an IC50 value (130 μM) that compares favorably to the commercially used control compounds kojic acid (46 μM) and arbutin (100 μM). These are the first natural products reported from a bacterium belonging to the genus Thalassotalea. PMID:26824128

  17. Purification and characterization of variants of acyl-CoA-binding protein in the bovine liver.

    PubMed Central

    Jensen, M S; Højrup, P; Rasmussen, J T; Knudsen, J

    1992-01-01

    Four differently modified forms of acyl-CoA-binding protein (ACBP) were identified in ACBP purified from bovine liver. The majority of the purified ACBP was focused at pH 5.9 in isoelectric focusing and could be shown to be N-acetylated ACBP without any further modifications. Two minor peaks were focused at pH 5.25 and 4.85 respectively. Mass spectrometry and sequence determination showed that the pI 5.25 form was acetylated at Lys18 and that the pI 4.85 form was malonylated in the same position. Furthermore, it could be shown that non-enzymic glycosylation occurred during purification. The acetylated and malonylated variants of ACBP were only found in adult cattle. Images Fig. 5. PMID:1622397

  18. The enzymes associated with denitrification

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  19. Giardia fatty acyl-CoA synthetases as potential drug targets

    PubMed Central

    Guo, Fengguang; Ortega-Pierres, Guadalupe; Argüello-García, Raúl; Zhang, Haili; Zhu, Guan

    2015-01-01

    Giardiasis caused by Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the leading causes of diarrheal parasitic diseases worldwide. Although limited drugs to treat giardiasis are available, there are concerns regarding toxicity in some patients and the emerging drug resistance. By data-mining genome sequences, we observed that G. intestinalis is incapable of synthesizing fatty acids (FA) de novo. However, this parasite has five long-chain fatty acyl-CoA synthetases (GiACS1 to GiACS5) to activate FA scavenged from the host. ACS is an essential enzyme because FA need to be activated to form acyl-CoA thioesters before they can enter subsequent metabolism. In the present study, we performed experiments to explore whether some GiACS enzymes could serve as drug targets in Giardia. Based on the high-throughput datasets and protein modeling analyses, we initially studied the GiACS1 and GiACS2, because genes encoding these two enzymes were found to be more consistently expressed in varied parasite life cycle stages and when interacting with host cells based on previously reported transcriptome data. These two proteins were cloned and expressed as recombinant proteins. Biochemical analysis revealed that both had apparent substrate preference toward palmitic acid (C16:0) and myristic acid (C14:0), and allosteric or Michaelis–Menten kinetics on palmitic acid or ATP. The ACS inhibitor triacsin C inhibited the activity of both enzymes (IC50 = 1.56 μM, Ki = 0.18 μM for GiACS1, and IC50 = 2.28 μM, Ki = 0.23 μM for GiACS2, respectively) and the growth of G. intestinalis in vitro (IC50 = 0.8 μM). As expected from giardial evolutionary characteristics, both GiACSs displayed differences in overall folding structure as compared with their human counterparts. These observations support the notion that some of the GiACS enzymes may be explored as drug targets in this parasite. PMID:26257723

  20. Enzyme Action in the Regulation of Plant Hormone Responses*

    PubMed Central

    Westfall, Corey S.; Muehler, Ashley M.; Jez, Joseph M.

    2013-01-01

    Plants synthesize a chemically diverse range of hormones that regulate growth, development, and responses to environmental stresses. The major classes of plant hormones are specialized metabolites with exquisitely tailored perception and signaling systems, but equally important are the enzymes that control the dose and exposure to the bioactive forms of these molecules. Here, we review new insights into the role of enzyme families, including the SABATH methyltransferases, the methylesterases, the GH3 acyl acid-amido synthetases, and the hormone peptidyl hydrolases, in controlling the biosynthesis and modifications of plant hormones and how these enzymes contribute to the network of chemical signals responsible for plant growth, development, and environmental adaptation. PMID:23709222

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

    PubMed Central

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

    2014-01-01

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

  2. Novel B(12)-dependent acyl-CoA mutases and their biotechnological potential.

    PubMed

    Cracan, Valentin; Banerjee, Ruma

    2012-08-01

    The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5'-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a triose phosphate isomerase (TIM)-barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA; and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals. PMID:22803641

  3. Novel B12-dependent Acyl-CoA Mutases and their Biotechnological Potential†

    PubMed Central

    Valentin, Cracan; Banerjee, Ruma

    2012-01-01

    The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5′-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a TIM barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA, and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals. PMID:22803641

  4. Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

    PubMed Central

    Kemp, Louise E.; Rusch, Marion; Adibekian, Alexander; Bullen, Hayley E.; Graindorge, Arnault; Freymond, Céline; Rottmann, Matthias; Braun-Breton, Catherine; Baumeister, Stefan; Porfetye, Arthur T.; Vetter, Ingrid R.; Hedberg, Christian; Soldati-Favre, Dominique

    2013-01-01

    In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein. PMID:23913689

  5. Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds. [Lunaria annua L. ; Sinapis alba L

    SciTech Connect

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

    1990-10-01

    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-{sup 14}C)oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and (2-{sup 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-{sup 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-{sup 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.

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

    PubMed

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

    2015-09-01

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

  7. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  8. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  9. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  10. Draft Genome Sequence of the Thermophile Thermus filiformis ATCC 43280, Producer of Carotenoid-(Di)glucoside-Branched Fatty Acid (Di)esters and Source of Hyperthermostable Enzymes of Biotechnological Interest

    PubMed Central

    Mandelli, Fernanda; Oliveira Ramires, Brenda; Couger, Matthew Brian; Paixão, Douglas A. A.; Camilo, Cesar M.; Polikarpov, Igor; Prade, Rolf

    2015-01-01

    Here, we present the draft genome sequence of Thermus filiformis strain ATCC 43280, a thermophile bacterium capable of producing glycosylated carotenoids acylated with branched fatty acids and enzymes of biotechnological potential. PMID:25977443

  11. Draft Genome Sequence of the Thermophile Thermus filiformis ATCC 43280, Producer of Carotenoid-(Di)glucoside-Branched Fatty Acid (Di)esters and Source of Hyperthermostable Enzymes of Biotechnological Interest.

    PubMed

    Mandelli, Fernanda; Oliveira Ramires, Brenda; Couger, Matthew Brian; Paixão, Douglas A A; Camilo, Cesar M; Polikarpov, Igor; Prade, Rolf; Riaño-Pachón, Diego M; Squina, Fabio M

    2015-01-01

    Here, we present the draft genome sequence of Thermus filiformis strain ATCC 43280, a thermophile bacterium capable of producing glycosylated carotenoids acylated with branched fatty acids and enzymes of biotechnological potential. PMID:25977443

  12. Synthesis and antihyperglycemic activity of novel N-acyl-2-arylethylamines and N-acyl-3-coumarylamines.

    PubMed

    Dwivedi, Atma P; Kumar, Shailesh; Varshney, Vandana; Singh, Amar B; Srivastava, Arvind K; Sahu, Devi P

    2008-04-01

    A series of novel N-acyl-2-arylethylamines and N-acyl-3-coumarylamines were synthesized and evaluated for their antihyperglycemic activity. Compounds 3g and 6d exhibited lowering of postprandial plasma glucose by 30.7%, 23.3% in SLM and 25.6%, 25.4% in STZ models respectively which is significant compared to metformin and glybenclamide. Other compounds exhibited moderate to good activity ranging from 19.5% to 32.8% in SLM and 3.26% to 25.4% in STZ models. PMID:18353644

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

    PubMed

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

    2016-08-01

    The medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that catalyzes the first step of mitochondrial fatty acid β-oxidation (mFAO) pathway. Its deficiency is the most common genetic disorder of mFAO. Many of the MCAD disease-causing variants, including the most common p.K304E variant, show loss of function due to protein misfolding. Herein, we used molecular dynamics simulations to provide insights into the structural stability and dynamic behavior of MCAD wild-type (MCADwt) and validate a structure that would allow reliable new studies on its variants. Our results revealed that in both proteins the flavin adenine dinucleotide (FAD) has an important structural role on the tetramer stability and also in maintaining the volume of the enzyme catalytic pockets. We confirmed that the presence of substrate changes the dynamics of the catalytic pockets and increases FAD affinity. A comparison between the porcine MCADwt (pMCADwt) and human MCADwt (hMCADwt) structures revealed that both proteins are essentially similar and that the reversion of the double mutant E376G/T255E of hMCAD enzyme does not affect the structure of the protein neither its behavior in simulation. Our validated hMCADwt structure is crucial for complementing and accelerating the experimental studies aiming for the discovery and development of potential stabilizers of MCAD variants as candidates for the treatment of MCAD deficiency (MCADD). PMID:26992026

  14. Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region

    SciTech Connect

    Chakravarty, R.; Rice, R.H.

    1989-01-05

    The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH/sub 2/ or COOH terminus.

  15. Mouse very long-chain acyl-CoA synthetase in X-linked adrenoleukodystrophy.

    PubMed

    Heinzer, Ann K; Kemp, Stephan; Lu, Jyh-Feng; Watkins, Paul A; Smith, Kirby D

    2002-08-01

    X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by accumulation of very long-chain fatty acids (VLCFA). This accumulation has been attributed to decreased VLCFA beta-oxidation and peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity. The X-ALD gene, ABCD1, encodes a peroxisomal membrane ATP binding cassette transporter, ALDP, that is hypothesized to affect VLCS activity in peroxisomes by direct interaction with the VLCS enzyme. Recently, a VLCS gene that encodes a protein with significant sequence identity to known rat and human peroxisomal VLCS protein has been identified in mice. We find that the mouse VLCS gene (Vlcs) encodes an enzyme (Vlcs) with VLCS activity that localizes to peroxisomes and is expressed in X-ALD target tissues. We show that the expression of Vlcs in the peroxisomes of X-ALD mouse fibroblasts improves VLCFA beta-oxidation in these cells, implying a role for this enzyme in the biochemical abnormality of X-ALD. X-ALD mice, which accumulate VLCFA in tissues, show no change in the expression of Vlcs, the subcellular localization of Vlcs, or general peroxisomal VLCS activity. These observations imply that ALDP is not necessary for the proper expression or localization of Vlcs protein, and the control of VLCFA levels does not depend on the direct interaction of Vlcs and ALDP. PMID:12048192

  16. The enzyme complex citramalate lyase from Clostridium tetanomorphum.

    PubMed

    Buckel, W; Bobi, A

    1976-04-15

    1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between

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

    PubMed

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

    2015-09-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. A new acylated isoflavone glucoside from Pterocarpus santalinus.

    PubMed

    Krishnaveni, K S; Srinivasa Rao, J V

    2000-09-01

    Phytochemical investigation on the constituents of heartwood of Pterocarpus santalinus resulted in the isolation of a new acylated isoflavone glucoside. The structure of the new compound was elucidated on the basis of spectral studies as 4',5-dihydroxy-7-O-methyl isoflavone 3'-O-D-(3''-E-cinnamoyl)glucoside. PMID:10993243

  20. Novel triterpenoid acyl esters and alkaloids from Anoectochilus roxburghii.

    PubMed

    Han, Mei-Hua; Yang, Xiu-Wei; Jin, Yan-Ping

    2008-01-01

    Two novel sorghumol acyl esters, sorghumol 3-O-Z-p-coumarate and sorghumol 3-O-E-p-coumarate, and a novel alkaloid, anoectochine, were isolated from the whole plants of Anoectochilus roxburghii along with one known triterpenoid, sorghumol. Their structures were established by their detailed spectral studies, including two-dimensional NMR ((1)H-(1)H COSY, HSQC and HMBC). PMID:18435530

  1. Lubricity characteristics of seed oils modified by acylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

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

  4. The enzymes of bacterial census and censorship.

    PubMed

    Fast, Walter; Tipton, Peter A

    2012-01-01

    N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication. PMID:22099187

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

    PubMed Central

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

    2014-01-01

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

  6. Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media.

    PubMed

    Zeuner, Birgitte; Kontogeorgis, Georgios M; Riisager, Anders; Meyer, Anne S

    2012-02-15

    Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources. PMID:22154740

  7. Enhanced Enzymatic Preparation of Biodiesel Using Ricinoleic Acid as Acyl Donor: Optimization Using Response Surface Methodology.

    PubMed

    Wang, Ping; Sun, Shangde

    2016-09-01

    Castor oil methyl ester is a kind of biodiesel from castor oil. However, in those previous methods for biodiesel preparation using castor oil as feedstock, glycerol was the main by-product, which had a strong blocking effect on the immobilized enzyme activity and affected the mass transfer of reaction system. For avoiding the negative effect of glycerol on the enzymatic esterification, biodiesel was prepared using ricinoleic acid (RA) as acyl donor. Enzyme screening was also studied, and the effects of reaction temperature, molar ratio of ricinoleic acid and methanol, enzyme load, and reaction time, on the preparation of castor methyl ester were also evaluated. Response surface methodology (RSM) was used to optimize the interaction effect of reaction variables (reaction temperature (30-70°C), enzyme load (2-7%; relative to the weight of total substrates), molar ratio of methanol to ricinoleic acid (2:1-10:1), and reaction time (0.5-2.5 h)) on the acid value (AV) and the degree of esterification (DE). Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of AV and DE. The optimum preparation conditions were as follows: reaction temperature, 48.2°C; enzyme load, 5.8%; molar ratio of methanol to ricinoleic acid, 5.56:1; reaction time, 2.36 h. Under these conditions, the AV and DE of the esterification reaction are 10.36±1.05 mgKOH/g and 94.03±0.60%, respectively. The relationship between initial reaction rate and temperature was also established, and the activation energy (Ea) of the enzymatic esterification is 33.87 KJ/mol. PMID:27477073

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

    PubMed

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

    2015-02-01

    Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants. PMID:25553535

  9. Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations.

    PubMed

    Reid, J D; Hussain, S; Sreedharan, S K; Bailey, T S; Pinitglang, S; Thomas, E W; Verma, C S; Brocklehurst, K

    2001-07-15

    The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over<8 ps) out of papain and caricain but 'lingers' around the active centre of actinidin. Uniquely in actinidin, an Asp142-Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 A) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically. PMID:11439083

  10. Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations.

    PubMed Central

    Reid, J D; Hussain, S; Sreedharan, S K; Bailey, T S; Pinitglang, S; Thomas, E W; Verma, C S; Brocklehurst, K

    2001-01-01

    The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over<8 ps) out of papain and caricain but 'lingers' around the active centre of actinidin. Uniquely in actinidin, an Asp142-Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 A) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically. PMID:11439083

  11. Bacterial Acyl-CoA Mutase Specifically Catalyzes Coenzyme B12-dependent Isomerization of 2-Hydroxyisobutyryl-CoA and (S)-3-Hydroxybutyryl-CoA*

    PubMed Central

    Yaneva, Nadya; Schuster, Judith; Schäfer, Franziska; Lede, Vera; Przybylski, Denise; Paproth, Torsten; Harms, Hauke; Müller, Roland H.; Rohwerder, Thore

    2012-01-01

    Coenzyme B12-dependent acyl-CoA mutases are radical enzymes catalyzing reversible carbon skeleton rearrangements in carboxylic acids. Here, we describe 2-hydroxyisobutyryl-CoA mutase (HCM) found in the bacterium Aquincola tertiaricarbonis as a novel member of the mutase family. HCM specifically catalyzes the interconversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA. Like isobutyryl-CoA mutase, HCM consists of a large substrate- and a small B12-binding subunit, HcmA and HcmB, respectively. However, it is thus far the only acyl-CoA mutase showing substrate specificity for hydroxylated carboxylic acids. Complete loss of 2-hydroxyisobutyric acid degradation capacity in hcmA and hcmB knock-out mutants established the central role of HCM in A. tertiaricarbonis for degrading substrates bearing a tert-butyl moiety, such as the fuel oxygenate methyl tert-butyl ether (MTBE) and its metabolites. Sequence analysis revealed several HCM-like enzymes in other bacterial strains not related to MTBE degradation, indicating that HCM may also be involved in other pathways. In all strains, hcmA and hcmB are associated with genes encoding for a putative acyl-CoA synthetase and a MeaB-like chaperone. Activity and substrate specificity of wild-type enzyme and active site mutants HcmA I90V, I90F, and I90Y clearly demonstrated that HCM belongs to a new subfamily of B12-dependent acyl-CoA mutases. PMID:22433853

  12. Bacterial acyl-CoA mutase specifically catalyzes coenzyme B12-dependent isomerization of 2-hydroxyisobutyryl-CoA and (S)-3-hydroxybutyryl-CoA.

    PubMed

    Yaneva, Nadya; Schuster, Judith; Schäfer, Franziska; Lede, Vera; Przybylski, Denise; Paproth, Torsten; Harms, Hauke; Müller, Roland H; Rohwerder, Thore

    2012-05-01

    Coenzyme B(12)-dependent acyl-CoA mutases are radical enzymes catalyzing reversible carbon skeleton rearrangements in carboxylic acids. Here, we describe 2-hydroxyisobutyryl-CoA mutase (HCM) found in the bacterium Aquincola tertiaricarbonis as a novel member of the mutase family. HCM specifically catalyzes the interconversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA. Like isobutyryl-CoA mutase, HCM consists of a large substrate- and a small B(12)-binding subunit, HcmA and HcmB, respectively. However, it is thus far the only acyl-CoA mutase showing substrate specificity for hydroxylated carboxylic acids. Complete loss of 2-hydroxyisobutyric acid degradation capacity in hcmA and hcmB knock-out mutants established the central role of HCM in A. tertiaricarbonis for degrading substrates bearing a tert-butyl moiety, such as the fuel oxygenate methyl tert-butyl ether (MTBE) and its metabolites. Sequence analysis revealed several HCM-like enzymes in other bacterial strains not related to MTBE degradation, indicating that HCM may also be involved in other pathways. In all strains, hcmA and hcmB are associated with genes encoding for a putative acyl-CoA synthetase and a MeaB-like chaperone. Activity and substrate specificity of wild-type enzyme and active site mutants HcmA I90V, I90F, and I90Y clearly demonstrated that HCM belongs to a new subfamily of B(12)-dependent acyl-CoA mutases. PMID:22433853

  13. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  14. N-Acylsaccharins: Stable Electrophilic Amide-Based Acyl Transfer Reagents in Pd-Catalyzed Suzuki-Miyaura Coupling via N-C Cleavage.

    PubMed

    Liu, Chengwei; Meng, Guangrong; Liu, Yongmei; Liu, Ruzhang; Lalancette, Roger; Szostak, Roman; Szostak, Michal

    2016-09-01

    The development of efficient catalytic methods for N-C bond cleavage in amides remains an important synthetic challenge. The first Pd-catalyzed Suzuki-Miyaura cross-coupling of N-acylsaccharins with boronic acids by selective N-C bond activation is reported. The reaction enables preparation of a variety of functionalized diaryl and alkyl-aryl ketones with broad functional group tolerance and in good to excellent yields. Of general interest, N-acylsaccharins serve as new, highly reactive, bench-stable, economical, amide-based, electrophilic acyl transfer reagents via acyl-metal intermediates. Mechanistic studies strongly support the amide N-C(O) bond twist as the enabling feature of N-acylsaccharins in the N-C bond cleavage. PMID:27513821

  15. X-ray, FT-IR, NMR and PM5 structural studies and antibacterial activity of unexpectedly stable salinomycin-benzotriazole intermediate ester

    NASA Astrophysics Data System (ADS)

    Huczyński, Adam; Janczak, Jan; Antoszczak, Michał; Stefańska, Joanna; Brzezinski, Bogumil

    2012-08-01

    The unexpectedly stable benzotriazole ester of salinomycin (SAL-HOBt) - an intermediate product of the amidation reaction of salinomycin has been isolated and structurally characterised (using a single crystal) by X-ray, FT-IR, NMR and semiempirical methods. The results of the X-ray and spectroscopic studies demonstrated that this intermediate ester exist in the solid state and in solution exclusively as the stable O-acyl form. The molecular structure of SAL-HOBt is stabilised by relatively weak intramolecular hydrogen bonds. The PM5 calculation of possible structures of SAL-HOBt has shown that the O-acyl form is more energetically favourable than its N-oxide-N-acyl isomers. The antimicrobial tests show that SAL-HOBt is active against Gram-positive bacteria and clinical isolates methicillin-resistant Staphylococcus aureus (MIC = 1-2 μg/ml).

  16. Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase

    PubMed Central

    Ye, Zhixia; Musiol, Ewa M; Weber, Tilmann; Williams, Gavin J

    2014-01-01

    SUMMARY Protein interactions between acyl carrier proteins (ACP’s) and trans-acting acyltransferase domains (trans-AT’s) are critical for regioselective extender unit installation by many polyketide synthases. Yet, little is known regarding the specificity of these interactions, particularly for trans-AT’s with unusual extender unit specificities. Currently, the best-studied trans-AT with non-malonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed a new assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a non-cognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions, and for engineering modular polyketide synthases to produce analogues. PMID:24726832

  17. Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions.

    PubMed

    Ngo, Tien Anh; Nakata, Eiji; Saimura, Masayuki; Morii, Takashi

    2016-03-01

    We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes. PMID:26881296

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase

    PubMed Central

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D.; Thinon, Emmanuelle; Rodgers, Ursula R.; Owens, Raymond J.; Magee, Anthony I.; Tate, Edward W.

    2016-01-01

    In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI”) class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015) [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors. PMID:27077078

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

    PubMed

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

    2016-01-01

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

  2. Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase.

    PubMed

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D; Thinon, Emmanuelle; Rodgers, Ursula R; Owens, Raymond J; Magee, Anthony I; Tate, Edward W

    2016-06-01

    In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed "RU-SKI") class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article "Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase" (Lanyon-Hogg et al., 2015) [1]. (1)H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors. PMID:27077078

  3. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  4. Time-resolved heme protein intermediates

    NASA Astrophysics Data System (ADS)

    Rousseau, Denis

    2005-03-01

    To determine the enzymatic mechanisms of heme proteins, it is necessary to identify the intermediates along the catalytic pathway and measure the times of their formation and decay. Resonance Raman scattering spectra are especially powerful for obtaining such information as the electronic structure of the heme group and the nature of the ligand coordinated to the heme iron atom may be monitored. The oxygen intermediates of two physiologically important enzymes will be presented. Nitric oxide synthase (NOS) uses oxygen to convert arginine to NO and citrulline; and cytochrome c oxidase (CcO) reduces oxygen to water to support oxidative phosphorylation. The fate or the oxygen in each of these enzymes has been followed by resonance Raman scattering. In NOS the oxygen is slowly converted to an activated species that then reacts fast, whereas in CcO the oxygen is rapidly converted to a reactive species that subsequently reacts slowly. The properties of the intermediates and the origin of the differences between these enzymes will be discussed.

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

    PubMed

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

    2015-08-01

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

  6. The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase

    PubMed Central

    Ma, Qingjun; Zhao, Xin; Eddine, Ali Nasser; Geerlof, Arie; Li, Xinping; Cronan, John E.; Kaufmann, Stefan H. E.; Wilmanns, Matthias

    2006-01-01

    Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4′-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB. PMID:16735476

  7. Topology of 1-Acyl-sn-glycerol-3-phosphate Acyltransferases SLC1 and ALE1 and Related Membrane-bound O-Acyltransferases (MBOATs) of Saccharomyces cerevisiae*

    PubMed Central

    Pagac, Martin; de la Mora, Hector Vazquez; Duperrex, Cécile; Roubaty, Carole; Vionnet, Christine; Conzelmann, Andreas

    2011-01-01

    In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed. PMID:21849510

  8. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    PubMed Central

    Nanson, Jeffrey D.; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  9. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

    PubMed

    Nanson, Jeffrey D; Himiari, Zainab; Swarbrick, Crystall M D; Forwood, Jade K

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  10. Characterization of N-Acyl Phosphatidylethanolamine-Specific Phospholipase-D Isoforms in the Nematode Caenorhabditis elegans

    PubMed Central

    Harrison, Neale; Lone, Museer A.; Kaul, Tiffany K.; Reis Rodrigues, Pedro; Ogungbe, Ifedayo Victor; Gill, Matthew S.

    2014-01-01

    N-acylethanolamines are an important class of lipid signaling molecules found in many species, including the nematode Caenorhabditis elegans (C. elegans) where they are involved in development and adult lifespan. In mammals, the relative activity of the biosynthetic enzyme N-acyl phosphatidylethanolamine-specific phospholipase-D and the hydrolytic enzyme fatty acid amide hydrolase determine N-acylethanolamine levels. C. elegans has two N-acyl phosphatidylethanolamine-specific phospholipase-D orthologs, nape-1 and nape-2, that are likely to have arisen from a gene duplication event. Here, we find that recombinant C. elegans NAPE-1 and NAPE-2 are capable of generating N-acylethanolamines in vitro, confirming their functional conservation. In vivo, they exhibit overlapping expression in the pharynx and the nervous system, but are also expressed discretely in these and other tissues, suggesting divergent roles. Indeed, nape-1 over-expression results in delayed growth and shortened lifespan only at 25°C, while nape-2 over-expression results in significant larval arrest and increased adult lifespan at 15°C. Interestingly, deletion of the N-acylethanolamine degradation enzyme faah-1 exacerbates nape-1 over-expression phenotypes, but suppresses the larval arrest phenotype of nape-2 over-expression, suggesting that faah-1 is coupled to nape-2, but not nape-1, in a negative feedback loop. We also find that over-expression of either nape-1 or nape-2 significantly enhances recovery from the dauer larval stage in the insulin signaling mutant daf-2(e1368), but only nape-1 over-expression reduces daf-2 adult lifespan, consistent with increased levels of the N-acylethanolamine eicosapentaenoyl ethanolamine. These results provide evidence that N-acylethanolamine biosynthetic enzymes in C. elegans have conserved function and suggest a temperature-dependent, functional divergence between the two isoforms. PMID:25423491

  11. Radical SAM catalysis via an organometallic intermediate with an Fe-[5'-C]-deoxyadenosyl bond.

    PubMed

    Horitani, Masaki; Shisler, Krista; Broderick, William E; Hutcheson, Rachel U; Duschene, Kaitlin S; Marts, Amy R; Hoffman, Brian M; Broderick, Joan B

    2016-05-13

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes. PMID:27174986

  12. A multisubstrate assay for lipases/esterases: assessing acyl chain length selectivity by reverse-phase high-performance liquid chromatography.

    PubMed

    Divakar, K; Gautam, Pennathur

    2014-03-01

    Lipases and esterases are hydrolytic enzymes and are known to hydrolyze esters with unique substrate specificity and acyl chain length selectivity. We have developed a simple competitive multiple substrate assay for determination of acyl chain length selectivity of lipases/esterases using RP-HPLC with UV detection. A method for separation and quantification of 4-nitrophenyl fatty acid esters (C4-C18) was developed and validated. The chain length selectivity of five lipases and two esterases was determined in a multisubstrate reaction system containing equimolar concentrations of 4-nitrophenyl esters (C4-C18). This assay is simple, reproducible, and a useful tool for determining chain length selectivity of lipases/esterases. PMID:24316114

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

    PubMed

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

    2016-07-01

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

  14. A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase[W

    PubMed Central

    Dayan, Franck E.; Ferreira, Daneel; Wang, Yan-Hong; Khan, Ikhlas A.; McInroy, John A.; Pan, Zhiqiang

    2008-01-01

    Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the π-π stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD+. Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection. PMID:18467464

  15. Prolyl oligopeptidase inhibition by N-acyl-pro-pyrrolidine-type molecules.

    PubMed

    Kánai, Károly; Arányi, Péter; Böcskei, Zsolt; Ferenczy, György; Harmat, Veronika; Simon, Kálmán; Bátori, Sándor; Náray-Szabo, Gábor; Hermecz, István

    2008-12-11

    Three novel, N-acyl-pro-pyrrolidine-type, inhibitors of prolyl oligopeptidase (POP) with nanomolar activities were synthesized and their binding analyzed to the host enzyme in the light of X-ray diffraction and molecular modeling studies. We were interested in the alteration in the binding affinity at the S3 site as a function of the properties of the N-terminal group of the inhibitors. Our studies revealed that, for inhibitors with flat aromatic terminal groups, the optimal length of the linker chain is three C-C bonds, but this increases to four C-C bonds if there is a bulky group in the terminal position. Molecular dynamics calculations indicate that this is due to the better fit into the binding pocket. A 4-fold enhancement of the inhibitor activity upon replacement of the 4-CH2 group of the proline ring by CF2 is a consequence of a weak hydrogen bond formed between the fluorine atom and the hydroxy group of Tyr473 of the host enzyme. There is notably good agreement between the calculated and experimental free energies of binding; the average error in the IC50 values is around 1 order of magnitude. PMID:19006380

  16. The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple classes of acyl-CoA binding proteins are encoded by plant genomes, including a plant-unique class of predicted integral membrane-proteins. Transcript analysis revealed that both of the integral membrane-acyl-CoA binding proteins of Arabidopsis thaliana, ACBP1 and ACBP2, are expressed in al...

  17. 40 CFR 180.1207 - N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false N-acyl sarcosines and sodium N-acyl sarcosinates; exemption from the requirement of a tolerance. 180.1207 Section 180.1207 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD...

  18. New acylated anthocyanins from purple yam and their antioxidant activity.

    PubMed

    Moriya, Chiemi; Hosoya, Takahiro; Agawa, Sayuri; Sugiyama, Yasumasa; Kozone, Ikuko; Shin-Ya, Kazuo; Terahara, Norihiko; Kumazawa, Shigenori

    2015-01-01

    Purple yam (Dioscorea alata L.), which is widely distributed in tropical and subtropical regions, is characterized by its color and viscosity. Previous studies have shown that purple yams contain a variety of acylated anthocyanins that exhibit higher levels of antioxidant activity than the corresponding nonacylated compounds. In this study, the pigments found in purple yams from the Philippines (D. alata) were isolated and evaluated in terms of antioxidant activity. Four new acylated anthocyanins, alanins (1-4) were isolated from the MeOH extracts of purple yam, which were subsequently determined to be cyanidin (1, 2, and 4) and peonidin (3) type compounds, along with four known anthocyanins (5-8). The structures of 1-4 were determined by spectroscopic methods, including NMR and MS analyses. The antioxidant activities of anthocyanins 1-8 were investigated using oxygen radical absorbing capacity and ferric reducing antioxidant power assays. PMID:25848974

  19. Site-Selective Acylations with Tailor-Made Catalysts.

    PubMed

    Huber, Florian; Kirsch, Stefan F

    2016-04-18

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

  20. Mutations in p53 change phosphatidylinositol acyl chain composition

    PubMed Central

    Naguib, Adam; Bencze, Gyula; Engle, Dannielle; Chio, Iok I. C.; Herzka, Tali; Watrud, Kaitlin; Bencze, Szilvia; Tuveson, David A.; Pappin, Darryl J; Trotman, Lloyd C.

    2014-01-01

    Phosphatidylinositol phosphate (PIP) second messengers relay extracellular growth cues through the phosphorylation status of the inositol sugar, a signal transduction system that is deregulated in cancer. In stark contrast to PIP inositol head group phosphorylation, changes in phosphatidylinositol (PI) lipid acyl chains in cancer have remained ill-defined. Here, we apply a mass spectrometry-based method capable of unbiased high-throughput identification and quantification of cellular PI acyl chain composition. Using this approach we find that PI lipid chains represent a cell-specific fingerprint and are unperturbed by serum-mediated signaling in contrast to the inositol head group. We find that mutation of Trp53 results in PIs containing reduced-length fatty acid moieties. Our results suggest that the anchoring tails of lipid second messengers form an additional layer of PIP signaling in cancer that operates independently of PTEN/PI3-Kinase activity, but is instead linked somehow to p53. PMID:25543136

  1. A novel fatty Acyl-CoA Synthetase is required for pollen development and sporopollenin biosynthesis in Arabidopsis.

    PubMed

    de Azevedo Souza, Clarice; Kim, Sung Soo; Koch, Stefanie; Kienow, Lucie; Schneider, Katja; McKim, Sarah M; Haughn, George W; Kombrink, Erich; Douglas, Carl J

    2009-02-01

    Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by self-fertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein-ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes. PMID:19218397

  2. Characterization of oryza sativa acyl activating enzyme3 (OsAAE3)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxalate, the smallest of the dicarboxylic acids, is produced in many plants. This acid has been shown to play an important role in both plant physiology and defense, specifically in regards to metal detoxification, calcium regulation, sucking and chewing insect deterrence, and the production of calc...

  3. Intermediate Strength Gravitational Lensing

    SciTech Connect

    Irwin, John

    2005-03-17

    Weak lensing is found in the correlations of shear in {approx}10{sup 4} galaxy images, strong lensing is detected by the obvious distortion of a single galaxy image, whereas intermediate lensing requires detection of less obvious curvature in several neighboring galaxies. Small impact-parameter lensing causes a sextupole distortion whose orientation is correlated with the quadrupole distortion (shear). By looking within a field for the spatial correlation of this sextupole-quadrupole correlation, an intermediate lensing regime is observed. This technique requires correction for the sextupole as well as the quadrupole content of the PSF. We remove the HST PSF and uncover intermediate lensing in the Hubble deep fields. Correlations of the type expected are found.

  4. Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

    SciTech Connect

    Wu, Jing; Bu, Weishu; Sheppard, Kelly; Kitabatake, Makoto; Kwon, Suk-Tae; Söll, Dieter; Smith, Janet L.

    2010-08-17

    Many bacteria form Gln-tRNA{sup Gln} and Asn-tRNA{sup ASN} by conversion of the misacylated Glu-tRNA{sup Gln} and Asp-tRNA{sup ASN} species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction. A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn{sup 2+} site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn{sup 2+} binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA{sup Gln} or Asn-tRNA{sup ASN}.

  5. Acyl Chain Length of Phosphatidylserine Is Correlated with Plant Lifespan

    PubMed Central

    Tian, Xuejun; Li, Weiqi

    2014-01-01

    Plant lifespan is affected by factors with genetic and environmental bases. The laws governing these two factors and how they affect plant lifespan are unclear. Here we show that the acyl chain length (ACL) of phosphatidylserine (PS) is correlated with plant lifespan. Among the detected eight head-group classes of membrane lipids with lipidomics based on triple quadrupole tandem mass spectrometry, the ACL of PS showed high diversity, in contrast to the ACLs of the other seven classes, which were highly conserved over all stages of development in all plant species and organs and under all conditions that we studied. Further investigation found that acyl chains of PS lengthened during development, senescence, and under environmental stresses and that increasing length was accelerated by promoted- senescence. The acyl chains of PS were limited to a certain carbon number and ceased to increase in length when plants were close to death. These findings suggest that the ACL of PS can count plant lifespan and could be a molecular scale ruler for measuring plant development and senescence. PMID:25058060

  6. Gastrointestinal uptake of nasunin, acylated anthocyanin in eggplant.

    PubMed

    Ichiyanagi, Takashi; Terahara, Norihiko; Rahman, M Mamunur; Konishi, Tetsuya

    2006-07-26

    We previously showed that nasunin, acylated anthocyanins in eggplant peel, comprises two isomers, cis-nasunin and trans-nasunin. In this study, gastrointestinal absorption of cis- and trans-nasunins was studied in rats. Orally administered nasunins were quickly absorbed in their original acylated forms and maximally appeared in blood plasma after 15 min. When the maximum plasma concentration and area under the plasma concentration curve were normalized by orally administered dose (micromoles per kilogram), there was no significant difference in the uptake efficiency between two isomers and both exhibited a plasma level almost identical to that of delphinidin 3-O-beta-D-glucopyranoside. However, metabolites such as 4'-O-methyl analogues and extended glucuronides which were observed for delphinidin 3-O-beta-D-glucopyranoside and cyanidin 3-O-beta-D-glucopyranoside metabolisms were not detected in urine or blood plasma. Moreover, deacylated and glycolytic products of nasunins such as delphinidin 3-O-beta-D-glucopyranoside or delphinidin (aglycone) were also not detected in blood plasma even after oral administration for 8 h. These results indicated that nasunins were absorbed in their original acylated forms and exhibit a bioavailability almost identical to that of nonacylated anthocyanins. PMID:16848510

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

    PubMed

    Kamal, Md Arif; Raghunathan, V A

    2016-01-14

    The interdigitated phase of the lipid bilayer results when acyl chains from opposing monolayers fully interpenetrate such that the terminal methyl groups of the respective lipid chains are located at the interfacial region on the opposite sides of the bilayer. Usually, chain interdigitation is not encountered in a symmetric chain phosphatidylcholine (PC) membrane but can be induced under certain special conditions. In this article, we elucidate the contribution of small amphiphatic molecules in altering the physical properties of a symmetric chain PC bilayer membrane, which results in acyl chain interdigitation. Using small-angle X-ray scattering (SAXS), we have carried out a systematic investigation of the physical interactions of three naphthalene derivatives containing hydroxyl groups: β-naphthol, 2,3-dihydroxynaphthalene, and 2,7-dihydroxynaphthalene, with dipalmitoylphosphatidylcholine (DPPC) bilayers. On the basis of the diffraction patterns, we have determined the temperature-composition phase diagrams of these binary mixtures. The present study not only enables us to gain insight into the role played by small molecules in altering the packing arrangement of the acyl chains of the constituting PC lipids of the bilayer but also brings to light some important features that have not yet been reported hitherto. One such feature is the stabilization of the enigmatic asymmetric ripple phase over a wide temperature and concentration range. The results presented here strongly point toward a clear correlation between chain interdigitation and the stability of the ripple phase. PMID:26687052

  8. Acylation of 1,3-butadiene with acetylfluorosulfonate

    SciTech Connect

    Gavrishova, T.N.; Shastin, A.V.; Balenkova, E.S.; Novikov, N.A.

    1987-11-20

    1-Acetylbutadiene (I) is widely used in diene syntheses, and is also a valuable starting material for the preparation of otherwise difficulty accessible 1-substituted butadienes, which are then used in the synthesis of natural products. At the present time, however, there is no convenient one-step method for the preparation of acetylbutadiene (I). Acylation of butadiene (II) in the presence of Lewis acids is accompanied by extensive polymerization, and the maximum yield of acylated product in these reactions is only 10%. The authors have now successfully carried out the acylation of butadiene (II) with acetyfluorosulfonate. Workup of the reaction mixture with triethylamine gave acetylbutadiene (I) in 33% yield. The E-configuration of compound (I) was established based on the values of the spin-spin coupling constants for interaction of the olefinic protons, /sup 3/J(H/sup a/-H/sup b/) 15.4 Hz, which was determined by NMR spectroscopy. The presence of a characteristic absorption band at 965 cm/sup -1/ in the IR spectrum also confirms the E-configuration of compound (I).

  9. Serum lipids and acyl group composition of alcoholic patients.

    PubMed

    Sun, G Y; Rush, A; Chin, P C; Gorka, C; Lahiri, S; Wood, W G

    1988-01-01

    The lipid content and acyl group composition of serum from a group of alcoholic patients at a VA Medical Center were compared to control subjects sampled either from University of Missouri personnel or from subjects who were undergoing a preemployment physical examination at the same VA Medical Center. Plasma of alcoholic patients indicated an elevated triacylglycerol level (24-35%) as compared to both control groups. In addition, the acyl groups of triacylglycerols of alcoholic patients showed a markedly lower proportion of 18:2 and a higher proportion of 18:0 and 18:1 as compared to the control groups. The level of phosphatidylcholines in the plasma of alcoholic patients was not different from controls. However, acyl group composition of phosphatidylcholines from alcoholics indicated a lower proportion of 22:6 (n-3) as compared to controls. Although the cholesteryl ester level in serum was higher in alcoholics than in controls, the difference did not reach a level of significance. There was a similar decrease in 18:2 and an increase in 18:0 in cholesteryl esters of alcoholics as compared to controls. Results indicate that alcoholics in the United States show a similar change in certain serum lipids as reported for the Swedish alcoholics. This study also shows the complexities involved in selecting appropriate control groups to be compared with alcoholic patients. PMID:3395462

  10. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  11. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  12. Rigidifying Acyl Carrier Protein Domain in Iterative Type I PKS CalE8 Does Not Affect Its Function

    PubMed Central

    Lim, Jackwee; Sun, Huihua; Fan, Jing-Song; Hameed, Iman Fahim; Lescar, Julien; Liang, Zhao-Xun; Yang, Daiwen

    2012-01-01

    Acyl carrier protein (ACP) domains shuttle acyl intermediates among the catalytic domains of multidomain type I fatty acid synthase and polyketide synthase (PKS) systems. It is believed that the unique function of ACPs is associated with their dynamic property, but it remains to be fully elucidated what type of protein dynamics is critical for the shuttling domain. Using NMR techniques, we found that the ACP domain of iterative type I PKS CalE8 from Micromonospora echinospora is highly dynamic on the millisecond-second timescale. Introduction of an interhelical disulfide linkage in the ACP domain suppresses the dynamics on the millisecond-second timescale and reduces the mobility on the picosecond-nanosecond timescale. We demonstrate that the full-length PKS is fully functional upon rigidification of the ACP domain, suggesting that although the flexibility of the disordered terminal linkers may be important for the function of the ACP domain, the internal dynamics of the helical regions is not critical for that function. PMID:23009853

  13. Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver and other diseases: An update

    PubMed Central

    Yan, Sheng; Yang, Xue-Feng; Liu, Hao-Lei; Fu, Nian; Ouyang, Yan; Qing, Kai

    2015-01-01

    Long-chain acyl-CoA synthetase (ACSL) family members include five different ACSL isoforms, each encoded by a separate gene and have multiple spliced variants. ACSLs on endoplasmic reticulum and mitochondrial outer membrance catalyze fatty acids with chain lengths from 12 to 20 carbon atoms to form acyl-CoAs, which are lipid metabolic intermediates and involved in fatty acid metabolism, membrane modifications and various physiological processes. Gain- or loss-of-function studies have shown that the expression of individual ACSL isoforms can alter the distribution and amount of intracellular fatty acids. Changes in the types and amounts of fatty acids, in turn, can alter the expression of intracellular ACSLs. ACSL family members affect not only the proliferation of normal cells, but the proliferation of malignant tumor cells. They also regulate cell apoptosis through different signaling pathways and molecular mechanisms. ACSL members have individual functions in fatty acid metabolism in different types of cells depending on substrate preferences, subcellular location and tissue specificity, thus contributing to liver diseases and metabolic diseases, such as fatty liver disease, obesity, atherosclerosis and diabetes. They are also linked to neurological disorders and other diseases. However, the mechanisms are unclear. This review addresses new findings in the classification and properties of ACSLs and the fatty acid metabolism-associated effects of ACSLs in diseases. PMID:25834313

  14. Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    SciTech Connect

    Bera, Asim K.; Atanasova, Vesna; Gamage, Swarna; Robinson, Howard; Parsons, James F.

    2010-06-01

    The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

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

    PubMed Central

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

    1995-01-01

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

  16. Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts.

    PubMed

    Fukuoka, Tokuma; Morita, Tomotake; Konishi, Masaaki; Imura, Tomohiro; Kitamoto, Dai

    2007-07-01

    Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids. PMID:17417694

  17. Annotating enzymes of uncertain function: the deacylation of D-amino acids by members of the amidohydrolase superfamily.

    PubMed

    Cummings, Jennifer A; Fedorov, Alexander A; Xu, Chengfu; Brown, Shoshana; Fedorov, Elena; Babbitt, Patricia C; Almo, Steven C; Raushel, Frank M

    2009-07-14

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group

  18. Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily

    SciTech Connect

    Cummings, J.; Fedorov, A; Xu, C; Brown, S; Fedorov, E; Babbitt, P; Almo, S; Raushel, F

    2009-01-01

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} = 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250

  19. Hispanic American Heritage, Intermediate.

    ERIC Educational Resources Information Center

    Shepherd, Mike

    This resource book features the cultural heritage of Hispanics living within the United States and includes ideas, materials, and activities to be used with students in the intermediate grades and middle school. This book explores the definition of the term "Hispanic Americans" and suggests a multilayered population with a variety of cultural…

  20. English 200: Intermediate Composition

    ERIC Educational Resources Information Center

    Ritter, Kelly

    2005-01-01

    "English 200: Intermediate Composition" is a program elective for English majors and a writing-intensive elective for nonmajors at Southern Connecticut State University (SCSU), a comprehensive institution of 11,000 undergraduate and graduate (master's level) students. English 200 is described in the departmental course catalog as a course "in…

  1. Water oxidation: Intermediate identification

    NASA Astrophysics Data System (ADS)

    Cowan, Alexander J.

    2016-08-01

    The slow kinetics of light-driven water oxidation on haematite is an important factor limiting the material's efficiency. Now, an intermediate of the water-splitting reaction has been identified offering hope that the full mechanism will soon be resolved.

  2. INTERMEDIATE READINGS IN TAGALOG.

    ERIC Educational Resources Information Center

    BOWEN, J. DONALD, ED.

    THE SECOND IN A SERIES OF TEXTS DESIGNED TO HELP THE STUDENT ACHIEVE AN UNDERSTANDING OF FILIPINO CULTURE AND ACQUIRE ENOUGH PROFICIENCY IN TAGALOG TO COMMUNICATE EASILY AND MEANINGFULLY, THESE INTERMEDIATE READINGS ARE COORDINATED WITH THE EDITOR'S "BEGINNING TAGALOG" (ED 014 696). INCLUDED IN PART I ARE READINGS WRITTEN ESPECIALLY FOR THIS TEXT…

  3. GLOSSARY TO INTERMEDIATE HINDI.

    ERIC Educational Resources Information Center

    Wisconsin Univ., Madison. Indian Language and Area Center.

    INCLUDED IN THIS GLOSSARY ARE THE VOCABULARY ITEMS FOR THE READINGS IN "INTERMEDIATE HINDI." THE ITEMS ARE ARRANGED BY SELECTION IN SERIAL ORDER. EACH ENTRY INCLUDES NAGARI (DEVANAGARI) SCRIPT SPELLING, A NOTATION OF THE FORM CLASS, AND A SHORT ENGLISH GLOSS. THESE TWO VOLUMES ARE ALSO AVAILABLE AS A SET FOR $7.00 FROM THE COLLEGE PRINTING…

  4. SPACE: Intermediate Level Modules.

    ERIC Educational Resources Information Center

    Indiana State Dept. of Education, Indianapolis. Center for School Improvement and Performance.

    These modules were developed to assist teachers at the intermediate level to move away from extensive skill practice and toward more meaningful interdisciplinary learning. This packet, to be used by teachers in the summer Extended Learning Program, provides detailed thematic lesson plans matched to the Indiana Curriculum Proficiency Guide. The…

  5. Intermediate Cambodian Reader.

    ERIC Educational Resources Information Center

    Huffman, Franklin E., Ed.; Proum, Im, Ed.

    This book is a sequel to the "Cambodian System of Writing and Beginning Reader." It is intended to serve as an intermediate reader to develop the student's ability to the point of reading Cambodian texts with the aid of a dictionary. Part One of the book consists of 37 readings, graded in length and difficulty, and selected to provide a wide range…

  6. Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon.

    PubMed

    Yamada, M; Saier, M H

    1987-04-25

    The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene. PMID:3553176

  7. Metal-Free Route for the Synthesis of 4-Acyl-1,2,3-Triazoles from Readily Available Building Blocks.

    PubMed

    Thomas, Joice; Goyvaerts, Vince; Liekens, Sandra; Dehaen, Wim

    2016-07-11

    Functionalized 1,2,3-triazole heterocycles have been known for a long time and hold an extraordinary potential in diverse research areas ranging from medicinal chemistry to material science. However, the scope of therapeutically important 1-substituted 4-acyl-1H-1,2,3-triazoles is much less explored, probably due to the lack of synthetic methodologies of good scope and practicality. Here, we describe a practical and efficient one-pot multicomponent reaction for the synthesis of α-ketotriazoles from readily available building blocks such as methyl ketones, N,N-dimethylformamide dimethyl acetal, and organic azides with 100 % regioselectivity. This reaction is enabled by the in situ formation of an enaminone intermediate followed by its 1,3-dipolar cycloaddition reaction with an organic azide. We effectively utilized the developed strategy for the derivatization of various heterocycles and natural products, a protocol which is difficult or impossible to realize by other means. PMID:27172985

  8. Construction of efficient and effective transformation vectors for palmitoyl-acyl carrier protein thioesterase gene silencing in oil palm

    PubMed Central

    Bhore, Subhash Janardhan; Shah, Farida Habib

    2011-01-01

    Palm oil obtained from E. guineensis Jacq. Tenera is known to have about 44% of palmitic acid (C16:0). Palmitoyl-Acyl Carrier Protein Thioesterase (PATE) is one of the key enzymes involved in plastidial fatty acid biosynthesis; and it determines the level of the C16:0 assimilation in oilseeds. This enzyme's activity in oil palm is responsible for high (> 44 % in E. guineensis Jacq. Tenera and 25 % in E. oleifera) content of C16:0 in its oil. By post-transcriptional PATE gene silencing, C16:0 content can be minimized for nutritional value improvement of the palm oil. The objective of this study was the construction of novel transformation vectors for PATE gene silencing. Six different transformation vectors targeted against PATE gene were constructed using 619 bp long PATE gene (5' region) fragment (from GenBank AF507115). In one set of three transformation vectors, PATE gene fragment was fused with CaMV 35S promoter in antisense, intron-spliced inverted repeat (ISIR), and inverted repeat (IR) orientations to generate antisense mRNA and hair-pin RNAs (hpRNA). In another set of three transformation vectors with same design, CaMV 35S was replaced with Oil palm mesocarp tissue-specific promoter (MSP). The expression cassette of antisense, ISIR, and IR of PATE gene fragments were constructed in primary cloning vector, pHANNIBAL or its derivative/s. Finally, all 6 expression cassettes were sub-cloned into pCAMBIA 1301 which contains the Hygromycinr and the GUS reporter genes for transformant selection and transformation detection respectively. The results of the RE analyses of the constructs and sequence analyses of PATE and MSP shows and confirms the orientation, size and locations of all the components from constructs. We hypothesize that 4 (pISIRPATE-PC, pIRPATE-PC, pMISIRPATE-PC and pMIRPATE-PC) out of 6 transformation vectors constructed in this study will be efficient and effective in palmitoyl-ACP thioesterase gene silencing in oil palm. Abbreviations anti

  9. Insights into Sphingolipid Miscibility: Separate Observation of Sphingomyelin and Ceramide N-Acyl Chain Melting

    PubMed Central

    Leung, Sherry S.W.; Busto, Jon V.; Keyvanloo, Amir; Goñi, Félix M.; Thewalt, Jenifer

    2012-01-01

    Ceramide produced from sphingomyelin in the plasma membrane is purported to affect signaling through changes in the membrane’s physical properties. Thermal behavior of N-palmitoyl sphingomyelin (PSM) and N-palmitoyl ceramide (PCer) mixtures in excess water has been monitored by 2H NMR spectroscopy and compared to differential scanning calorimetry (DSC) data. The alternate use of either perdeuterated or proton-based N-acyl chain PSM and PCer in our 2H NMR studies has allowed the separate observation of gel-fluid transitions in each lipid in the presence of the other one, and this in turn has provided direct information on the lipids’ miscibility over a wide temperature range. The results provide further evidence of the stabilization of the PSM gel state by PCer. Moreover, overlapping NMR and DSC data reveal that the DSC-signals parallel the melting of the major component (PSM) except at intermediate (20 and 30 mol %) fractions of PCer. In such cases, the DSC endotherm reports on the presumably highly cooperative melting of PCer. Up to at least 50 mol % PCer, PSM and PCer mix ideally in the liquid crystalline phase; in the gel phase, PCer becomes incorporated into PSM:PCer membranes with no evidence of pure solid PCer. PMID:23260048

  10. Kinetic characterization of the inhibition of acyl coenzyme A: steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta).

    PubMed

    Sternberg, Robin M; LeBlanc, Gerald A

    2006-06-30

    Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17beta-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT (K(i)= approximately 9microM) and is a weaker, noncompetitive inhibitor of AEAT (K(i)= approximately 31microM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17beta-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17beta-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis-the fatty acid esterification of testosterone. PMID:16638618

  11. Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

    SciTech Connect

    Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent; Besra, Gurdyal S. Fütterer, Klaus

    2007-10-01

    Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

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

    PubMed

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

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription. PMID:24960695

  13. Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide.

    PubMed

    Mashima, Tetsuo; Sato, Shigeo; Okabe, Sachiko; Miyata, Satoshi; Matsuura, Masaaki; Sugimoto, Yoshikazu; Tsuruo, Takashi; Seimiya, Hiroyuki

    2009-08-01

    Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl-CoA synthetase (ACS), which converts long-chain fatty acids to acyl-CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c-resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax-induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well-known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non-toxic dose enhanced the antitumor efficacy of a low-dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide. PMID:19459852

  14. Intermediate water recovery system

    NASA Technical Reports Server (NTRS)

    Deckman, G.; Anderson, A. R. (Editor)

    1973-01-01

    A water recovery system for collecting, storing, and processing urine, wash water, and humidity condensates from a crew of three aboard a spacecraft is described. The results of a 30-day test performed on a breadboard system are presented. The intermediate water recovery system produced clear, sterile, water with a 96.4 percent recovery rate from the processed urine. Recommendations for improving the system are included.

  15. Plant Cytosolic Acyl-CoA-Binding Proteins.

    PubMed

    Ye, Zi-Wei; Chye, Mee-Len

    2016-01-01

    A gene family encoding six members of acyl-CoA-binding proteins (ACBP) exists in Arabidopsis and they are designated as AtACBP1-AtACBP6. They have been observed to play pivotal roles in plant lipid metabolism, consistent to the abilities of recombinant AtACBP in binding different medium- and long-chain acyl-CoA esters in vitro. While AtACBP1 and AtACBP2 are membrane-associated proteins with ankyrin repeats and AtACBP3 contains a signaling peptide for targeting to the apoplast, AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. They were verified to be subcellularly localized in the cytosol using diverse experimental methods, including cell fractionation followed by western blot analysis, immunoelectron microscopy and confocal laser-scanning microscopy using autofluorescence-tagged fusions. AtACBP4 (73.2 kDa) and AtACBP5 (70.1 kDa) are the largest, while AtACBP6 (10.4 kDa) is the smallest. Their binding affinities to oleoyl-CoA esters suggested that they can potentially transfer oleoyl-CoA esters from the plastids to the endoplasmic reticulum, facilitating the subsequent biosynthesis of non-plastidial membrane lipids in Arabidopsis. Recent studies on ACBP, extended from a dicot (Arabidopsis) to a monocot, revealed that six ACBP are also encoded in rice (Oryza sativa). Interestingly, three small rice ACBP (OsACBP1, OsACBP2 and OsACBP3) are present in the cytosol in comparison to one (AtACBP6) in Arabidopsis. In this review, the combinatory and distinct roles of the cytosolic AtACBP are discussed, including their functions in pollen and seed development, light-dependent regulation and substrate affinities to acyl-CoA esters. PMID:26662549

  16. Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube.

    PubMed

    Suma, Yanasinee; Lim, Heejun; Kwean, Oh Sung; Cho, Suyeon; Yang, Junwon; Kim, Yohan; Kang, Christina S; Kim, Han S

    2016-06-01

    This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60-79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use. PMID:26810145

  17. Purification of a Jojoba Embryo Fatty Acyl-Coenzyme A Reductase and Expression of Its cDNA in High Erucic Acid Rapeseed

    PubMed Central

    Metz, James G.; Pollard, Michael R.; Anderson, Lana; Hayes, Thomas R.; Lassner, Michael W.

    2000-01-01

    The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes. PMID:10712526

  18. An Ultrahigh Resolution Structure of TEM-1 beta-Lactamase Suggests a Role for Glu166 as the General Base in Acylation

    SciTech Connect

    Minasov, George; Wang, Xiaojun; Shoichet, Brian K.

    2010-03-08

    Although TEM-1 {beta}-lactamase is among the best studied enzymes, its acylation mechanism remains controversial. To investigate this problem, the structure of TEM-1 in complex with an acylation transition-state analogue was determined at ultrahigh resolution (0.85 {angstrom}) by X-ray crystallography. The quality of the data was such as to allow for refinement to an R-factor of 9.1% and an R{sub free} of 11.2%. In the resulting structure, the electron density features were clear enough to differentiate between single and double bonds in carboxylate groups, to identify multiple conformations that are occupied by residues and loops, and to assign 70% of the protons in the protein. Unexpectedly, even at pH 8.0 where the protein was crystallized, the active site residue Glu166 is clearly protonated. This supports the hypothesis that Glu166 is the general base in the acylation half of the reaction cycle. This structure suggests that Glu166 acts through the catalytic water to activate Ser70 for nucleophilic attack on the {beta}-lactam ring of the substrate. The hydrolytic mechanism of class A {beta}-lactamases, such as TEM-1, appears to be symmetrical, as are the serine proteases. Apart from its mechanistic implications, this atomic resolution structure affords an unusually detailed view of the structure, dynamics, and hydrogen-bonding networks of TEM-1, which may be useful for the design of inhibitors against this key antibiotic resistance target.

  19. The role of the β5-α11 loop in the active-site dynamics of acylated penicillin-binding protein A from Mycobacterium tuberculosis

    PubMed Central

    Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

    2012-01-01

    Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in M. tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of the apo enzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured second-order acylation rate constants for the antibiotics, imipenem, penicillin G and ceftriaxone. Of these, imipenem, which has demonstrable antitubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined and all show conformational differences in the β5-α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that PBPs in apo form can occupy different conformational states. PMID:22365933

  20. Relatedness of acyl carrier proteins shown by amino acid compositions.

    PubMed

    Walker, T A; Ernst-Fonberg, M L

    1982-01-01

    1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552. PMID:7128903

  1. Extraction of lanthanides with halogen substituted 4-acyl-pyrazolones

    SciTech Connect

    Huang, C.H.; Freiser, H.

    1983-01-01

    Equilibrium extraction behavior for a series of representative tervalent lanthanide ions, La, Pr, Eu, and Yb, using chloroform solutions containing halogenated derivatives of 4-acyl-1-phenyl-3-methyl-5-pyrazolone have been studied. The results demonstrate that these lanthanides are extracted as simple chelates, LnL/sub 3/. The equilibrium constants of these extraction reactions have been calculated. The relationships between the acid dissociation constants, K/sub a/, determined by a two-phase titration method, distribution constants, K/sub DR/, and the extraction equilibrium constants, K/sub ex/, are discussed. 14 refs., 5 figs., 5 tabs.

  2. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  3. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  4. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

    Krest, Courtney M.; Onderko, Elizabeth L.; Yosca, Timothy H.; Calixto, Julio C.; Karp, Richard F.; Livada, Jovan; Rittle, Jonathan; Green, Michael T.

    2013-01-01

    Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

  5. Genetics Home Reference: peroxisomal acyl-CoA oxidase deficiency

    MedlinePlus

    ... enzyme is found in sac-like cell structures (organelles) called peroxisomes, which contain a variety of enzymes ... 1 link) Health Topic: Leukodystrophies Genetic and Rare Diseases Information Center (1 link) Pseudoneonatal adrenoleukodystrophy Educational Resources ( ...

  6. Anion-π Enzymes

    PubMed Central

    2016-01-01

    In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pKa of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pKa of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization. PMID:27413782

  7. Anion-π Enzymes.

    PubMed

    Cotelle, Yoann; Lebrun, Vincent; Sakai, Naomi; Ward, Thomas R; Matile, Stefan

    2016-06-22

    In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pK a of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pK a of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization. PMID:27413782

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

    PubMed Central

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

    2014-01-01

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

  9. Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of β-oxidation cycle.

    PubMed

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

    2015-02-01

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

  10. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives' Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps.

    PubMed

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydro