Cloning and Characterization of Unusual Fatty Acid Desaturases from Anemone leveillei: Identification of an Acyl-Coenzyme A C20 Δ5-Desaturase Responsible for the Synthesis of Sciadonic Acid1

PubMed Central

Sayanova, Olga; Haslam, Richard; Venegas Caleron, Monica; Napier, Johnathan A.

2007-01-01

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Δ5,11,14; SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C20 Δ5cis-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Δ5-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C18 Δ9-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Δ5,11,14,17). Thus, AL10 acted only on C20 polyunsaturated fatty acids in a manner analogous to “front-end” desaturases. However, neither AL10 nor AL21 contain the cytochrome b5 domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Δ5-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Δ5-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Δ5, 18:1Δ5, and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Δ5-desaturases using acyl-CoA substrates. PMID:17384161

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

PubMed Central

Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

2003-01-01

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

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

PubMed

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

2015-09-01

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

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

PubMed Central

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

2015-01-01

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

Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene.

PubMed Central

Slocombe, S P; Piffanelli, P; Fairbairn, D; Bowra, S; Hatzopoulos, P; Tsiantis, M; Murphy, D J

1994-01-01

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151-155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo. PMID:8016261

Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cahoon, E.B.; Shanklin, J.; Ohlrogge, J.B.

1992-12-01

Little is known about the metabolic origin of petroselinic acid (18:1[Delta][sup 6cis]), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the [Delta][sup 9]-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriandermore » endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and [Delta][sup 4]-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase. 27 refs., 5 figs.« less

Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

PubMed

Monroig, Oscar; Navarro, Juan C; Dick, James R; Alemany, Frederic; Tocher, Douglas R

2012-08-01

Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

PubMed Central

Murakami, Akira; Nagao, Kohjiro; Juni, Naoto; Hara, Yuji; Umeda, Masato

2017-01-01

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid–dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid–dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline–dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a “di-proline motif,” which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids. PMID:28972163

Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera

PubMed Central

2008-01-01

Background Moths have evolved highly successful mating systems, relying on species-specific mixtures of sex pheromone components for long-distance mate communication. Acyl-CoA desaturases are key enzymes in the biosynthesis of these compounds and to a large extent they account for the great diversity of pheromone structures in Lepidoptera. A novel desaturase gene subfamily that displays Δ11 catalytic activities has been highlighted to account for most of the unique pheromone signatures of the taxonomically advanced ditrysian species. To assess the mechanisms driving pheromone evolution, information is needed about the signalling machinery of primitive moths. The currant shoot borer, Lampronia capitella, is the sole reported primitive non-ditrysian moth known to use unsaturated fatty-acid derivatives as sex-pheromone. By combining biochemical and molecular approaches we elucidated the biosynthesis paths of its main pheromone component, the (Z,Z)-9,11-tetradecadien-1-ol and bring new insights into the time point of the recruitment of the key Δ11-desaturase gene subfamily in moth pheromone biosynthesis. Results The reconstructed evolutionary tree of desaturases evidenced two ditrysian-specific lineages (the Δ11 and Δ9 (18C>16C)) to have orthologs in the primitive moth L. capitella despite being absent in Diptera and other insect genomes. Four acyl-CoA desaturase cDNAs were isolated from the pheromone gland, three of which are related to Δ9-desaturases whereas the fourth cDNA clusters with Δ11-desaturases. We demonstrated that this transcript (Lca-KPVQ) exclusively accounts for both steps of desaturation involved in pheromone biosynthesis. This enzyme possesses a Z11-desaturase activity that allows transforming the palmitate precursor (C16:0) into (Z)-11-hexadecenoic acid and the (Z)-9-tetradecenoic acid into the conjugated intermediate (Z,Z)-9,11-tetradecadienoic acid. Conclusion The involvement of a single Z11-desaturase in pheromone biosynthesis of a non-ditrysian moth species, supports that the duplication event leading to the origin of the Lepidoptera-specific Δ11-desaturase gene subfamily took place before radiation of ditrysian moths and their divergence from other heteroneuran lineages. Our findings uncover that this novel class of enzymes affords complex combinations of unique unsaturated fatty acyl-moieties of variable chain-lengths, regio- and stereo-specificities since early in moth history and contributes a notable innovation in the early evolution of moth-pheromones. PMID:18831750

Sex-related differences in the enhancing effects of perfluoro-octanoic acid on stearoyl-CoA desaturase and its influence on the acyl composition of phospholipid in rat liver. Comparison with clofibric acid and tiadenol.

PubMed Central

Kawashima, Y; Uy-Yu, N; Kozuka, H

1989-01-01

The effects of the peroxisome proliferators clofibric acid (p-chlorophenoxyisobutyric acid), tiadenol [2,2'-(decamethylenedithio)diethanol] and perfluoro-octanoic acid (PFOA) on hepatic stearoyl-CoA desaturation in male and female rats were compared. Treatment of male rats with the three peroxisome proliferators increased markedly the activity of stearoyl-CoA desaturase. Administration of clofibric acid or tiadenol to female rats increased greatly the hepatic activity of stearoyl-CoA desaturase, the extent of the increases being slightly less pronounced than those of male rats. In contrast with the other two peroxisome proliferators, however, PFOA did not change the activity of stearoyl-CoA desaturase in female rats. Hormonal manipulations revealed that this sex-related difference in the effect of PFOA on stearoyl-CoA desaturase activity is strongly dependent on testosterone. The increase in stearoyl-CoA desaturase activity by peroxisome proliferators was not accompanied by any notable increases in the microsomal content of cytochrome b5 or the activity of NADH: cytochrome b5 reductase. The administration of the peroxisome proliferators greatly altered the acyl composition of hepatic phosphatidylcholine and phosphatidylethanolamine (namely the proportions of C18:1 and C20:3,n-9 fatty acids increased in both phospholipids), and the alterations were partially associated with the increase in stearoyl-CoA desaturase activity. PMID:2574572

Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

PubMed Central

Kawashima, Y; Hirose, A; Kozuka, H

1986-01-01

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed. PMID:2874791

Identification and functional analysis of delta-9 desaturase, a key enzyme in PUFA Synthesis, isolated from the oleaginous diatom Fistulifera.

PubMed

Muto, Masaki; Kubota, Chihiro; Tanaka, Masayoshi; Satoh, Akira; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

2013-01-01

Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ(9) desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ(9) acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ(9) desaturase. Furthermore, the function of these Δ(9) desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ(9) acyl-CoA desaturases showed Δ(9) desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ(9) desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.

Evolution of the Insect Desaturase Gene Family with an Emphasis on Social Hymenoptera

PubMed Central

Helmkampf, Martin; Cash, Elizabeth; Gadau, Jürgen

2015-01-01

Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes. PMID:25425561

FATTY ACID DESATURASE4 of Arabidopsis encodes a protein distinct from characterized fatty acid desaturases.

PubMed

Gao, Jinpeng; Ajjawi, Imad; Manoli, Arthur; Sawin, Andrew; Xu, Changcheng; Froehlich, John E; Last, Robert L; Benning, Christoph

2009-12-01

Polar membrane glycerolipids occur in a mixture of molecular species defined by a polar head group and characteristic acyl groups esterified to a glycerol backbone. A molecular species of phosphatidylglycerol specific to chloroplasts of plants carries a Delta(3-trans) hexadecenoic acid in the sn-2 position of its core glyceryl moiety. The fad4-1 mutant of Arabidopsis thaliana missing this particular phosphatidylglycerol molecular species lacks the necessary fatty acid desaturase, or a component thereof. The overwhelming majority of acyl groups associated with membrane lipids in plants contains double bonds with a cis configuration. However, FAD4 is unusual because it is involved in the formation of a trans double bond introduced close to the carboxyl group of palmitic acid, which is specifically esterified to the sn-2 glyceryl carbon of phosphatidylglycerol. As a first step towards the analysis of this unusual desaturase reaction, the FAD4 gene was identified by mapping of the FAD4 locus and coexpression analysis with known lipid genes. FAD4 encodes a predicted integral membrane protein that appears to be unrelated to classic membrane bound fatty acid desaturases based on overall sequence conservation. However, the FAD4 protein contains two histidine motifs resembling those of metalloproteins such as fatty acid desaturases. FAD4 is targeted to the plastid. Overexpression of the cDNA in transgenic Arabidopsis led to increased accumulation of the Delta(3-trans) hexadecanoyl group in phosphatidylglycerol relative to wild type. Taken together these results are consistent with the hypothesis that FAD4 is the founding member of a novel class of fatty acid desaturases.

Development of markers for Delta9-Stearoyl-ACP-Desaturase (SAD) to screen for cold acclimation

USDA-ARS?s Scientific Manuscript database

Delta 9-Stearoyl-acyl carrier protein (ACP) desaturase (SAD) is an important enzyme of fatty acid biosynthesis in higher plants. Located in the plastid stroma, SAD catalyzes the desaturation of stearoyl-ACP to oleyl-ACP. SAD plays a key role in determining the ratio of saturated fatty acids to unsat...

Stearoyl-Acyl Carrier Protein Desaturase Mutations Uncover an Impact of Stearic Acid in Leaf and Nodule Structure.

PubMed

Lakhssassi, Naoufal; Colantonio, Vincent; Flowers, Nicholas D; Zhou, Zhou; Henry, Jason; Liu, Shiming; Meksem, Khalid

2017-07-01

Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the accumulation of seed stearic acid; however, no study has previously reported its involvement in leaf stearic acid content and impact on leaf structure and morphology. A subset of an ethyl methanesulfonate mutagenized population of soybean ( Glycine max ) 'Forrest' was screened to identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one nonsense and four missense Gmsacpd-c mutants were identified to have high levels of seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of the GmSACPD-C enzyme revealed that most of these mutations were localized near or at conserved residues essential for diiron ion coordination. Soybeans carrying Gmsacpd-c mutations at conserved residues showed the highest stearic acid content, and these mutations were found to have deleterious effects on nodule development and function. Interestingly, mutations at nonconserved residues show an increase in stearic acid content yet retain healthy nodules. Thus, random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics. Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts were significantly more abundant in soybeans with deleterious mutations at conserved residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase in leaf stearic acid content and an alteration of leaf structure and morphology in addition to differences in nitrogen-fixing nodule structure. © 2017 American Society of Plant Biologists. All Rights Reserved.

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

DOE PAGES

Sedeek, Khalid E. M.; Whittle, Edward; Guthörl, Daniela; ...

2016-05-19

Here, we show that mimicry illustrates the power of selection to produce phenotypic convergence in biology. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species. This involves mimicry of visual, tactile, and chemical signals of females, especially their sex pheromones. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius. A difference in alkene double-bond positions is responsible for reproductivemore » isolation between O. exaltata and closely related species, such as O. sphegodes. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5’s reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. In conclusion, this change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution.« less

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator's Sex Pheromone.

PubMed

Sedeek, Khalid E M; Whittle, Edward; Guthörl, Daniela; Grossniklaus, Ueli; Shanklin, John; Schlüter, Philipp M

2016-06-06

Mimicry illustrates the power of selection to produce phenotypic convergence in biology [1]. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species [2-4]. This involves mimicry of visual, tactile, and chemical signals of females [2-7], especially their sex pheromones [8-11]. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius [11-13]. A difference in alkene double-bond positions is responsible for reproductive isolation between O. exaltata and closely related species, such as O. sphegodes [13-16]. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5's reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. This change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sedeek, Khalid E. M.; Whittle, Edward; Guthörl, Daniela

Here, we show that mimicry illustrates the power of selection to produce phenotypic convergence in biology. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species. This involves mimicry of visual, tactile, and chemical signals of females, especially their sex pheromones. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius. A difference in alkene double-bond positions is responsible for reproductivemore » isolation between O. exaltata and closely related species, such as O. sphegodes. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5’s reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. In conclusion, this change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution.« less

Azide and acetate complexes plus two iron-depleted crystal structures of the di-iron enzyme delta9 stearoyl-acyl carrier protein desaturase. Implications for oxygen activation and catalytic intermediates.

PubMed

Moche, Martin; Shanklin, John; Ghoshal, Alokesh; Lindqvist, Ylva

2003-07-04

Delta9 stearoyl-acyl carrier protein (ACP) desaturase is a mu-oxo-bridged di-iron enzyme, which belongs to the structural class I of large helix bundle proteins and that catalyzes the NADPH and O2-dependent formation of a cis-double bond in stearoyl-ACP. The crystal structures of complexes with azide and acetate, respectively, as well as the apoand single-iron forms of Delta9 stearoyl-ACP desaturase from Ricinus communis have been determined. In the azide complex, the ligand forms a mu-1,3-bridge between the two iron ions in the active site, replacing a loosely bound water molecule. The structure of the acetate complex is similar, with acetate bridging the di-iron center in the same orientation with respect to the di-iron center. However, in this complex, the iron ligand Glu196 has changed its coordination mode from bidentate to monodentate, the first crystallographic observation of a carboxylate shift in Delta9 stearoyl-ACP desaturase. The two complexes are proposed to mimic a mu-1,2 peroxo intermediate present during catalytic turnover. There are striking structural similarities between the di-iron center in the Delta9 stearoyl-ACP desaturase-azide complex and in the reduced rubrerythrin-azide complex. This suggests that Delta9 stearoyl-ACP desaturase might catalyze the formation of water from exogenous hydrogen peroxide at a low rate. From the similarity in iron center structure, we propose that the mu-oxo-bridge in oxidized desaturase is bound to the di-iron center as in rubrerythrin and not as reported for the R2 subunit of ribonucleotide reductase and the hydroxylase subunit of methane monooxygenase. The crystal structure of the one-iron depleted desaturase species demonstrates that the affinities for the two iron ions comprising the di-iron center are not equivalent, Fe1 being the higher affinity site and Fe2 being the lower affinity site.

A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone

PubMed Central

Serra, Montserrat; Piña, Benjamin; Abad, José Luis; Camps, Francisco; Fabriàs, Gemma

2007-01-01

The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from palmitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final Δ13 desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1Δ/ole1Δ yeast mutants (both elongase 1 and Δ9 desaturase-deficient) for functional assays. The construct fully rescued the Δole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed Δ11 desaturase, Δ11 acetylenase, and Δ13 desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth. PMID:17921252

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases.

PubMed

Zhang, Yao; Luan, Xiao; Zhang, Huaiyuan; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

2017-06-21

γ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides. To enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain. These findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.

Plant fatty acid hydroxylases

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank

2001-01-01

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

The Crystal Structure of the Ivy delta4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guy,J.; Whittle, E.; Kumaran, D.

2007-01-01

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the {Delta}{sup 4} desaturation of 16:0-ACP and the{Delta}{sup 9} desaturation of 18:0-ACP and further desaturates{Delta}{sup 9}-16:1 or {Delta}{sup 9}-18:1 to the corresponding {Delta}{sup 4,9} dienes. The crystal structure of the enzyme has been solved to 1.95{angstrom} resolution, and both the iron-iron distance of 3.2{angstrom} and the presence of a {mu}-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase;more » His{sup 227} coordination to Fe2 is lost, and the side chain of Glu{sup 224}, which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.« less

Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice.

PubMed

Jiang, Chang-Jie; Shimono, Masaki; Maeda, Satoru; Inoue, Haruhiko; Mori, Masaki; Hasegawa, Morifumi; Sugano, Shoji; Takatsuji, Hiroshi

2009-07-01

Fatty acids and their derivatives play important signaling roles in plant defense responses. It has been shown that suppressing a gene for stearoyl acyl carrier protein fatty-acid desaturase (SACPD) enhances the resistance of Arabidopsis (SSI2) and soybean to multiple pathogens. In this study, we present functional analyses of a rice homolog of SSI2 (OsSSI2) in disease resistance of rice plants. A transposon insertion mutation (Osssi2-Tos17) and RNAi-mediated knockdown of OsSSI2 (OsSSI2-kd) reduced the oleic acid (18:1) level and increased that of stearic acid (18:0), indicating that OsSSI2 is responsible for fatty-acid desaturase activity. These plants displayed spontaneous lesion formation in leaf blades, retarded growth, slight increase in endogenous free salicylic acid (SA) levels, and SA/benzothiadiazole (BTH)-specific inducible genes, including WRKY45, a key regulator of SA/BTH-induced resistance, in rice. Moreover, the OsSSI2-kd plants showed markedly enhanced resistance to the blast fungus Magnaporthe grisea and leaf-blight bacteria Xanthomonas oryzae pv. oryzae. These results suggest that OsSSI2 is involved in the negative regulation of defense responses in rice, as are its Arabidopsis and soybean counterparts. Microarray analyses identified 406 genes that were differentially expressed (>or=2-fold) in OsSSI2-kd rice plants compared with wild-type rice and, of these, approximately 39% were BTH responsive. Taken together, our results suggest that induction of SA-responsive genes, including WRKY45, is likely responsible for enhanced disease resistance in OsSSI2-kd rice plants.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris [Portola Valley, CA; Broun, Pierre [Burlingame, CA; van de Loo, Frank [Weston, AU; Boddupalli, Sekhar S [Manchester, MI

2011-08-23

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

2005-08-30

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Genes affecting novel seed constituents in Limnanthes alba Benth: transcriptome analysis of developing embryos and a new genetic map of meadowfoam

PubMed Central

Cooper, Laurel D.; Kishore, Venkata K.; Knapp, Steven J.; Kling, Jennifer G.

2015-01-01

The seed oil of meadowfoam, a new crop in the Limnanthaceae family, is highly enriched in very long chain fatty acids that are desaturated at the Δ5 position. The unusual oil is desirable for cosmetics and innovative industrial applications and the seed meal remaining after oil extraction contains glucolimnanthin, a methoxylated benzylglucosinolate whose degradation products are herbicidal and anti-microbial. Here we describe EST analysis of the developing seed transcriptome that identified major genes involved in biosynthesis and assembly of the seed oil and in glucosinolate metabolic pathways. mRNAs encoding acyl-CoA Δ5 desaturase were notably abundant. The library was searched for simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). Fifty-four new SSR markers and eight candidate gene markers were developed and combined with previously developed SSRs to construct a new genetic map for Limnanthes alba. Mapped genes in the lipid biosynthetic pathway encode 3-ketoacyl-CoA synthase (KCS), Δ5 desaturase (Δ5DS), lysophosphatidylacyl-acyl transferase (LPAT), and acyl-CoA diacylglycerol acyl transferase (DGAT). Mapped genes in glucosinolate biosynthetic and degradation pathways encode CYP79A, myrosinase (TGG), and epithiospecifier modifier protein (ESM). The resources developed in this study will further the domestication and improvement of meadowfoam as an oilseed crop. PMID:26038713

Conserved Function of ACYL-ACYL CARRIER PROTEIN DESATURASE 5 on Seed Oil and Oleic Acid Biosynthesis between Arabidopsis thaliana and Brassica napus.

PubMed

Jin, Changyu; Li, Dong; Gao, Chenhao; Liu, Kaige; Qi, Shuanghui; Duan, Shaowei; Li, Zixiong; Gong, Jingyun; Wang, Jianjun; Hai, Jiangbo; Chen, Mingxun

2017-01-01

Previous studies have shown that several ACYL-ACYL CARRIER PROTEIN DESATURASE (AtAAD) members in Arabidopsis thaliana are responsible for oleic acid (C18:1) biosynthesis. Limited research has been conducted on another member, AtAAD5, and its paralog BnAAD5 in the closely related and commercially important plant, Brassica napus . Here, we found that AtAAD5 was predominantly and exclusively expressed in developing embryos at the whole seed developmental stages. The aad5 mutation caused a significant decrease in the amounts of oil and C18:1, and a considerable increase in the content of stearic acid (C18:0) in mature seeds, suggesting that AtAAD5 functioned as an important facilitator of seed oil biosynthesis. We also cloned the full-length coding sequence of BnAAD5-1 from the A3 subgenome of the B. napus inbred line L111. We showed that ectopic expression of BnAAD5-1 in the A. thaliana aad5-2 mutant fully complemented the phenotypes of the mutant, such as lower oil content and altered contents of C18:0 and C18:1. These results help us to better understand the functions of AAD members in A. thaliana and B. napus and provide a promising target for genetic manipulation of B. napus .

The Mycobacterium tuberculosis desaturase DesA1 (Rv0824c) is a Ca{sup 2+} binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeruva, Veena C., E-mail: veenachaitanya@ccmb.res.in; Savanagouder, Mamata; Khandelwal, Radhika

The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome butmore » is unique in containing a βγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca{sup 2+} binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and {sup 45}CaCl{sub 2} overlay, we demonstrate that Ca{sup 2+} binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several βγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca{sup 2+} dependent variation in surface phenotype, revealing a functional role for Ca{sup 2+}in DesA1 activity. This study represents the first identification of a Ca{sup 2+} binding βγ-crystallin in M.tb, emphasizing the implicit role of Ca{sup 2+} in the pathogenesis of M.tb. - Highlights: • Mycobacterium tuberculosis DesA1 is an essential acyl-ACP desaturase. • DesA1 was identified to contain a βγ-crystallin Greek key signature motif. • Ca{sup 2+} binds to DesA1 with an affinity of 53 μM and induces changes in its conformation. • M. smegmatis overexpressing M.tb DesA1 shows a Ca{sup 2+} dependent phenotype. • Targetting the Ca{sup 2+} dependent function of DesA1 could be of therapeutic value.« less

RNA-Seq Analysis of Developing Pecan (Carya illinoinensis) Embryos Reveals Parallel Expression Patterns among Allergen and Lipid Metabolism Genes.

PubMed

Mattison, Christopher P; Rai, Ruhi; Settlage, Robert E; Hinchliffe, Doug J; Madison, Crista; Bland, John M; Brashear, Suzanne; Graham, Charles J; Tarver, Matthew R; Florane, Christopher; Bechtel, Peter J

2017-02-22

The pecan nut is a nutrient-rich part of a healthy diet full of beneficial fatty acids and antioxidants, but can also cause allergic reactions in people suffering from food allergy to the nuts. The transcriptome of a developing pecan nut was characterized to identify the gene expression occurring during the process of nut development and to highlight those genes involved in fatty acid metabolism and those that commonly act as food allergens. Pecan samples were collected at several time points during the embryo development process including the water, gel, dough, and mature nut stages. Library preparation and sequencing were performed using Illumina-based mRNA HiSeq with RNA from four time points during the growing season during August and September 2012. Sequence analysis with Trinotate software following the Trinity protocol identified 133,000 unigenes with 52,267 named transcripts and 45,882 annotated genes. A total of 27,312 genes were defined by GO annotation. Gene expression clustering analysis identified 12 different gene expression profiles, each containing a number of genes. Three pecan seed storage proteins that commonly act as allergens, Car i 1, Car i 2, and Car i 4, were significantly up-regulated during the time course. Up-regulated fatty acid metabolism genes that were identified included acyl-[ACP] desaturase and omega-6 desaturase genes involved in oleic and linoleic acid metabolism. Notably, a few of the up-regulated acyl-[ACP] desaturase and omega-6 desaturase genes that were identified have expression patterns similar to the allergen genes based upon gene expression clustering and qPCR analysis. These findings suggest the possibility of coordinated accumulation of lipids and allergens during pecan nut embryogenesis.

Identification of the Binding Region of the [2Fe-2S] Ferredoxin in Stearoyl-Acyl Carrier Protein Desaturase

PubMed Central

Sobrado, Pablo; Lyle, Karen S.; Kaul, Steven P.; Turco, Michelle M.; Arabshahi, Ida; Marwah, Ashok; Fox, Brian G.

2008-01-01

Stearoyl-acyl carrier protein desaturase (Δ9D) catalyzes the O2 and 2e- dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e- are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Δ9D complex by use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches and molecular docking studies. Treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Δ9D contribute to the cross-linking. The single substitutions of K60A, K56A, and K230A on Δ9D decreased the kcat/KM for Fd by 4-, 22- and 2,400-fold, respectively, as compared to wt Δ9D and a K41A substitution. The double substitution K56A/K60A decreased the kcat/KM for Fd by 250-fold, while the triple mutation K56A/K60A/K230A decreased the kcat/KM for Fd by at least 700,000-fold. These results strongly implicate the triad of K56, K60 and K230 of Δ9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd near to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix-7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Δ9D and other protein-protein complexes. PMID:16605252

The acyl desaturase CER17 is involved in producing wax unsaturated primary alcohols and cutin monomers

USDA-ARS?s Scientific Manuscript database

We report here n-6 mono-unsaturated primary alcohols (the C26:1, C28:1, and C30:1 homologues) in the cuticular waxes of Arabidopsis inflorescence stem, a class of wax compound not previously reported in Arabidopsis. Further, we used mutation and transgenic complementation analyses to demonstrate tha...

Cellular and Molecular Responses of Dunaliella tertiolecta by Expression of a Plant Medium Chain Length Fatty Acid Specific Acyl-ACP Thioesterase

PubMed Central

Lin, Huixin; Shen, Hui; Lee, Yuan K.

2018-01-01

Metabolic engineering of microalgae to accumulate high levels of medium chain length fatty acids (MCFAs) has met with limited success. Traditional approaches employ single introduction of MCFA specific acyl-ACP thioesterases (TEs), but our current research in transgenic Dunaliella tertiolecta line has highlighted that, there is no single rate-limiting approach that can effectively increase MCFA levels. Here, we explore the accumulation of MCFAs in D. tertiolecta after transgenic expression of myristic acid biased TE (C14TE). We observe that the MCFA levels were negatively correlated to the fatty acid (FA) synthesis genes, ketoacyl-ACP synthase II (KASII), stearoyl-CoA-9-desaturase (Δ9D), and oleoyl-CoA-12-desaturase (Δ12D). To further examine the molecular mechanism of MCFA accumulation in microalgae, we investigate the transcriptomic dynamics of the MCFA producing strain of D. tertiolecta. At the transcript level, enhanced MCFA accumulation primarily involved up-regulation of photosynthetic genes and down-regulation of genes from central carbon metabolic processes, resulting in an overall decrease in carbon precursors for FA synthesis. We additionally observe that MCFA specific peroxisomal β-oxidation gene (ACX3) was greatly enhanced to prevent excessive build-up of unusual MCFA levels. Besides, long chain acyl-CoA synthetase gene (LACS) was down-regulated, likely in attempt to control fatty acyl supply flux to FA synthesis cycle. This article provides a spatial regulation model of unusual FA accumulation in microalgae and a platform for additional metabolic engineering targeting pathways from FA synthesis, FA transport, and peroxisomal β-oxidation to achieve microalgae oils with higher levels of MCFAs. PMID:29670594

Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm.

PubMed

Rosli, Rozana; Amiruddin, Nadzirah; Ab Halim, Mohd Amin; Chan, Pek-Lan; Chan, Kuang-Lim; Azizi, Norazah; Morris, Priscilla E; Leslie Low, Eng-Ti; Ong-Abdullah, Meilina; Sambanthamurthi, Ravigadevi; Singh, Rajinder; Murphy, Denis J

2018-01-01

Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops.

Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm

PubMed Central

Rosli, Rozana; Amiruddin, Nadzirah; Ab Halim, Mohd Amin; Chan, Pek-Lan; Chan, Kuang-Lim; Azizi, Norazah; Morris, Priscilla E.; Leslie Low, Eng-Ti; Ong-Abdullah, Meilina; Sambanthamurthi, Ravigadevi; Singh, Rajinder

2018-01-01

Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops. PMID:29672525

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

PubMed

Zheng, X; Seiliez, I; Hastings, N; Tocher, D R; Panserat, S; Dickson, C A; Bergot, P; Teale, A J

2004-10-01

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

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

PubMed Central

Faergeman, N J; Knudsen, J

1997-01-01

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

Cognitive Function in Adolescence: Testing for Interactions Between Breast-Feeding and "FADS2" Polymorphisms

ERIC Educational Resources Information Center

Martin, Nicolas W.; Benyamin, Beben; Hansell, Narelle K.; Montgomery, Grant W.; Martin, Nicholas G.; Wright, Margaret J.; Bates, Timothy C.

2011-01-01

Objectives: Breast-fed C-allele carriers of the rs single nucleotide polymorphism in the fatty acyl desaturase 2 ("FADS2") gene have been reported to show a 6.4 to 7 IQ point advantage over formula-fed C-allele carriers, with no effect of breast-feeding in GG carriers. An Australian sample was examined to determine if an interaction between…

Molecular and functional characterization of a fads2 orthologue in the Amazonian teleost, Arapaima gigas.

PubMed

Lopes-Marques, Mónica; Ozório, Rodrigo; Amaral, Ricardo; Tocher, Douglas R; Monroig, Óscar; Castro, L Filipe C

2017-01-01

The Brazilian teleost Arapaima gigas is an iconic species of the Amazon. In recent years a significant effort has been put into the farming of arapaima to mitigate overfishing threats. However, little is known regarding the nutritional requirements of A. gigas in particular those for essential fatty acids including the long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ability to biosynthesize LC-PUFA is dependent upon the gene repertoire of fatty acyl desaturases (Fads) and elongases (Elovl), as well as their fatty acid specificities. In the present study we characterized both molecularly and functionally an orthologue of the desaturase fatty acid desaturase 2 (fads2) from A. gigas. The isolated sequence displayed the typical desaturase features, a cytochrome b 5 -domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. Functional characterization of A. gigas fads2 showed that, similar to other teleosts, the A. gigas fads2 exhibited a predominant Δ6 activity complemented with some capacity for Δ8 desaturation. Given that A. gigas belongs to one of the oldest teleostei lineages, the Osteoglossomorpha, these findings offer a significant insight into the evolution LC-PUFA biosynthesis in teleosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed Central

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-01-01

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions. Images PMID:8861937

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-08-15

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

A Bacillus subtilis Gene Induced by Cold Shock Encodes a Membrane Phospholipid Desaturase

PubMed Central

Aguilar, Pablo S.; Cronan, John E.; de Mendoza, Diego

1998-01-01

Bacillus subtilis grown at 37°C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37°C are transferred to 20°C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a Δ5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37°C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37°C, but a shift of cultures from 37 to 20°C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase. PMID:9555904

Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis

PubMed Central

Kachroo, Aardra; Venugopal, Srivathsa C.; Lapchyk, Ludmila; Falcone, Deane; Hildebrand, David; Kachroo, Pradeep

2004-01-01

Stearoyl-acyl-carrier-protein-desaturase-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is a key step, which regulates levels of unsaturated fatty acids in cells. We previously showed that stearoyl-acyl-carrier-protein-desaturase mutants ssi2/fab2 carrying a loss-of-function mutation in the plastidial glycerol-3-phosphate (G3P) acyltransferase (act1) have elevated 18:1 levels and are restored in their altered defense signaling. Because G3P is required for the acylation of 18:1 by G3P acyltransferase, it was predicted that reduction of G3P levels should increase 18:1 levels and thereby revert ssi2-triggered phenotypes. Here we show that a mutation in G3P dehydrogenase restores both salicylic acid- and jasmonic acid-mediated phenotypes of ssi2 plants. The G3P dehydrogenase gene was identified by map-based cloning of the ssi2 suppressor mutant rdc8 (gly1-3) and confirmed by epistatic analysis of ssi2 with gly1-1. Restoration of ssi2-triggered phenotypes by the gly1-3 mutation was age-dependent and correlated with the levels of 18:1. Regeneration of G3P pools by glycerol application in ssi2 and ssi2 gly1-3 plants caused a marked reduction in the 18:1 levels, which rendered these plants hypersensitive to glycerol. This hypersensitivity in ssi2 was rescued by the act1 mutation. Furthermore, overexpression of the ACT1 gene resulted in enhanced sensitivity to glycerol. Glycerol application also lowered the 18:1 content in SSI2 plants and converted these into ssi2-mimics. Our results show that 18:1 levels in plastids are regulated by means of acylation with G3P, and a balance between G3P and 18:1 is critical for the regulation of salicylic acid- and jasmonic acid-mediated signaling pathways. PMID:15044700

Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein

PubMed Central

Monroig, Óscar; de Llanos, Rosa; Varó, Inmaculada; Hontoria, Francisco; Tocher, Douglas R.; Puig, Sergi; Navarro, Juan C.

2017-01-01

Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 (∆520:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 (∆1120:1) to ∆5,1120:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs. PMID:28335553

Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein.

PubMed

Monroig, Óscar; de Llanos, Rosa; Varó, Inmaculada; Hontoria, Francisco; Tocher, Douglas R; Puig, Sergi; Navarro, Juan C

2017-03-21

Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C 18 chain lengths. Scd was unable to desaturate 20:1 n- 15 ( ∆5 20:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1 n- 9 ( ∆11 20:1) to ∆5,11 20:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5 n- 3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C 24 ) PUFAs.

Enhancing freezing tolerance of Brassica napus L. by overexpression of a stearoyl-acyl carrier protein desaturase gene (SAD) from Sapium sebiferum (L.) Roxb.

PubMed

Peng, Dan; Zhou, Bo; Jiang, Yueqiao; Tan, XiaoFeng; Yuan, DeYi; Zhang, Lin

2018-07-01

Sapium sebiferum (L.) Roxb. is an important woody oil tree and traditional herbal medicine in China. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids in oil; these fatty acids greatly influence the freezing tolerance of plants. However, it remains unclear whether freezing tolerance can be regulated by the expression level of SsSAD in S. sebiferum L. Our research indicated that SsSAD expression in S. sebiferum L. increased under freezing stress. To further confirm this result, we constructed a pEGAD-SsSAD vector and transformed it into B. napus L. W10 by Agrobacterium tumefaciens-mediated transformation. Transgenic plants that overexpressed the SsSAD gene exhibited significantly higher linoleic (18:2) and linolenic acid (18:3) content and advanced freezing tolerance. These results suggest that SsSAD overexpression in B. napus L. can increase the content of polyunsaturated fatty acids (PUFAs) such as linoleic (18:2) and linolenic acid (18:3), which are likely pivotal in improving freezing tolerance in B. napus L. plants. Thus, SsSAD overexpression could be useful in the production of freeze-tolerant varieties of B. napus L. Copyright © 2018 Elsevier B.V. All rights reserved.

Ethylene and 1-MCP regulate major volatile biosynthetic pathways in apple fruit.

PubMed

Yang, Xiaotang; Song, Jun; Du, Lina; Forney, Charles; Campbell-Palmer, Leslie; Fillmore, Sherry; Wismer, Paul; Zhang, Zhaoqi

2016-03-01

The effects of ethylene and 1-methylcyclopropene (1-MCP) on apple fruit volatile biosynthesis and gene expression were investigated. Statistical analysis identified 17 genes that changed significantly in response to ethylene and 1-MCP treatments. Genes encoding branched-chain amino acid aminotransferase (BCAT), aromatic amino acid aminotransferase (ArAT) and amino acid decarboxylases (AADC) were up-regulated during ripening and further enhanced by ethylene treatment. Genes related to fatty acid synthesis and metabolism, including acyl-carrier-proteins (ACPs), malonyl-CoA:ACP transacylase (MCAT), acyl-ACP-desaturase (ACPD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC2), β-oxidation, acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECHD), acyl-CoA dehydrogenase (ACAD), and alcohol acyltransferases (AATs) also increased during ripening and in response to ethylene treatment. Allene oxide synthase (AOS), alcohol dehydrogenase 1 (ADH1), 3-ketoacyl-CoA thiolase and branched-chain amino acid aminotransferase 2 (BCAT2) decreased in ethylene-treated fruit. Treatment with 1-MCP and ethylene generally produced opposite effects on related genes, which provides evidence that regulation of these genes is ethylene dependent. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

A New F131V Mutation in Chlamydomonas Phytoene Desaturase Locates a Cluster of Norflurazon Resistance Mutations near the FAD-Binding Site in 3D Protein Models

PubMed Central

Suarez, Julio V.; Banks, Stephen; Thomas, Paul G.; Day, Anil

2014-01-01

The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM). The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505) but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene. PMID:24936791

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L.

PubMed

Zhang, Yufan; Maximova, Siela N; Guiltinan, Mark J

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance.

The Genetic Basis of Pollinator Adaptation in a Sexually Deceptive Orchid

PubMed Central

Xu, Shuqing; Schlüter, Philipp M.; Grossniklaus, Ueli; Schiestl, Florian P.

2012-01-01

In plants, pollinator adaptation is considered to be a major driving force for floral diversification and speciation. However, the genetic basis of pollinator adaptation is poorly understood. The orchid genus Ophrys mimics its pollinators' mating signals and is pollinated by male insects during mating attempts. In many species of this genus, chemical mimicry of the pollinators' pheromones, especially of alkenes with different double-bond positions, plays a key role for specific pollinator attraction. Thus, different alkenes produced in different species are probably a consequence of pollinator adaptation. In this study, we identify genes that are likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturases (SAD), in three closely related Ophrys species, O. garganica, O. sphegodes, and O. exaltata. Combining floral odor and gene expression analyses, two SAD homologs (SAD1/2) showed significant association with the production of (Z)-9- and (Z)-12-alkenes that were abundant in O. garganica and O. sphegodes, supporting previous biochemical data. In contrast, two other newly identified homologs (SAD5/6) were significantly associated with (Z)-7-alkenes that were highly abundant only in O. exaltata. Both molecular evolutionary analyses and pollinator preference tests suggest that the alkenes associated with SAD1/2 and SAD5/6 are under pollinator-mediated divergent selection among species. The expression patterns of these genes in F1 hybrids indicate that species-specific expression differences in SAD1/2 are likely due to cis-regulation, while changes in SAD5/6 are likely due to trans-regulation. Taken together, we report a genetic mechanism for pollinator-mediated divergent selection that drives adaptive changes in floral alkene biosynthesis involved in reproductive isolation among Ophrys species. PMID:22916031

Bioinformatics analysis of the ς-carotene desaturase gene in cabbage (Brassica oleracea var. capitata)

NASA Astrophysics Data System (ADS)

Sun, Bo; Zheng, Aihong; Jiang, Min; Xue, Shengling; Zhang, Fen; Tang, Haoru

2018-04-01

ς-carotene desaturase (ZDS) is an important enzyme in carotenoid biosynthesis. Here, the Brassica oleracea var. capitata ZDS (BocZDS) gene sequences were obtained from Brassica database (BRAD), and preformed for bioinformatics analysis. The BocZDS gene mapped to Scaffold000363, and contains an open reading frame of 1,686 bp that encodes a 561-amino acid protein with a calculated molecular mass of 62.00 kD and an isoelectric point (pI) of 8.2. Subcellular localization predicted the BocZDS gene was in the chloroplast. The conserved domain of the BocZDS protein is PLN02487, indicating that it belongs the member of zeta-carotene desaturase. Homology analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B. oleracea var. oleracea, B. napus, and B. rapa. The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in cabbage.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L

PubMed Central

Zhang, Yufan; Maximova, Siela N.; Guiltinan, Mark J.

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance. PMID:25926841

Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana

PubMed Central

Liénard, Marjorie A; Wang, Hong-Lei; Lassance, Jean-Marc; Löfstedt, Christer

2014-01-01

Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies. PMID:24862548

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

PubMed Central

Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

2016-01-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

PubMed

Kalinger, Rebecca S; Pulsifer, Ian P; Rowland, Owen

2018-06-01

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and β-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 β-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/β-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Phosphatidic Acid Synthesis in Bacteria

PubMed Central

Yao, Jiangwei; Rock, Charles O.

2012-01-01

Membrane phospholipid synthesis is a vital facet of bacterial physiology. Although the spectrum of phospholipid headgroup structures produced by bacteria is large, the key precursor to all of these molecules is phosphatidic acid (PtdOH). Glycerol-3-phosphate derived from the glycolysis via glycerol-phosphate synthase is the universal source for the glycerol backbone of PtdOH. There are two distinct families of enzymes responsible for the acylation of the 1-position of glycerol-3-phosphate. The PlsB acyltransferase was discovered in Escherichia coli, and homologs are present in many eukaryotes. This protein family primarily uses acyl-acyl carrier protein (ACP) endproducts of fatty acid synthesis as acyl donors, but may also use acyl-CoA derived from exogenous fatty acids. The second protein family, PlsY, is more widely distributed in bacteria and utilizes the unique acyl donor, acyl-phosphate, which is produced from acyl-ACP by the enzyme PlsX. The acylation of the 2-position is carried out by members of the PlsC protein family. All PlsCs use acyl-ACP as the acyl donor, although the PlsCs of the γ-proteobacteria also may use acyl-CoA. Phospholipid headgroups are precursors in the biosynthesis of other membrane-associated molecules and the diacylglycerol product of these reactions is converted to PtdOH by one of two distinct families of lipid kinases. The central importance of the de novo and recycling pathways to PtdOH in cell physiology suggest these enzymes are suitable targets for the development of antibacterial therapeutics in Gram-positive pathogens. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:22981714

Disruption of a horizontally transferred phytoene desaturase abolishes carotenoid accumulation and diapause in Tetranychus urticae

PubMed Central

Bryon, Astrid; Kurlovs, Andre H.; Greenhalgh, Robert; Riga, Maria; Grbić, Miodrag; Tirry, Luc; Osakabe, Masahiro; Vontas, John; Clark, Richard M.; Van Leeuwen, Thomas

2017-01-01

Carotenoids underlie many of the vibrant yellow, orange, and red colors in animals, and are involved in processes ranging from vision to protection from stresses. Most animals acquire carotenoids from their diets because de novo synthesis of carotenoids is primarily limited to plants and some bacteria and fungi. Recently, sequencing projects in aphids and adelgids, spider mites, and gall midges identified genes with homology to fungal sequences encoding de novo carotenoid biosynthetic proteins like phytoene desaturase. The finding of horizontal gene transfers of carotenoid biosynthetic genes to three arthropod lineages was unprecedented; however, the relevance of the transfers for the arthropods that acquired them has remained largely speculative, which is especially true for spider mites that feed on plant cell contents, a known source of carotenoids. Pigmentation in spider mites results solely from carotenoids. Using a combination of genetic approaches, we show that mutations in a single horizontally transferred phytoene desaturase result in complete albinism in the two-spotted spider mite, Tetranychus urticae, as well as in the citrus red mite, Panonychus citri. Further, we show that phytoene desaturase activity is essential for photoperiodic induction of diapause in an overwintering strain of T. urticae, consistent with a role for this enzyme in provisioning provitamin A carotenoids required for light perception. Carotenoid biosynthetic genes of fungal origin have therefore enabled some mites to forgo dietary carotenoids, with endogenous synthesis underlying their intense pigmentation and ability to enter diapause, a key to the global distribution of major spider mite pests of agriculture. PMID:28674017

Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis

PubMed Central

Koschmieder, Julian; Brausemann, Anton; Drepper, Friedel; Rodriguez-Franco, Marta; Ghisla, Sandro; Warscheid, Bettina; Einsle, Oliver; Beyer, Peter

2015-01-01

Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FADred, while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds. PMID:26147209

Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids.

PubMed

Beld, Joris; Abbriano, Raffaela; Finzel, Kara; Hildebrand, Mark; Burkart, Michael D

2016-04-01

In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes. We fed various odd and short chain fatty acids to the bacterium Escherichia coli, cyanobacterium Synechocystis sp. PCC 6803, green microalga Chlamydomonas reinhardtii and diatom Thalassiosira pseudonana. Major differences were observed, specifically in the ability among species to incorporate and elongate short chain fatty acids. We demonstrate that E. coli, C. reinhardtii, and T. pseudonana can produce longer fatty acid products from short chain precursors (C3 and C5), while Synechocystis sp. PCC 6803 lacks this ability. However, Synechocystis can incorporate and elongate longer chain fatty acids due to acyl-acyl carrier protein synthetase (AasS) activity, and knockout of this protein eliminates the ability to incorporate these fatty acids. In addition, expression of a characterized AasS from Vibrio harveyii confers a similar capability to E. coli. The ability to desaturate exogenously added fatty acids was only observed in Synechocystis and C. reinhardtii. We further probed fatty acid metabolism of these organisms by feeding desaturase inhibitors to test the specificity of long-chain fatty acid desaturases. In particular, supplementation with thia fatty acids can alter fatty acid profiles based on the location of the sulfur in the chain. We show that coupling sensitive gas chromatography mass spectrometry to supplementation of unnatural fatty acids can reveal major differences between fatty acid metabolism in various organisms. Often unnatural fatty acids have antibacterial or even therapeutic properties. Feeding of short precursors now gives us easy access to these extended molecules.

In Vivo Lipid "Tag and Track" Approach Shows Acyl Editing of Plastid Lipids and Chloroplast Import of Phosphatidylglycerol Precursors in Arabidopsis thaliana.

PubMed

Hurlock, Anna K; Wang, Kun; Takeuchi, Tomomi; Horn, Patrick J; Benning, Christoph

2018-06-19

In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexity in vivo, we developed an in vivo lipid "tag and track" method. Essentially, we probed lipid metabolism in Arabidopsis thaliana by expressing a coding sequence for a fatty acid desaturase from Physcomitrella patens (Δ6D). This enzyme places a double bond after the 6 th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at its sn-2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to "tag and track" the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence of ER-derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this "tag and track" method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Genome sequencing-assisted identification and the first functional validation of N-acyl-homoserine-lactone synthases from the Sphingomonadaceae family

PubMed Central

Gan, Han Ming; Dailey, Lucas K.; Halliday, Nigel; Williams, Paul; Hudson, André O.

2016-01-01

Background Members of the genus Novosphingobium have been isolated from a variety of environmental niches. Although genomics analyses have suggested the presence of genes associated with quorum sensing signal production e.g., the N-acyl-homoserine lactone (AHL) synthase (luxI) homologs in various Novosphingobium species, to date, no luxI homologs have been experimentally validated. Methods In this study, we report the draft genome of the N-(AHL)-producing bacterium Novosphingobium subterraneum DSM 12447 and validate the functions of predicted luxI homologs from the bacterium through inducible heterologous expression in Agrobacterium tumefaciens strain NTL4. We developed a two-dimensional thin layer chromatography bioassay and used LC-ESI MS/MS analyses to separate, detect and identify the AHL signals produced by the N. subterraneum DSM 12447 strain. Results Three predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of each luxI homologs followed by LC-ESI MS/MS and two-dimensional reverse phase thin layer chromatography bioassays followed by bioluminescent ccd camera imaging indicate that the three LuxI homologs are able to produce a variety of medium-length AHL compounds. New insights into the LuxI phylogeny was also gleemed as inferred by Bayesian inference. Discussion This study significantly adds to our current understanding of quorum sensing in the genus Novosphingobium and provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genus Novosphingobium and more generally the family Sphingomonadaceae. PMID:27635318

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

The GmFAD7 gene family from soybean: identification of novel genes and tissue-specific conformations of the FAD7 enzyme involved in desaturase activity.

PubMed

Andreu, Vanesa; Lagunas, Beatriz; Collados, Raquel; Picorel, Rafael; Alfonso, Miguel

2010-07-01

The FAD7 gene encodes a omega3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean omega3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular omega3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity.

Identification and Analysis of a Gene from Calendula officinalis Encoding a Fatty Acid Conjugase

PubMed Central

Qiu, Xiao; Reed, Darwin W.; Hong, Haiping; MacKenzie, Samuel L.; Covello, Patrick S.

2001-01-01

Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Δ12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes. PMID:11161042

A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

PubMed Central

Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

2000-01-01

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

A Specific Hepatic Transfer RNA for Phosphoserine*

PubMed Central

Mäenpää, Pekka H.; Bernfield, Merton R.

1970-01-01

Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

An integrative “omics” approach identifies new candidate genes to impact aroma volatiles in peach fruit

PubMed Central

2013-01-01

Background Ever since the recent completion of the peach genome, the focus of genetic research in this area has turned to the identification of genes related to important traits, such as fruit aroma volatiles. Of the over 100 volatile compounds described in peach, lactones most likely have the strongest effect on fruit aroma, while esters, terpenoids, and aldehydes have minor, yet significant effects. The identification of key genes underlying the production of aroma compounds is of interest for any fruit-quality improvement strategy. Results Volatile (52 compounds) and gene expression (4348 genes) levels were profiled in peach fruit from a maturity time-course series belonging to two peach genotypes that showed considerable differences in maturation characteristics and postharvest ripening. This data set was analyzed by complementary correlation-based approaches to discover the genes related to the main aroma-contributing compounds: lactones, esters, and phenolic volatiles, among others. As a case study, one of the candidate genes was cloned and expressed in yeast to show specificity as an ω-6 Oleate desaturase, which may be involved in the production of a precursor of lactones/esters. Conclusions Our approach revealed a set of genes (an alcohol acyl transferase, fatty acid desaturases, transcription factors, protein kinases, cytochromes, etc.) that are highly associated with peach fruit volatiles, and which could prove useful in breeding or for biotechnological purposes. PMID:23701715

Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.

PubMed

Levine, L; Gjika, H B; Van Vunakis, H

1989-11-30

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold.

PubMed

Gao, Jinpeng; Wallis, James G; Browse, John

2015-09-01

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C-6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes. © 2015 American Society of Plant Biologists. All Rights Reserved.

Structure of a bacterial toxin-activating acyltransferase.

PubMed

Greene, Nicholas P; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2015-06-09

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.

Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba

PubMed Central

2013-01-01

Background Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil. Results Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase. Conclusions Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished relative to their abundance in the total FA pool. PMID:24308551

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

PubMed

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

2014-10-15

Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. Copyright © 2014 Elsevier Inc. All rights reserved.

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)-Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis.

PubMed

Hemmerling, Franziska; Lebe, Karen E; Wunderlich, Johannes; Hahn, Frank

2018-03-08

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of peptide .alpha.-thioesters

DOEpatents

Camarero, Julio A [Livermore, CA; Mitchell, Alexander R [Livermore, CA; De Yoreo, James J [Clayton, CA

2008-08-19

Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

Pollination by sexual deception-it takes chemistry to work.

PubMed

Bohman, Björn; Flematti, Gavin R; Barrow, Russell A; Pichersky, Eran; Peakall, Rod

2016-08-01

Semiochemicals are of paramount importance in sexually deceptive plants. These plants sexually lure specific male insects as pollinators by chemical and physical mimicry of the female of the pollinator. The strategy has evolved repeatedly in orchids, with a wide diversity of insect groups exploited. Chemical communication systems confirmed by field bioassays include: alkenes and alkanes in bee pollinated Ophrys species, keto-acid and hydroxy-acids in scoliid wasp pollinated O. speculum, and cyclohexanediones and pyrazines in thynnine wasp pollinated Chiloglottis and Drakaea orchids, respectively. In Ophrys, stearoyl-acyl carrier protein desaturase (SAD) enzymes have been confirmed to control species level variation in alkene double bond position. The production of cyclohexanediones in Chiloglottis unexpectedly depends on UVB light, a phenomenon unknown for other plant specialised metabolites. Potential biosynthetic pathways for other systems are explored, and alternative approaches to further accelerate chemical discovery in sexually deceptive plants are proposed. Copyright © 2016 Elsevier Ltd. All rights reserved.

A link between lipid metabolism and epithelial-mesenchymal transition provides a target for colon cancer therapy

PubMed Central

Sánchez-Martínez, Ruth; Álvarez-Fernández, Mónica; Vargas, Teodoro; Molina, Susana; García, Belén; Herranz, Jesús; Moreno-Rubio, Juan; Reglero, Guillermo; Pérez-Moreno, Mirna; Feliu, Jaime; Malumbres, Marcos; de Molina, Ana Ramírez

2015-01-01

The alterations in carbohydrate metabolism that fuel tumor growth have been extensively studied. However, other metabolic pathways involved in malignant progression, demand further understanding. Here we describe a metabolic acyl-CoA synthetase/stearoyl-CoA desaturase ACSL/SCD network causing an epithelial-mesenchymal transition (EMT) program that promotes migration and invasion of colon cancer cells. The mesenchymal phenotype produced upon overexpression of these enzymes is reverted through reactivation of AMPK signaling. Furthermore, this network expression correlates with poorer clinical outcome of stage-II colon cancer patients. Finally, combined treatment with chemical inhibitors of ACSL/SCD selectively decreases cancer cell viability without reducing normal cells viability. Thus, ACSL/SCD network stimulates colon cancer progression through conferring increased energetic capacity and invasive and migratory properties to cancer cells, and might represent a new therapeutic opportunity for colon cancer treatment. PMID:26451612

Engineering acyl carrier protein to enhance production of shortened fatty acids.

PubMed

Liu, Xueliang; Hicks, Wade M; Silver, Pamela A; Way, Jeffrey C

2016-01-01

The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris. We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.

The effects of space flight on some rat liver enzymes regulating carbohydrate and lipid metabolism

NASA Technical Reports Server (NTRS)

Abraham, S.; Lin, C. Y.; Klein, H. P.; Volkmann, C.

1981-01-01

The effects of space flight conditions on the activities of certain enzymes regulating carbohydrate and lipid metabolism in rat liver are investigated in an attempt to account for the losses in body weight observed during space flight despite preflight caloric consumption. Liver samples were analyzed for the activities of 32 cytosolic and microsomal enzymes as well as hepatic glycogen and individual fatty acid levels for ground control rats and rats flown on board the Cosmos 936 biosatellite under normal space flight conditions and in centrifuges which were sacrificed upon recovery or 25 days after recovery. Significant decreases in the activities of glycogen phosphorylase, alpha-glycerol phosphate acyl transferase, diglyceride acyl transferase, aconitase and 6-phosphogluconate dehydrogenase and an increase in palmitoyl CoA desaturase are found in the flight stationary relative to the flight contrifuged rats upon recovery, with all enzymes showing alterations returning to normal values 25 days postflight. The flight stationary group is also observed to be characterized by more than twice the amount of liver glycogen of the flight centrifuged group as well as a significant increase in the ratio of palmitic to palmitoleic acid. Results thus indicate metabolic changes which may be involved in the mechanism of weight loss during weightlessness, and demonstrate the equivalence of centrifugation during space flight to terrestrial gravity.

ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana1

PubMed Central

Sautron, Emeline; Boudiere, Laurence; Michaud, Morgane; Dubots, Emmanuelle; Albrieux, Catherine; Marechal, Eric; Jouhet, Juliette

2016-01-01

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature. PMID:26620528

Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, W.; Shanklin, J.; Yu, X.-H.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. Inmore » addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30 C), MS2 exhibits a K{sub m} for 16:0-Acyl Carrier Protein of 23.3 {+-} 4.0 {mu}m, a V{sub max} of 38.3 {+-} 4.5 nmol mg{sup -1} min{sup -1}, and a catalytic efficiency/K{sub m} of 1,873 m{sup -1} s{sup -1}. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.« less

[Cloning and functional characterization of phytoene desaturase in Andrographis paniculata].

PubMed

Shen, Qin-qin; Li, Li-xia; Zhan, Peng-lin; Wang, Qiang

2015-10-01

A full-length cDNA of phytoene desaturase (PDS) gene from Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank: KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD ( H) -binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway.

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

PubMed Central

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

2010-01-01

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

Effects of water temperature and diets containing palm oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes of rainbow trout (Oncorhynchus mykiss).

PubMed

Tocher, Douglas R; Fonseca-Madrigal, Jorge; Dick, James R; Ng, Wing-Keong; Bell, J Gordon; Campbell, Patrick J

2004-01-01

Food grade fisheries have reached their sustainable limits while aquaculture production has increased to meet consumer demands. However, for growth in aquaculture to continue and utilise sustainable, feeding ingredients, alternatives to fish oil (FO), the predominant lipid component of fish diets, must be developed. Therefore, there is currently considerable interest in the regulation of fatty acid metabolism in fish in order to determine strategies for the best use of plant oils in diets for commercially important cultured fish species. Plant oils are characteristically rich in C18 polyunsaturated fatty acids (PUFA) but devoid of C20 and C22 highly unsaturated fatty acids (HUFA) found in FO. The fatty acyl desaturase enzyme activities involved in the biosynthesis of HUFA from PUFA are known to be under nutritional regulation and can be increased in fish fed diets rich in plant oils. However, fatty acid desaturase activity is also known to be modulated by water temperature in fish. The present study aimed to investigate the interaction between water temperature and diet in the regulation of fatty acid metabolism in rainbow trout. Trout, acclimatized to 7, 11 or 15 degrees C, were fed for 4 weeks on diets in which the FO was replaced in a graded manner by palm oil. At the end of the trial, fatty acyl desaturation/elongation and beta-oxidation activities were determined in isolated hepatocytes and intestinal enterocytes using [1-14C]18:3n-3 as substrate, and samples of liver were collected for analysis of lipid and fatty acid composition. The most obvious effect of temperature was that fatty acid desaturation/elongation and beta-oxidation were reduced in both hepatocytes and intestinal enterocytes from fish maintained at the highest water temperature (15 degrees C). There were differences between the two tissues with the highest desaturation/elongation and beta-oxidation activities tending to be in fish held at 11 degrees C in the case of hepatocytes, but 7 degrees C in enterocytes. Correlations between fatty acid metabolism and dietary palm oil were most clearly observed in desaturation/elongation activities in both hepatocytes and enterocytes at 11 degrees C. The highest beta-oxidation activities were generally observed in fish fed FO alone in both hepatocytes and enterocytes with palm oil having differential effects in the two cell types.

Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

PubMed

Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

2015-03-01

The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

PubMed

Metz, J G; Pollard, M R; Anderson, L; Hayes, T R; Lassner, M W

2000-03-01

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.

The β-Ketoacyl-CoA Synthase HvKCS1, Encoded by Cer-zh, Plays a Key Role in Synthesis of Barley Leaf Wax and Germination of Barley Powdery Mildew.

PubMed

Li, Chao; Haslam, Tegan M; Krüger, Anna; Schneider, Lizette M; Mishina, Kohei; Samuels, Lacey; Yang, Hongxing; Kunst, Ljerka; Schaffrath, Ulrich; Nawrath, Christiane; Chen, Guoxiong; Komatsuda, Takao; von Wettstein-Knowles, Penny

2018-04-01

The cuticle coats the primary aerial surfaces of land plants. It consists of cutin and waxes, which provide protection against desiccation, pathogens and herbivores. Acyl cuticular waxes are synthesized via elongase complexes that extend fatty acyl precursors up to 38 carbons for downstream modification pathways. The leaves of 21 barley eceriferum (cer) mutants appear to have less or no epicuticular wax crystals, making these mutants excellent tools for identifying elongase and modification pathway biosynthetic genes. Positional cloning of the gene mutated in cer-zh identified an elongase component, β-ketoacyl-CoA synthase (CER-ZH/HvKCS1) that is one of 34 homologous KCSs encoded by the barley genome. The biochemical function of CER-ZH was deduced from wax and cutin analyses and by heterologous expression in yeast. Combined, these experiments revealed that CER-ZH/HvKCS1 has a substrate specificity for C16-C20, especially unsaturated, acyl chains, thus playing a major role in total acyl chain elongation for wax biosynthesis. The contribution of CER-ZH to water barrier properties of the cuticle and its influence on the germination of barley powdery mildew fungus were also assessed.

ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use.

PubMed

Wang, Mingxuan; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Wei; Chen, Yong Q

2013-12-01

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.

Lipid metabolism in Rhodnius prolixus: Lessons from the genome.

PubMed

Majerowicz, David; Calderón-Fernández, Gustavo M; Alves-Bezerra, Michele; De Paula, Iron F; Cardoso, Lívia S; Juárez, M Patricia; Atella, Georgia C; Gondim, Katia C

2017-01-05

The kissing bug Rhodnius prolixus is both an important vector of Chagas' disease and an interesting model for investigation into the field of physiology, including lipid metabolism. The publication of this insect genome will bring a huge amount of new molecular biology data to be used in future experiments. Although this work represents a promising scenario, a preliminary analysis of the sequence data is necessary to identify and annotate the genes involved in lipid metabolism. Here, we used bioinformatics tools and gene expression analysis to explore genes from different genes families and pathways, including genes for fat breakdown, as lipases and phospholipases, and enzymes from β-oxidation, fatty acid metabolism, and acyl-CoA and glycerolipid synthesis. The R. prolixus genome encodes 31 putative lipase genes, including 21 neutral lipases and 5 acid lipases. The expression profiles of some of these genes were analyzed. We were able to identify nine phospholipase A2 genes. A variety of gene families that participate in fatty acid synthesis and modification were studied, including fatty acid synthase, elongase, desaturase and reductase. Concerning the synthesis of glycerolipids, we found a second isoform of glycerol-3-phosphate acyltransferase that was ubiquitously expressed throughout the organs. Finally, all genes involved in fatty acid β-oxidation were identified, but not a long-chain acyl-CoA dehydrogenase. These results provide fundamental data to be used in future research on insect lipid metabolism and its possible relevance to Chagas' disease transmission. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Hepatic delta 6-desaturase activity in lean and genetically obese ob/ob mice.

PubMed Central

Hughes, S; York, D A

1985-01-01

Hepatic delta 6-desaturase activity is primarily located in the mitochondrial fraction in mice. Both delta 6- and delta 5-desaturase activities are increased in the liver of young (6-week-old) obese mice. The increase in hepatic delta 6-desaturase activity in obese mice does not occur until weaning. Neither restriction of food intake nor hyperinsulinaemia normalize hepatic delta 6-desaturase activity of obese mice. Both cold acclimation and tri-iodothyronine (30 micrograms/day per kg) decreased hepatic delta 6-desaturase activity of obese mice to levels observed in lean mice, whereas the increase in activity in obese mice was still maintained after the induction of hypothyroidism. PMID:3977836

Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

USDA-ARS?s Scientific Manuscript database

In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

Effects of saturated and unsaturated fatty acids on estimated desaturase activities during a controlled dietary intervention.

PubMed

Warensjö, Eva; Risérus, Ulf; Gustafsson, Inga-Britt; Mohsen, Rawya; Cederholm, Tommy; Vessby, Bengt

2008-12-01

Direct measurement of desaturase activities are difficult to obtain in humans. Consequently, surrogate measures of desaturase activity (estimated desaturase activities) have been frequently used in observational studies, and estimated Delta(9)- (or stearoyl-CoA-desaturase (SCD)), Delta(6)- and Delta(5)-desaturase activities have been associated with cardiometabolic disease. Data on how the markers of desaturase activities are modified by changes in dietary fat quality are lacking and therefore warrant examination. In a two-period (three weeks) strictly controlled cross-over study, 20 subjects (six women and 14 men) consumed a diet high in saturated fat (SAT-diet) and a rapeseed oil diet (RO-diet), rich in oleic acid (OA), linoleic acid (LA) and alpha-linolenic acid (ALA). Estimated desaturase activities were calculated as precursor to product FA ratios in serum cholesteryl esters and phospholipids. The estimated SCD [16:1 n-7/16:0] and Delta(6)-desaturase [20:3 n-6/18:2 n-6] was significantly higher while Delta(5)-desaturase [20:4 n-6/20:3 n-6] was significantly lower in the SAT-diet (P<0.001 for all), compared to the RO-diet. The serum proportions of palmitic, stearic, palmitoleic and dihomo-gamma-linolenic acids were significantly higher in the SAT-diet while the proportions of LA and ALA were significantly higher in the RO-diet. This is the first study to demonstrate that surrogate measures of desaturase activities change as a consequence of an alteration in dietary fat quality. Both the [16:1/16:0]-ratio and 16:1 seem to reflect changes in saturated fat intake and may be useful markers of saturated fat intake in Western countries.

Sex Pheromone Evolution Is Associated with Differential Regulation of the Same Desaturase Gene in Two Genera of Leafroller Moths

PubMed Central

Albre, Jérôme; Liénard, Marjorie A.; Sirey, Tamara M.; Schmidt, Silvia; Tooman, Leah K.; Carraher, Colm; Greenwood, David R.; Löfstedt, Christer; Newcomb, Richard D.

2012-01-01

Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation. PMID:22291612

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

PubMed Central

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

2014-01-01

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

Genome-Wide Association Study of Genetic Control of Seed Fatty Acid Biosynthesis in Brassica napus

PubMed Central

Gacek, Katarzyna; Bayer, Philipp E.; Bartkowiak-Broda, Iwona; Szala, Laurencja; Bocianowski, Jan; Edwards, David; Batley, Jacqueline

2017-01-01

Fatty acids and their composition in seeds determine oil value for nutritional or industrial purposes and also affect seed germination as well as seedling establishment. To better understand the genetic basis of seed fatty acid biosynthesis in oilseed rape (Brassica napus L.) we applied a genome-wide association study, using 91,205 single nucleotide polymorphisms (SNPs) characterized across a mapping population with high-resolution skim genotyping by sequencing (SkimGBS). We identified a cluster of loci on chromosome A05 associated with oleic and linoleic seed fatty acids. The delineated genomic region contained orthologs of the Arabidopsis thaliana genes known to play a role in regulation of seed fatty acid biosynthesis such as Fatty acyl-ACP thioesterase B (FATB) and Fatty Acid Desaturase (FAD5). This approach allowed us to identify potential functional genes regulating fatty acid composition in this important oil producing crop and demonstrates that this approach can be used as a powerful tool for dissecting complex traits for B. napus improvement programs. PMID:28163710

Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10.

PubMed

Jiang, Xianzhang; Liu, Hongjiao; Niu, Yongchao; Qi, Feng; Zhang, Mingliang; Huang, Jianzhong

2017-03-01

To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ 6 -desaturase gene (Md6) from Mucor sp. and its 5'-upstream sequence was functionally identified in Saccharomyces cerevisiae. Expression of the Δ 6 -fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5'-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between -919 to -784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ 6 -desaturase. The activity of Δ 6 -desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of -434 with forward or reverse orientations respectively. To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ 6 -desaturase reported so far and the sequence between -919 to -784 bp is an enhancer for Δ 6 -desaturase activity.

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

Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs

PubMed Central

Surm, Joachim M.; Prentis, Peter J.; Pavasovic, Ana

2015-01-01

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs. PMID:26308548

Mutant fatty acid desaturase and methods for directed mutagenesis

DOEpatents

Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

2008-01-29

The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

PubMed

Monroig, Oscar; Zheng, Xiaozhong; Morais, Sofia; Leaver, Michael J; Taggart, John B; Tocher, Douglas R

2010-09-01

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology. 2010 Elsevier B.V. All rights reserved.

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos.

PubMed

Hsieh, S L; Liao, W L; Kuo, C M

2001-12-01

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%-64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.

Host-virus shift of the sphingolipid pathway along an Emiliania huxleyi bloom: survival of the fattest.

PubMed

Pagarete, António; Allen, Michael J; Wilson, William H; Kimmance, Susan A; de Vargas, Colomban

2009-11-01

The interactions between viruses and phytoplankton play a key role in shaping the ecological and evolutionary dynamics of oceanic ecosystems. One of the most fascinating examples of horizontal gene transfer between a eukaryotic host and its virus is a de novo sphingolipid biosynthesis pathway (SBP) found in the genomes of both Emiliania huxleyi and its coccolithovirus EhV-86. Here, we focus on a natural E. huxleyi/coccolithovirus system off the coast of Norway and investigate the dynamics of host and virus homologous gene expression for two of the most important sphingolipid biosynthesis enzymes, serine palmitoyl transferase (SPT) and dihydroceramide desaturase (DCD). Transcriptional dynamics display three defined stages along E. huxleyi bloom formation and decline, with the coccolithovirus transcripts taking over and controlling the SBP in stages 2 and 3. The observed patterns fit the hypothesis according to which viral sphingolipids are involved in the timing and physical processes of virion release from the host cells. This study provides a unique insight into the transcriptional interplay of homologous metabolic pathways between virus and host during temporal progression of oceanic E. huxleyi blooms.

Fatty Acid Desaturases, Polyunsaturated Fatty Acid Regulation, and Biotechnological Advances

PubMed Central

Lee, Je Min; Lee, Hyungjae; Kang, SeokBeom; Park, Woo Jung

2016-01-01

Polyunsaturated fatty acids (PUFAs) are considered to be critical nutrients to regulate human health and development, and numerous fatty acid desaturases play key roles in synthesizing PUFAs. Given the lack of delta-12 and -15 desaturases and the low levels of conversion to PUFAs, humans must consume some omega-3 and omega-6 fatty acids in their diet. Many studies on fatty acid desaturases as well as PUFAs have shown that fatty acid desaturase genes are closely related to different human physiological conditions. Since the first front-end desaturases from cyanobacteria were cloned, numerous desaturase genes have been identified and animals and plants have been genetically engineered to produce PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Recently, a biotechnological approach has been used to develop clinical treatments for human physiological conditions, including cancers and neurogenetic disorders. Thus, understanding the functions and regulation of PUFAs associated with human health and development by using biotechnology may facilitate the engineering of more advanced PUFA production and provide new insights into the complexity of fatty acid metabolism. PMID:26742061

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.

Identification and characterization of a novel N-acyl-homoserine lactonase gene in Sphingomonas ursincola isolated from industrial cooling water systems.

PubMed

Morohoshi, Tomohiro; Sato, Niina; Iizumi, Taro; Tanaka, Airi; Ikeda, Tsukasa

2017-05-01

Biofilm formation by bacteria is one of the main causes of fouling in industrial cooling water systems. In many gram-negative bacteria, biofilm formation is regulated by N-acyl-homoserine lactone (AHL)-mediated quorum sensing. In this study, we isolated three AHL-degrading bacteria from cooling water systems and identified them as Sphingomonas ursincola. The draft genome sequence of S. ursincola A1 revealed the presence of an AHL-degrading gene homolog, designated qsdS. The qsdS region was also amplified by PCR from the genomes of the other two S. ursincola strains, SF1 and SF8. Escherichia coli DH5α harboring a QsdS-expressing plasmid showed high degradative activity against AHLs with short and 3-oxo-substituted acyl chains. High-performance liquid chromatography analysis revealed that QsdS is an AHL lactonase, an enzyme that catalyzes AHL ring opening. Furthermore, heterologous expression of QsdS in Pseudomonas aeruginosa PAO1 resulted in degradation of endogenous AHLs and interfered with the quorum-sensing-regulated phenotype. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction*

PubMed Central

Abe, Tomoko; Hashimoto, Yoshiteru; Zhuang, Ye; Ge, Yin; Kumano, Takuto; Kobayashi, Michihiko

2016-01-01

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical” S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed. PMID:26586916

Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum.

PubMed

Saito, T; Ochiai, H

1999-10-01

cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.

Conversion of hexadecanoic acid to hexadecenoic acid by rat Delta 6-desaturase.

PubMed

Guillou, Hervé; Rioux, Vincent; Catheline, Daniel; Thibault, Jean-Nöel; Bouriel, Monique; Jan, Sophie; D'Andrea, Sabine; Legrand, Philippe

2003-03-01

A higher content of C16:1 n-10 has recently been reported in the preputial gland of mice with a targeted disruption of the gene encoding stearoyl-CoA desaturase 1 (SCD1-/- mice) when compared with wild-type mice. This result has provided the first physiological evidence for the presence and regulation of a palmitoyl-CoA Delta 6-desaturase in mammals. To investigate the putative involvement of the known Delta 6-desaturase (FADS2) in this process, COS-7 cells expressing rat Delta 6-desaturase were incubated with C16:0. Transfected cells were able to synthesize C16:1 n-10, while nontransfected cells did not produce any C16:1 n-10. Evidence is therefore presented that the rat Delta 6-desaturase, which acts on the 18- and 24-carbon fatty acids of the n-6 and n-3 series, is also able to catalyze palmitic acid Delta 6 -desaturation.

FadD Is Required for Utilization of Endogenous Fatty Acids Released from Membrane Lipids ▿ †

PubMed Central

Pech-Canul, Ángel; Nogales, Joaquina; Miranda-Molina, Alfonso; Álvarez, Laura; Geiger, Otto; Soto, María José; López-Lara, Isabel M.

2011-01-01

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth. PMID:21926226

A More Desirable Balanced Polyunsaturated Fatty Acid Composition Achieved by Heterologous Expression of Δ15/Δ4 Desaturases in Mammalian Cells

PubMed Central

Zhu, Guiming; Ou, Qin; Zhang, Tao; Jiang, Xudong; Sun, Guozhi; Zhang, Ning; Wang, Kunfu; Fang, Heng; Wang, Mingfu; Sun, Jie; Ge, Tangdong

2013-01-01

Arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway–a further elongation step from docosapentaenoic acid (DPA), a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage), coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line). Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs) in transgenic HEK293 cells made a more desirable fatty acid composition – a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health. PMID:24391980

A Novel Glucosylation Reaction on Anthocyanins Catalyzed by Acyl-Glucose–Dependent Glucosyltransferase in the Petals of Carnation and Delphinium[C][W

PubMed Central

Matsuba, Yuki; Sasaki, Nobuhiro; Tera, Masayuki; Okamura, Masachika; Abe, Yutaka; Okamoto, Emi; Nakamura, Haruka; Funabashi, Hisakage; Takatsu, Makoto; Saito, Mikako; Matsuoka, Hideaki; Nagasawa, Kazuo; Ozeki, Yoshihiro

2010-01-01

Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose–dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose–dependent glucosyltransferases. PMID:20971893

Acyltransferases in Bacteria

PubMed Central

Röttig, Annika

2013-01-01

SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue. PMID:23699259

Preparation of Some Homologous TEMPO Nitroxides and Oxoammonium Salts; Notes on the NMR Spectroscopy of Nitroxide Free Radicals; Observed Radical Nature of Oxoammonium Salt Solutions Containing Trace Amounts of Corresponding Nitroxides in an Equilibrium Relationship.

PubMed

Bobbitt, James M; Eddy, Nicholas A; Cady, Clyde X; Jin, Jing; Gascon, Jose A; Gelpí-Dominguez, Svetlana; Zakrzewski, Jerzy; Morton, Martha D

2017-09-15

Three new homologous TEMPO oxoammonium salts and three homologous nitroxide radicals have been prepared and characterized. The oxidation properties of the salts have been explored. The direct 13 C NMR and EPR spectra of the nitroxide free radicals and the oxoammonium salts, along with TEMPO and its oxoammonium salt, have been successfully measured with little peak broadening of the NMR signals. In the spectra of all ten compounds (nitroxides and corresponding oxoammonium salts), the carbons in the 2,2,6,6-tetramethylpiperidine core do not appear, implying paramagnetic properties. This unpredicted overall paramagnetism in the oxoammonium salt solutions is explained by a redox equilibrium as shown between oxoammonium salts and trace amounts of corresponding nitroxide. This equilibrium is confirmed by electron interchange reactions between nitroxides with an N-acetyl substituent and oxoammonium salts with longer acyl side chains.

A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry.

PubMed

Guy, Jodie E; Abreu, Isabel A; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-11-14

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

A single mutation in the castor Δ9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

PubMed Central

Guy, Jodie E.; Abreu, Isabel A.; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-01-01

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by ≈2 × 103-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme. PMID:17088542

The Drosophila protein palmitoylome: Characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases

PubMed Central

Bannan, Barbra A.; Van Etten, Jamie; Kohler, John A.; Tsoi, Yui; Hansen, Nicole M.; Sigmon, Stacey; Fowler, Elizabeth; Buff, Haley; Williams, Tiffany S.; Ault, Jeffrey G.; Glaser, Robert L.; Korey, Christopher A.

2010-01-01

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation. PMID:18719403

Functional characterisation of two cytochrome b5-fusion desaturases from Anemone leveillei: the unexpected identification of a fatty acid Delta6-desaturase.

PubMed

Whitney, Heather M; Michaelson, Louise V; Sayanova, Olga; Pickett, John A; Napier, Johnathan A

2003-10-01

The Ranunculaceae are known to accumulate a wide range of unusual fatty acids in their seed lipids, and this variability has been advocated as a taxonomic marker. The Anemone species, Anemone leveillei L. and Anemone rivularis Buch.-Ham., have previously been reported to accumulate Delta5-desaturated fatty acids in their seed tissue [K. Aitzetmüller (1995) Plant Syst Evol 9:229-240]. Two cDNAs, AL1 and AL2, with similarity to plant cytochrome b5-fusion "front-end" desaturases were isolated from developing seeds of A. leveillei and their function identified by expression in Saccharomyces cerevisiae. AL2 was characterised as a sphingolipid long-chain-base Delta8-desaturase, while AL1 acted as a fatty acid desaturase. However, AL1 did not produce Delta5-desaturated fatty acids as expected; instead, when expressed in transgenic S. cerevisiae or Arabidopsis thaliana this enzyme was functionally characterised as a Delta6-desaturase. Northern analysis confirmed the expression of this gene in seed tissue and leaf tissue of A. leveillei, though Delta6-desaturated fatty acids were found to accumulate only in the leaf tissue. The unexpected characterisation of a Delta6-desaturase in A. leveillei has implications for the use of fatty acids in chemotaxonomic studies. This is also the first report of a higher-plant Delta6-desaturase from a family other than the Boraginaceae.

A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid.

PubMed Central

Pereira, Suzette L; Huang, Yung-Sheng; Bobik, Emil G; Kinney, Anthony J; Stecca, Kevin L; Packer, Jeremy C L; Mukerji, Pradip

2004-01-01

Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans. In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors. Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase. This gene was isolated by PCR amplification of an S. diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases. Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA. This activity differs from that of the known omega3-desaturases from any organism. Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast. The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops. PMID:14651475

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis.

PubMed

Rodríguez, Sergio; Hao, Guixia; Liu, Weitian; Piña, Benjamín; Rooney, Alejandro P; Camps, Francisco; Roelofs, Wendell L; Fabriàs, Gemma

2004-12-01

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Delta11 and Delta9 desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Delta9 desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

PubMed

Thambugala, Dinushika; Cloutier, Sylvie

2014-11-01

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Bioengineering resistance to phytoene desaturase inhibitors in Arabidopsis thaliana

USDA-ARS?s Scientific Manuscript database

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all...

International Conference on Langmuir-Blodgett Films (5th) Held in Paris, France on 26-30 August 1991, Abstracts Booklet

DTIC Science & Technology

1991-08-01

Fojihh LN.. aIsetiki, LA. Bazaheu*o, .Kaimx BP30 CI Photo-amplified stroget n y with Incorporation of non-amphiphilic compounds polyion complexed p...donor- films of N-acyl-p-nitroaniline and its acceptor organic compounds homologous amphiphile D. Zhu, C. Yang, Yu. Liu, Y. Xu Y..Mixamo, K, Kauiu, T...R. Kenn, H. MChwald, K. Kjaer, J. LB films of polyheterocyclic compounds with Als-Nielsen long alkyl chains HLNAkabam K. Fukuda, M. Ikeda, K

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, Xiaomang; Li, Danyang; Chen, Dilong

Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose,more » insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. - Highlights: • We investigated the anti-steatotic effect of mangiferin (MA) in fructose-fed SHR. • MA (15 mg/kg/day for 7 weeks) ameliorated fructose-induced fatty liver in SHR. • MA inhibited hepatic DGAT2 expression at the mRNA and protein levels. • MA did not affect expression of the genes responsible for fatty acid synthesis. • MA ameliorates fructose-induced fatty liver by inhibiting hepatic DGAT2 in rats.« less

[The isozymes of stearil-coenzymeA-desaturase and insulin activity in the light of phylogenetic theory of pathology. Oleic fatty acid and realization of biologic functions of trophology and locomotion].

PubMed

2013-11-01

The formation of function of isozymes of stearil-coenzymeA-desaturases occured at the different stages of phylogeny under realization of biologic function of trophology (stearil-coenzymeA-desaturase 1) and biologic function of locomotion, insulin system (stearil-coenzymeA-desaturase 2) billions years later. The stearil-coenzymeA-desaturase 1 transforms in C 18:1 oleic fatty acid only exogenous C 16:0 palmitinic saturated fatty acid. The stearil-coenzymeA-desaturase 2 transforms only endogenic palmitinic saturated fatty acid, synthesized form glucose. The biologic role of insulin is in energy support of biologic function of locomotion. Insulin through expressing stearil-coenzymeA-desaturase 2 transforms energetically non-optimal palmitinic variation of metabolism of substrates into highly effective oleic variation for cells' groundwork of energy (saturated fatty acid and mono fatty acid). The surplus of palmitinic saturated fatty acid in food is enabled in pathogenesis of resistance to insulin and derangement of synthesis of hormone by beta-cells of islets. The resistance to insulin and diabetes mellitus are primarily the derangement of metabolism of saturated fatty acids with mono fatty acids, energy problems of organism and only afterwards the derangement of metabolism of carbohydrates. It is desirable to restrict food intake of exogenous palmitinic saturated fatty acid. The reasons are low expression of independent of insulin stearil-coenzymeA-desaturase 2, marked lipotoxicity of polar form of palmitinic saturated fatty acid and synthesis of non-optimal palmitinic triglycerides instead of physiologic and more energetically more effective oleic triglycerides.

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

PubMed Central

Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

1995-01-01

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker

USDA-ARS?s Scientific Manuscript database

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all...

Mutant fatty acid desaturase

DOEpatents

Shanklin, John; Cahoon, Edgar B.

2004-02-03

The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

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

PubMed

Tucci, Sara; Flögel, Ulrich; Spiekerkoetter, Ute

2015-07-01

Medium-chain triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders. Previously it was shown that long-term MCT supplementation strongly affects lipid metabolism in mice. We here investigate sex-specific effects in mice with very-long-chain-acyl-CoA dehydrogenase (VLCAD) deficiency in response to a long-term MCT modified diet. We quantified blood lipids, acylcarnitines, glucose, insulin and free fatty acids, as well as tissue triglycerides in the liver and skeletal muscle under a control and an MCT diet over 1 year. In addition, visceral and hepatic fat content and muscular intramyocellular lipids (IMCL) were assessed by in vivo(1)H magnetic resonance spectroscopy (MRS) techniques. The long-term application of an MCT diet induced a marked alteration of glucose homeostasis. However, only VLCAD-/- female mice developed a severe metabolic syndrome characterized by marked insulin resistance, dyslipidemia, severe hepatic and visceral steatosis, whereas VLCAD-/- males seemed to be protected and only presented with milder insulin resistance. Moreover, the highly saturated MCT diet is associated with a decreased hepatic stearoyl-CoA desaturase 1 (SCD1) activity in females aggravating the harmful effects of a saturated MCT diet. Long-term MCT supplementation deeply affects lipid metabolism in a sexual dimorphic manner resulting in a severe metabolic syndrome only in female mice. These findings are striking since the first signs of insulin resistance already occur in female VLCAD-/- mice during their reproductive period. How these metabolic adaptations are finally regulated needs to be determined. More important, the relevance of these findings for humans under these dietary modifications needs to be investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

How did nature engineer the highest surface lipid accumulation among plants? Exceptional expression of acyl-lipid-associated genes for the assembly of extracellular triacylglycerol by Bayberry ( Myrica pensylvanica ) fruits

DOE PAGES

Simpson, Jeffrey P.; Thrower, Nicholas; Ohlrogge, John B.

2016-02-09

Bayberry (Myrica pensylvanica) fruits are covered with a remarkably thick layer of crystalline wax consisting of triacylglycerol (TAG) and diacylglycerol (DAG) esterified exclusively with saturated fatty acids. As the only plant known to accumulate soluble glycerolipids as a major component of surface waxes, Bayberry represents a novel system to investigate neutral lipid biosynthesis and lipid secretion by vegetative plant cells. The assembly of Bayberry wax is distinct from conventional TAG and other surface waxes, and instead proceeds through a pathway related to cutin synthesis (Simpson and Ohlrogge, 2016). In this study, microscopic examination revealed that the fruit tissue that producesmore » and secretes wax (Bayberry knobs) is fully developed before wax accumulates and that wax is secreted to the surface without cell disruption. Comparison of transcript expression to genetically related tissues (Bayberry leaves, M. rubra fruits), cutin-rich tomato and cherry fruit epidermis, and to oil-rich mesocarp and seeds, revealed exceptionally high expression of 13 transcripts for acyl-lipid metabolism together with down-regulation of fatty acid oxidases and desaturases. The predicted protein sequences of the most highly expressed lipid-related enzyme-encoding transcripts in Bayberry knobs are 100% identical to the sequences from Bayberry leaves,which do not produce surface DAG or TAG. Together, these results indicate that TAG biosynthesis and secretion in Bayberry is achieved by both up and down-regulation of a small subset of genes related to the biosynthesis of cutin and saturated fatty acids, and also implies that modifications in gene expression, rather than evolution of new gene functions, was the major mechanism by which Bayberry evolved its specialized lipid metabolism.« less

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

PubMed

Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

2014-03-15

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. Copyright © 2013 Elsevier B.V. All rights reserved.

Iron, Oxidative Stress, and Δ9 Stearoyl-CoenzymeA Desaturase Index (C16:1/C16:0): An Analysis Applying the National Health and Nutrition Examination Survey 2003–04

PubMed Central

Wu, Yue; Baylin, Ana; Colacino, Justin A

2018-01-01

Abstract Background Stearoyl-coenzyme A desaturase (SCD) is a key enzyme in fatty acid metabolism, and elevated SCD activity is associated with multiple adverse health outcomes. Diet, hormone levels, and environmental exposures are potential factors affecting SCD activity. Less is known about the relationship between micronutrients, including iron, and SCD activity. Objective The aim of this study was to investigate the association between serum ferritin level, a biomarker of circulating iron levels, and the Δ9 desaturase index (C16:1/C16:0), a biomarker of estimated SCD activity, among women in the United States. Methods The association between serum ferritin and the Δ9 desaturase index was assessed in a cross-sectional study of 447 female participants, aged 20–49 y, from NHANES 2003–2004. The multivariate analyses were performed utilizing generalized linear modeling, adjusting for potential confounders. Mediation of the relationship between serum ferritin and Δ9 desaturase index by γ-glutamyltranspeptidase (GGT), a biomarker of oxidative stress, was also assessed. Results Increased ferritin was significantly associated with a higher Δ9 desaturase index. Adjusting for waist circumference, age, race, and cotinine levels, an interquartile range increase in serum ferritin corresponded to 3.92% (95% CI: 0.88%, 7.05%) higher Δ9 desaturase index. GGT, the biomarker used to measure oxidative stress level, did not appear to mediate the association between ferritin and Δ9 desaturase index. After stratifying by pregnancy status, these associations were limited to nonpregnant individuals. Conclusions Elevated SCD activity may be associated with increased iron storage inside the human body; the association did not appear to be mediated via oxidative stress, as estimated by GGT levels.

Characterization of Stearoyl-CoA Desaturases from a Psychrophilic Antarctic Copepod, Tigriopus kingsejongensis.

PubMed

Jung, Woongsic; Kim, Eun Jae; Han, Se Jong; Choi, Han-Gu; Kim, Sanghee

2016-10-01

Stearoyl-CoA desaturase is a key regulator in fatty acid metabolism that catalyzes the desaturation of stearic acid to oleic acid and controls the intracellular levels of monounsaturated fatty acids (MUFAs). Two stearoyl-CoA desaturases (SCD, Δ9 desaturases) genes were identified in an Antarctic copepod, Tigriopus kingsejongensis, that was collected in a tidal pool near the King Sejong Station, King George Island, Antarctica. Full-length complementary DNA (cDNA) sequences of two T. kingsejongensis SCDs (TkSCDs) were obtained from next-generation sequencing and isolated by reverse transcription PCR. DNA sequence lengths of the open reading frames of TkSCD-1 and TkSCD-2 were determined to be 1110 and 681 bp, respectively. The molecular weights deduced from the corresponding genes were estimated to be 43.1 kDa (TkSCD-1) and 26.1 kDa (TkSCD-2). The amino acid sequences were compared with those of fatty acid desaturases and sterol desaturases from various organisms and used to analyze the relationships among TkSCDs. As assessed by heterologous expression of recombinant proteins in Escherichia coli, the enzymatic functions of both stearoyl-CoA desaturases revealed that the amount of C16:1 and C18:1 fatty acids increased by greater than 3-fold after induction with isopropyl β-D-thiogalactopyranoside. In particular, C18:1 fatty acid production increased greater than 10-fold in E. coli expressing TkSCD-1 and TkSCD-2. The results of this study suggest that both SCD genes from an Antarctic marine copepod encode a functional desaturase that is capable of increasing the amounts of palmitoleic acid and oleic acid in a prokaryotic expression system.

Effect of seafood mediated PCB exposure on desaturase activity and PUFA profile in Faroese septuagenarians.

PubMed

Tøttenborg, Sandra Søgaard; Choi, Anna L; Bjerve, Kristian S; Weihe, Pal; Grandjean, Philippe

2015-07-01

Polychlorinated biphenyl (PCB) exposure may affect serum concentrations of polyunsaturated fatty acids (PUFAs) by inhibiting desaturases ∆5 and ∆6 that drive their synthesis from precursor fatty acids. Such changes in the composition of fatty acids may affect cardiovascular disease risk, which is thought to increase at elevated PCB exposures. This population-based cross-sectional study examined 712 Faroese men and women aged 70-74 years. The serum phospholipid fraction of fasting blood samples was used to determine the PUFA profile, including linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosatrienoic acid, and other relevant fatty acids. Ratios between precursor and metabolite fatty acids were used as proxies for ∆5 and ∆6 desaturase activity. Tertiles of serum-PCB concentrations were used in multiple regression analyses to determine the association between the exposure and desaturase activity. In multiple regression models, PCB exposure was inversely related to the estimated Δ6 desaturase activity resulting in accumulation of precursor fatty acids and decrease in the corresponding product PUFAs. A positive association between PCB and Δ5 desaturation was also found. A relative increase in EA was also observed, though only in the third tertile of PCB exposure. Non-linear relationships between the exposure and the desaturase activity were not found. Consuming fish and seafood may not be translated into beneficial fatty acid profiles if the diet simultaneously causes exposure to PCBs. Although the desaturase estimates were likely influenced by dietary intakes of product PUFAs, the association between PCB exposure and ∆6 desaturase activity is plausible and may affect cardiovascular disease risk. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi.

PubMed

Kotajima, Tomonori; Shiraiwa, Yoshihiro; Suzuki, Iwane

2014-10-01

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25°C, E. huxleyi produces mainly 14:0, 18:4n-3, 18:5n-3 and 22:6n-3. When the cells were transferred from 25°C to 15°C, the amount of unsaturated fatty acids, i.e. 18:1n-9, 18:3n-3 and 18:5n-3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ∆15 fatty acid desaturase, EhDES15, involved in the production of n-3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ∆6 desaturase-like domain, but it does not contain a cytochrome b5 domain nor typical His-boxes found in ∆15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n-3 fatty acid species, which are produced at low levels in wild-type cells grown at 30°C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ∆15 desaturases. These results suggested the gene encodes a novel ∆15 desaturase responsible for the synthesis of 18:3n-3 from 18:2n-6 in E. huxleyi. Copyright © 2014 Elsevier B.V. All rights reserved.

Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

PubMed

Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

2014-01-22

Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes. © 2013 Elsevier Ltd. All rights reserved.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

NASA Astrophysics Data System (ADS)

Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

2016-07-01

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

PubMed Central

Bi, Hongkai; Wang, Haihong; Cronan, John E.

2015-01-01

SUMMARY In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids. PMID:23972938

Dietary fish oil replacement by linseed oil: Effect on growth, nutrient utilization, tissue fatty acid composition and desaturase gene expression in silver barb (Puntius gonionotus) fingerlings.

PubMed

Nayak, Madhusmita; Saha, Ashis; Pradhan, Avinash; Samanta, Mrinal; Giri, Shiba Shankar

2017-03-01

Silver barb (Puntius gonionotus) is considered a promising medium carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of total or partial substitution of dietary fish oil (FO) with linseed oil (LO) on growth, nutrient utilization, whole-body composition, muscle and liver fatty acid composition. Fish (12.1±0.4g of initial body weight) were fed for 60days with five experimental iso-proteinous, iso-lipidic and iso-caloric diets in which FO (control diet) was replaced by 33.3%, 50%, 66.7% and 100% LO. Final weight, weight gain, percent weight gain, SGR decreased linearly (p<0.001) with increasing LO levels in the diets. Dietary LO substitution levels did not significantly (p>0.05) affect the feed conversion ratio (FCR), protein efficiency ratio (PER) and whole body proximate composition. Furthermore, enhanced level of LO increased α-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) and decreased eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n3) in muscle and liver. To understand the molecular mechanism of long chain-polyunsaturated fatty acid (LC-PUFA) biosynthesis, we cloned and characterized the fatty acyl Δ6 desaturase (Δ6 fad) cDNA and investigated its expression in various organs/tissues following replacement of FO with LO in the diet. The full-length Δ6 fad cDNA was 2056bp encoding 444 amino acids and was widely expressed in various organs/tissues. Replacement of FO with LO increased the expression of Δ6 fad mRNA in liver, muscle and intestine but no significant difference was found in the brain. Copyright © 2016 Elsevier Inc. All rights reserved.

Hepatic fatty acid biosynthesis is more responsive to protein than carbohydrate in rainbow trout during acute stimulations.

PubMed

Dai, Weiwei; Panserat, Stéphane; Kaushik, Sadasivam; Terrier, Frédéric; Plagnes-Juan, Elisabeth; Seiliez, Iban; Skiba-Cassy, Sandrine

2016-01-01

The link between dietary carbohydrate/protein and de novo lipogenesis (DNL) remains debatable in carnivorous fish. We aimed to evaluate and compare the response of hepatic lipogenic gene expression to dietary carbohydrate intake/glucose and dietary protein intake/amino acids (AAs) during acute stimulations using both in vivo and in vitro approaches. For the in vivo trial, three different diets and a controlled-feeding method were employed to supply fixed amount of dietary protein or carbohydrate in a single meal; for the in vitro trial, primary hepatocytes were stimulated with a low or high level of glucose (3 mM or 20 mM) and a low or high level of AAs (one-fold or four-fold concentrated AAs). In vitro data showed that a high level of AAs upregulated the expression of enzymes involved in DNL [fatty acid synthase (FAS) and ATP citrate lyase (ACLY)], lipid bioconversion [elongation of very long chain fatty acids like-5 (Elovl5), Elovl2, Δ6 fatty acyl desaturase (D6D) and stearoyl-CoA desaturase-1 (SCD1)], NADPH production [glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME)], and transcriptional factor sterol regulatory element binding protein 1-like, while a high level of glucose only elevated the expression of ME. Data in trout liver also showed that high dietary protein intake induced higher lipogenic gene expression (FAS, ACLY, and Elovl2) regardless of dietary carbohydrate intake, while high carbohydrate intake markedly suppressed the expression of acetyl-CoA carboxylase (ACC) and Elovl5. Overall, we conclude that, unlike rodents or humans, hepatic fatty acid biosynthetic gene expression in rainbow trout is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations. This discrepancy probably represents one important physiological and metabolic difference between carnivores and omnivores. Copyright © 2016 the American Physiological Society.

Rapid-mix and chemical quench studies of ferredoxin-reduced stearoyl-acyl carrier protein desaturase.

PubMed

Lyle, Karen S; Haas, Jeffrey A; Fox, Brian G

2003-05-20

Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (approximately 95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O(2) and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for approximately 90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over approximately 1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) approximately 1.5) and k(linear) (D)k(linear) approximately 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.

Molecular association of normal alkanoic acids with their thallium(I) salts: a new homologous series of fatty acid metal soaps.

PubMed

Fernández-García, M; García, M V; Redondo, M I; Cheda, J A; Fernández-García, M; Westrum, E F; Fernández-Martín, F

1997-02-01

A new homologous series of thallium(I) hydrogen dialkanoates, fatty acid thallium soaps, from the dipropane up to the ditetradecane is reported for the first time. This association with 1:1 stoichiometry is the only one exhibited by the thallium derivatives. They have been prepared by solidification of molten mixtures with equimolar proportions of acid and corresponding neutral salt, through crystallization from an anhydrous ethanolic solution of the mixture has also been successful in getting pure compounds with largest chain lengths. Vibrational spectroscopies clearly characterize these crystalline compounds as very strong hydrogen bonding systems. Assignations of active modes in proton and carbon nuclear magnetic resonance spectrometry (NMR) (in ethanol) and infrared (IR) and Raman spectra (in solid state) are reported. According to X-ray diffraction (XRD) they have monomolecular lamellar structures with the acyl chains arranged up and down to the cation/H-bond network in a methyl-to-methyl fashion, and vertically oriented to the basal plane. The acyl chains present all-trans conformation and alternating configuration (perpendicular orthorhombic subcell), like the beta'-phases of other kinds of lipids. Lamellar thickness is reported for the six room-temperature crystalline members. The molecular compounds present polymorphism, one crystal/crystal transition at temperatures close to the peritectical melting. Phase transition thermodynamics are also given and discussed with respect to their acid and salt parents. Their incongruent melting involves nearly 90% of the total enthalpic increments of both constituents' melting processes, making these compounds potential thermal energy storage materials.

Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

PubMed

Kurdrid, Pavinee; Subudhi, Sanjukta; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Hongsthong, Apiradee

2007-12-01

When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity

PubMed Central

Wang, Yun; Botolin, Daniela; Xu, Jinghua; Christian, Barbara; Mitchell, Ernestine; Jayaprakasam, Bolleddula; Nair, Muraleedharan; Peters, Jeffery M.; Busik, Julia; Olson, L. Karl; Jump, Donald B.

2009-01-01

Fatty acid elongases and desaturases play an important role in hepatic and whole body lipid composition. We examined the role that key transcription factors played in the control of hepatic elongase and desaturase expression. Studies with peroxisome proliferator-activated receptor α (PPARα)-deficient mice establish that PPARα was required for WY14643-mediated induction of fatty acid elongase-5 (Elovl-5), Elovl-6, and all three desaturases [Δ5 desaturase (Δ5D), Δ6D, and Δ9D]. Increased nuclear sterol-regulatory element binding protein-1 (SREBP-1) correlated with enhanced expression of Elovl-6, Δ5D, Δ6D, and Δ9D. Only Δ9D was also regulated independently by liver X receptor (LXR) agonist. Glucose induction of L-type pyruvate kinase, Δ9D, and Elovl-6 expression required the carbohydrate-regulatory element binding protein/MAX-like factor X (ChREBP/MLX) heterodimer. Suppression of Elovl-6 and Δ9D expression in livers of streptozotocin-induced diabetic rats and high fat-fed glucose-intolerant mice correlated with low levels of nuclear SREBP-1. In leptin-deficient obese mice (Lepob/ob), increased SREBP-1 and MLX nuclear content correlated with the induction of Elovl-5, Elovl-6, and Δ9D expression and the massive accumulation of monoun-saturated fatty acids (18:1,n-7 and 18:1,n-9) in neutral lipids. Diabetes- and obesity-induced changes in hepatic lipid composition correlated with changes in elongase and desaturase expression. In conclusion, these studies establish a role for PPARα, LXR, SREBP-1, ChREBP, and MLX in the control of hepatic fatty acid elongase and desaturase expression and lipid composition. PMID:16790840

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alloatti, Andres; Gupta, Shreedhara; Gualdron-Lopez, Melisa

2011-08-26

Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes namedmore » desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.« less

A novel selection signature in stearoyl-coenzyme A desaturase (SCD) gene for enhanced milk fat content in Bubalus bubalis.

PubMed

Maryam, J; Babar, M E; Bao, Zhang; Nadeem, A

2016-10-01

Modern molecular interventions are dynamic gears for breeding animals with superior genetic make-up. These scientific efforts lead us toward sustainable dairy herds with improved milk production in terms of yield and quality. Many of candidate genes have been dissected at molecular level, and suitable genetic markers have been identified in cattle, but this work has not been validated in buffaloes so far. Stearoyl-coenzyme A desaturase (SCD) has been a potential candidate gene for fat content of milk. Genomic analysis of SCD revealed a total of six variations that were identified through DNA sequencing of animals with lower and higher butter fat %age. After statistical analysis, genotype AB of p.K158I could be associated (P value <0.0001) with higher milk fat %age (10.5 ± 0.5464). This SNP was validated on larger data set by cleaved amplified polymorphic sequences (CAPS) by using DdeI. To scrutinize the functional consequences of p.K158I, 3D protein structure of SCD was predicted by homology modeling and this variation was found located in the vicinity of functional domain and a part of transmembrane helix of this membrane integrated protein. This is a first report toward genetic screening of SCD gene at molecular level in buffalo. This report illustrates the implication of SCD gene and in particular p.K158I variation, in imparting its effect on milk fat %age, which can be targeted in selection of superior dairy buffaloes.

Biosynthesis of 10,12-dienoic fatty acids by a bifunctional Delta11 desaturase in Spodoptera littoralis.

PubMed

Serra, Montserrat; Piña, Benjami; Bujons, Jordi; Camps, Francisco; Fabriàs, Gemma

2006-08-01

In the biosynthetic pathway of Spodoptera littoralis sex pheromone, (E,E)-10,12-tetradecadienoic acid is produced from (Z)-11-tetradecenoic acid by desaturation and concomitant migration of the precursor double bond. With the aim of identifying the enzyme involved in this biotransformation, yeast Deltaelo1/Deltaole mutants, which are both elongase 1 and Delta9 desaturase-deficient, were transformed with the S. littoralis Delta11 desaturase gene using a Cu+2 inducible expression vector. The transformants produced a recombinant polyhistidine-tagged Delta11 desaturase that could be detected by immunoblotting from cell lysates. Lipid analysis revealed that besides producing large quantities of C11-monounsaturated fatty acids, mainly (Z)-11-hexadecenoic acid, (E,E)-10,12-tetradecadienoic acid and minor amounts of (E,Z)-10,12-hexadecadienoic acid were also produced, as well as very low quantities of another tetradecadienoate, which was tentatively identified as the (E,Z)-10,12-tetradecadienoic isomer. None of these dienes was detected with the Delta11 desaturase gene of Trichoplusia ni, which does not produce conjugated dienes as pheromone components. We conclude that the Delta11 desaturase of S. littoralis is a bifunctional enzyme with both Delta11 and Delta10,12 desaturation activities. The relationship between the substrate structure and the stereochemical outcome of the reaction is discussed.

Contribution of two ζ-carotene desaturases to the poly-cis desaturation pathway in the cyanobacterium Nostoc PCC 7120.

PubMed

Breitenbach, Jürgen; Bruns, Marius; Sandmann, Gerhard

2013-07-01

The presence of two completely unrelated ζ-carotene desaturases CrtQa and CrtQb in some Nostoc strains is unique. CrtQb is the ζ-carotene desaturase, which was acquired by almost all cyanobacteria. The additional CrtQa can be regarded as an evolutionary relict of the CrtI desaturase present in non-photosynthetic bacteria. By reconstruction of the carotene desaturation pathway, we showed that both enzymes from Nostoc PCC 7120 were active. However, they differed in their preferred utilization of ζ-carotene Z isomers. CrtQa converted ζ-carotene isomers that were poorly metabolized by CrtQb. In this respect, CrtQa complemented the reactions of CrtQb, which is an advantage avoiding dead ends in the poly-cis desaturation pathway. In addition to ζ-carotene desaturation, CrtQa still possesses the Z to E isomerase function of the ancestral desaturase CrtI. Biochemical characterization showed that CrtQb is an enzyme with one molecule of tightly bound FAD and acts as a dehydrogenase transferring hydrogen to oxidized plastoquinone.

Feeding steam-pelleted rapeseed affects expression of genes involved in hepatic lipid metabolism and fatty acid composition of chicken meat.

PubMed

Li, S; Vestergren, A Schiller; Wall, H; Trattner, S; Pickova, J; Ivarsson, E

2017-08-01

This study investigated the dietary effect of steam-pelleted rapeseed (RS) diets with different inclusion levels on the fatty acid composition of chicken meat and the expression of lipid metabolism-related genes in the liver. Experimental diets included 6 different wheat-soybean meal based diets either in nonpelleted or steam-pelleted form supplemented with 80, 160, and 240 g RS/kg feed and one nonpelleted wheat-soybean meal based diet without RS supplementation as the control. These diets were fed to newly hatched broiler chickens (Ross 308) for 34 days. Compared to the control diet, steam-pelleted diets containing 160 or 240 g/kg RS significantly increased the content of omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) in the breast and drumstick, while their meat yields were not affected. Moreover, the mRNA levels of fatty acid desaturase 1 (FADS1) and acyl-coenzyme A oxidase 1 (ACOX1) in their livers increased. Therefore, steam-pelleted diets with 160 or 240 g/kg RS can be used to increase the n-3 LC-PUFA content in chicken meat without compromising meat yield. © 2017 Poultry Science Association Inc.

Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

PubMed

Breitenbach, J; Zhu, C; Sandmann, G

2001-11-01

Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

PAQR-2 Regulates Fatty Acid Desaturation during Cold Adaptation in C. elegans

PubMed Central

Svensk, Emma; Ståhlman, Marcus; Andersson, Carl-Henrik; Johansson, Maja; Borén, Jan; Pilon, Marc

2013-01-01

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant. PMID:24068966

PAQR-2 regulates fatty acid desaturation during cold adaptation in C. elegans.

PubMed

Svensk, Emma; Ståhlman, Marcus; Andersson, Carl-Henrik; Johansson, Maja; Borén, Jan; Pilon, Marc

2013-01-01

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant.

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in localized combination of conserved residues.

Plasma fatty acid composition, estimated desaturase activities, and their relation with the metabolic syndrome in a population at high risk of cardiovascular disease.

PubMed

Mayneris-Perxachs, Jordi; Guerendiain, Marcela; Castellote, Ana I; Estruch, Ramón; Covas, María Isabel; Fitó, Montserrat; Salas-Salvadó, Jordi; Martínez-González, Miguel A; Aros, Fernando; Lamuela-Raventós, Rosa M; López-Sabater, M Carmen

2014-02-01

The metabolic syndrome (MetS) is a clustering of various metabolic abnormalities which is associated with increased risk of cardiovascular disease (CVD) and type 2 diabetes mellitus. Due to its increasing prevalence, it has become an important public health concern. Altered fatty acid (FA) composition and desaturase activities have been associated with several metabolic diseases, including MetS. The aim of the present study was to evaluate the relationship of the plasma FA profile and desaturase activities with the MetS in a Mediterranean population at high risk of CVD. Baseline data from 427 participants aged 55-80 years who took part in the interventional PREDIMED study were obtained. Individual FA was determined in plasma and desaturase activities were estimated from product/precursor ratios. Odds ratios (OR) and partial correlation coefficients were used to examine these relations with MetS and its components, respectively. We found higher levels of C14:0, C16:0, C16:1n-7, estimated Δ(9)- or stearoyl-CoA desaturase (SCD), and estimated Δ(6) desaturase (D6D), and lower levels of C18:2n-6 in people with MetS compared to those without it. After adjustment for several confounders, only higher quartiles of C14:0, C16:0, C16:1n-7, and D6D were found to be associated with an increasing prevalence of MetS, while higher quartiles of C18:2n-6 were inversely associated with MetS. High proportions of C14:0, C16:0, C16:1n-7, C20:3n-6, SCD, and D6D, and decreased proportions of C18:2n-6 and estimated Δ(5)-desaturase (D5D) were associated with adverse profiles of several metabolic risk factors. Women showed more unhealthy FA pattern and lipid profiles than men, but only among those with MetS. A FA composition and estimated desaturase activities consisting in high levels of SFA, SCD and D6D, and low levels of PUFA and D5D are associated with increased MetS probability and are characteristic of people presenting MetS, especially women. These findings support those observed in non-Mediterranean populations in which an altered FA profile and estimated desaturase activities are associated with MetS. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

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

PubMed

Haslam, Tegan M; Haslam, Richard; Thoraval, Didier; Pascal, Stéphanie; Delude, Camille; Domergue, Frédéric; Fernández, Aurora Mañas; Beaudoin, Frédéric; Napier, Johnathan A; Kunst, Ljerka; Joubès, Jérôme

2015-03-01

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function. © 2015 American Society of Plant Biologists. All Rights Reserved.

ECERIFERUM2-LIKE Proteins Have Unique Biochemical and Physiological Functions in Very-Long-Chain Fatty Acid Elongation1[OPEN

PubMed Central

Haslam, Tegan M.; Haslam, Richard; Thoraval, Didier; Pascal, Stéphanie; Delude, Camille; Domergue, Frédéric; Fernández, Aurora Mañas; Beaudoin, Frédéric; Napier, Johnathan A.; Kunst, Ljerka; Joubès, Jérôme

2015-01-01

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function. PMID:25596184

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

PubMed

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

2016-09-13

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

Increasing dietary EPA and DHA influence estimated fatty acid desaturase activity in systemic organs which is reflected in the red blood cell in mice.

PubMed

Davidson, Emily A; Pickens, C Austin; Fenton, Jenifer I

2018-03-01

Delta-5 (D5D) and delta-6 (D6D) desaturase are key enzymes in fatty acid (FA) metabolism. Dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may alter tissue FA composition via D5D and D6D. The purpose was to determine the relationship between dietary EPA + DHA, estimated desaturase activities of various tissues and the reflection of desaturase activity in the red blood cell (RBC). Mice were fed diets with increasing percent of energy from EPA + DHA. Phospholipid FA composition of heart, muscle, spleen, lung, adipose tissues and RBC were analysed. D5D and D6D enzyme activity estimates (EAE) were calculated as the ratio of 20:4/20:3 and 20:3/18:2, respectively. D5D EAE decreased in all tissues, except muscle, with increasing dietary EPA + DHA. RBC D5D EAE positively correlated with D5D EAE in all tissues. RBC D6D EAE positively correlated with muscle and inversely correlated with adipose D6D EAE. Our findings suggest differential influence of dietary EPA + DHA upon tissue desaturase activities.

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

PubMed

Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

2010-07-01

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.

PubMed Central

Peelman, F.; Vinaimont, N.; Verhee, A.; Vanloo, B.; Verschelde, J. L.; Labeur, C.; Seguret-Mace, S.; Duverger, N.; Hutchinson, G.; Vandekerckhove, J.; Tavernier, J.; Rosseneu, M.

1998-01-01

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates. PMID:9541390

Factors affecting the palmitoyl-coenzyme A desaturase of Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Klein, H. P.; Volkmann, C. M.

1975-01-01

The activity and stability of the palmitoyl-coenzyme A (CoA) desaturase complex of Saccharomyces cerevisiae was influenced by several factors. Cells, grown nonaerobically and then incubated with glucose, either in air or under N2, showed a marked increase in desaturase activity. Cycloheximide, added during such incubations, prevented the increase in activity, suggesting de novo synthesis. The stability of the desaturase from cells grown nonaerobically was affected by subsequent treatment of the cells; enzyme from freshly harvested cells, or from cells that were then shaken under nitrogen, readily lost activity upon washing or during density gradient analysis, whereas aerated cells, in the presence or absence of glucose, yielded stable enzyme preparations. The loss of activity in nonaerobic preparations could be reversed by adding soluble supernatant from these homogenates and could be prevented by growing the cells in the presence of palmitoleic acid and ergosterol, but not with several other lipids tested.

Effect of varying dietary levels of LC-PUFA and vegetable oil sources on performance and fatty acids of Senegalese sole post larvae: puzzling results suggest complete biosynthesis pathway from C18 PUFA to DHA.

PubMed

Navarro-Guillén, Carmen; Engrola, Sofia; Castanheira, Filipa; Bandarra, Narcisa; Hachero-Cruzado, Ismael; Tocher, Douglas R; Conceição, Luís E C; Morais, Sofia

2014-01-01

Lipid nutrition of marine fish larvae has focused on supplying essential fatty acids (EFA) at high levels to meet requirements for survival, growth and development. However, some deleterious effects have been reported suggesting that excessive supply of EFA might result in insufficient supply of energy substrates, particularly in species with lower EFA requirements such as Senegalese sole (Solea senegalensis). This study addressed how the balance between EFA and non-EFA (better energy sources) affects larval performance, body composition and metabolism and retention of DHA, by formulating enrichment emulsions containing two different vegetable oil sources (olive oil or soybean oil) and three DHA levels. DHA positively affected growth and survival, independent of oil source, confirming that for sole post-larvae it is advantageous to base enrichments on vegetable oils supplying higher levels of energy, and supplement these with a DHA-rich oil. In addition, body DHA levels were generally comparable considering the large differences in their dietary supply, suggesting that the previously reported ∆4 fatty acyl desaturase (fad) operates in vivo and that DHA was synthesized at physiologically significant rates through a mechanism involving transcriptional up-regulation of ∆4fad, which was significantly up-regulated in the low DHA treatments. Furthermore, data suggested that DHA biosynthesis may be regulated by an interaction between dietary n-3 and n-6 PUFA, as well as by levels of LC-PUFA, and this may, under certain nutritional conditions, lead to DHA production from C18 precursors. The molecular basis of putative fatty acyl ∆5 and ∆6 desaturation activities remains to be fully determined as thorough searches have found only a single (∆4) Fads2-type transcript. Therefore, further studies are required but this might represent a unique activity described within vertebrate fads. © 2013.

How did nature engineer the highest surface lipid accumulation among plants? Exceptional expression of acyl-lipid-associated genes for the assembly of extracellular triacylglycerol by Bayberry (Myrica pensylvanica) fruits.

PubMed

Simpson, Jeffrey P; Thrower, Nicholas; Ohlrogge, John B

2016-09-01

Bayberry (Myrica pensylvanica) fruits are covered with a remarkably thick layer of crystalline wax consisting of triacylglycerol (TAG) and diacylglycerol (DAG) esterified exclusively with saturated fatty acids. As the only plant known to accumulate soluble glycerolipids as a major component of surface waxes, Bayberry represents a novel system to investigate neutral lipid biosynthesis and lipid secretion by vegetative plant cells. The assembly of Bayberry wax is distinct from conventional TAG and other surface waxes, and instead proceeds through a pathway related to cutin synthesis (Simpson and Ohlrogge, 2016). In this study, microscopic examination revealed that the fruit tissue that produces and secretes wax (Bayberry knobs) is fully developed before wax accumulates and that wax is secreted to the surface without cell disruption. Comparison of transcript expression to genetically related tissues (Bayberry leaves, M. rubra fruits), cutin-rich tomato and cherry fruit epidermis, and to oil-rich mesocarp and seeds, revealed exceptionally high expression of 13 transcripts for acyl-lipid metabolism together with down-regulation of fatty acid oxidases and desaturases. The predicted protein sequences of the most highly expressed lipid-related enzyme-encoding transcripts in Bayberry knobs are 100% identical to the sequences from Bayberry leaves, which do not produce surface DAG or TAG. Together, these results indicate that TAG biosynthesis and secretion in Bayberry is achieved by both up and down-regulation of a small subset of genes related to the biosynthesis of cutin and saturated fatty acids, and also implies that modifications in gene expression, rather than evolution of new gene functions, was the major mechanism by which Bayberry evolved its specialized lipid metabolism. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation1[C][W

PubMed Central

Orzaez, Diego; Medina, Aurora; Torre, Sara; Fernández-Moreno, Josefina Patricia; Rambla, José Luis; Fernández-del-Carmen, Asun; Butelli, Eugenio; Martin, Cathie; Granell, Antonio

2009-01-01

Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruit-specific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening. PMID:19429602

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed.

PubMed

Liu, Hua Liang; Yin, Zhi Jie; Xiao, Li; Xu, Yi Nong; Qu, Le Qing

2012-05-01

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g⁻¹ to 8.57 mg g⁻¹ and 10.06 mg g⁻¹, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.

Genome-Wide Survey and Characterization of Fatty Acid Desaturase Gene Family in Brassica napus and Its Parental Species.

PubMed

Xue, Yufei; Chen, Baojun; Wang, Rui; Win, Aung Naing; Li, Jiana; Chai, Yourong

2018-02-01

Rapeseed (Brassica napus) is an important oilseed crop worldwide, and fatty acid (FA) compositions determine the nutritional and economic value of its seed oil. Fatty acid desaturases (FADs) play a pivotal role in regulating FA compositions, but to date, no comprehensive genome-wide analysis of FAD gene family in rapeseed and its parent species has been reported. In this study, using homology searches, 84, 45, and 44 FAD genes were identified in rapeseed, Brassica rapa, and Brassica oleracea genomes, respectively. These FAD genes were unevenly located in 17 chromosomes and 2 scaffolds of rapeseed, 9 chromosomes and 1 scaffold of B. rapa, and all the chromosomes of B. oleracea. Phylogenetic analysis showed that the soluble and membrane-bound FADs in the three Brassica species were divided into four and six subfamilies, respectively. Generally, the soluble FADs contained two conserved histidine boxes, while three highly conserved histidine boxes were harbored in membrane-bound FADs. Exon-intron structure, intron phase, and motif composition and position were highly conserved in each FAD subfamily. Putative subcellular locations of FAD proteins in three Brassica species were consistent with those of corresponding known FADs. In total, 25 of simple sequence repeat (SSR) loci were found in FAD genes of the three Brassica species. Transcripts of selected FAD genes in the three species were examined in various organs/tissues or stress treatments from NCBI expressed sequence tag (EST) database. This study provides a critical molecular basis for quality improvement of rapeseed oil and facilitates our understanding of key roles of FAD genes in plant growth and development and stress response.

Heart rate is associated with markers of fatty acid desaturation: the GOCADAN study

PubMed Central

Ebbesson, Sven O.E.; Lopez-Alvarenga, Juan C.; Okin, Peter M.; Devereux, Richard B.; Tejero, Maria Elizabeth; Harris, William S.; Ebbesson, Lars O.E.; MacCluer, Jean W.; Wenger, Charlotte; Laston, Sandra; Fabsitz, Richard R.; Kennish, John; Howard, William J.; Howard, Barbara V.; Umans, Jason; Comuzzie, Anthony G.

2012-01-01

Objectives To determine if heart rate (HR) is associated with desaturation indexes as HR is associated with arrhythmia and sudden death. Study design A community based cross-sectional study of 1214 Alaskan Inuit. Methods Data of FA concentrations from plasma and red blood cell membranes from those ≥35 years of age (n =819) were compared to basal HR at the time of examination. Multiple linear regression with backward stepwise selection was employed to analyze the effect of the desaturase indexes on HR, after adjustment for relevant covariates. Results The Δ5 desaturase index (Δ5-DI) measured in serum has recently been associated with a protective role for cardiovascular disease. This index measured here in plasma and red blood cells showed a negative correlation with HR. The plasma stearoyl-CoA-desaturase (SCD) index, previously determined to be related to cardiovascular disease (CVD) mortality, on the other hand, was positively associated with HR, while the Δ6 desaturase index (Δ6-DI) had no significant effect on HR. Conclusion Endogenous FA desaturation is associated with HR and thereby, in the case of SCD, possibly with arrhythmia and sudden death, which would at least partially explain the previously observed association between cardiovascular mortality and desaturase activity. PMID:22456045

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

PubMed Central

Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

1999-01-01

Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

INHIBITION OF FATTY ACID DESATURASES IN Drosophila melanogaster LARVAE BLOCKS FEEDING AND DEVELOPMENTAL PROGRESSION.

PubMed

Wang, Yiwen; da Cruz, Tina Correia; Pulfemuller, Alicia; Grégoire, Stéphane; Ferveur, Jean-François; Moussian, Bernard

2016-05-01

Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, we inhibited fatty acid desaturases using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid desaturase1 gene. Based on these data, we propose that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. We also hypothesize that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program. © 2016 Wiley Periodicals, Inc.

Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis*

PubMed Central

Phetsuksiri, Benjawan; Jackson, Mary; Scherman, Hataichanok; McNeil, Michael; Besra, Gurdyal S.; Baulard, Alain R.; Slayden, Richard A.; DeBarber, Andrea E.; Barry, Clifton E.; Baird, Mark S.; Crick, Dean C.; Brennan, Patrick J.

2016-01-01

The thiourea isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 μg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Δ9-stearoyl desaturase as the drug target. Development of a cell-free assay for Δ9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Δ9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Δ9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Δ9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development. PMID:14559907

Correlation of polyunsaturated fatty acids with the cold adaptation of Rhodotorula glutinis.

PubMed

He, Jing; Yang, Zhaojie; Hu, Binbin; Ji, Xiuling; Wei, Yunlin; Lin, Lianbing; Zhang, Qi

2015-11-01

This study aimed to investigate the correlation between the cold adaptation of Rhodotorula glutinis YM25079 and the membrane fluidity, content of polyunsaturated fatty acids and mRNA expression level of the Δ(12)-desaturase gene. The optimum temperature for YM25079 growth was analysed first, then the composition changes of membrane lipid in YM25079 were detected by GC-MS and membrane fluidity was evaluated by 1-anilinonaphthalene-8-sulphonate (ANS) fluorescence. Meanwhile, the encoding sequence of Δ(12)-fatty acid desaturase in YM25079 was cloned and further transformed into Saccharomyces cerevisiae INVScl for functional analysis. The mRNA expression levels of Δ(12)-fatty acid desaturase at 15°C and 25°C were analysed by real-time PCR. YM25079 could grow at 5-30°C, with the optimum temperature of 15°C. The membrane fluidity of YM25079 was not significantly reduced when the culture temperature decreased from 25°C to 15°C, but the content of polyunsaturated fatty acids (PUFAs), including linoleic acid and α-Linolenic acid increased significantly from 29.4% to 55.39%. Furthermore, a novel Δ(12)-fatty acid desaturase gene YM25079RGD12 from YM25079 was successfully identified and characterized, and the mRNA transcription level of the Δ(12)-desaturase gene was about five-fold higher in YM25079 cells grown at 15°C than that at 25°C. These results suggests that the cold adaptation of Rhodotorula glutinis YM25079 might result from higher expression of genes, especially the Δ(12)-fatty acid desaturase gene, during polyunsaturated fatty acids biosynthesis, which increased the content of PUFAs in the cell membrane and maintained the membrane fluidity at low temperature. Copyright © 2015 John Wiley & Sons, Ltd.

WHEAT GERM CELL-FREE TRANSLATION, PURIFICATION, AND ASSEMBLY OF A FUNCTIONAL HUMAN STEAROYL-COA DESATURASE COMPLEX

PubMed Central

Goren, Michael A.; Fox, Brian G.

2008-01-01

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b5 led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed. PMID:18765284

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.

PubMed

Goren, Michael A; Fox, Brian G

2008-12-01

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.

Pomegranate seed oil influences the fatty acids profile and reduces the activity of desaturases in livers of Sprague-Dawley rats.

PubMed

Białek, Agnieszka; Stawarska, Agnieszka; Bodecka, Joanna; Białek, Małgorzata; Tokarz, Andrzej

2017-07-01

The aim of our study was to compare the influence of diet supplementation with pomegranate seed oil - as conjugated linolenic acids (CLnA) source, or conjugated linoleic acids (CLA) and to examine the mechanism of their activity. The content of fatty acids, levels of biomarkers of lipids' oxidation and the activity of key enzymes catalyzing lipids metabolism were measured. Obtained results revealed that conjugated fatty acids significantly decrease the activity of Δ5-desaturase (p=0.0001) and Δ6-desaturase (p=0.0008) and pomegranate seed oil reduces their activity in the most potent way. We confirmed that diet supplementation with pomegranate seed oil - a rich source of punicic acid leads to the increase of cis-9, trans-11 CLA content in livers (p=0.0003). Lack of side effects and beneficial influence on desaturases activity and fatty acids profile claim pomegranate seed oil to become interesting alternative for CLA as functional food. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular structure, supramolecular organization and thermotropic phase behavior of N-acylglycine alkyl esters with matched acyl and alkyl chains.

PubMed

Reddy, S Thirupathi; Swamy, Musti J

2017-11-01

N-Acylglycines (NAGs), the endogenous single-tailed lipids present in rat brain and other mammalian tissues, play significant roles in cell physiology and exhibit interesting pharmacological properties. In the present study, a homologous series of N-acylglycine alkyl esters (NAGEs) with matched chains were synthesized and characterized. Results of differential scanning calorimetric studies revealed that all NAGEs exhibit a single sharp phase transition and that the transition enthalpy and entropy show a linear dependence on the N-acyl and ester alkyl chain length. The structure of N-myristoylglycine myristyl ester (NMGME), solved by single-crystal X-ray diffraction, showed that the molecule adopts a linear geometry and revealed that the structure of N-myristoyl glycyl moiety in NMGME is identical to that in N-myristoylglycine. The molecules are packed in layers with the polar functional groups of the ester and amide functionalities located at the center of the layer. The crystal packing is stabilized by NH⋯O hydrogen bonds between the amide CO and NH groups of adjacent molecules as well as by CH⋯O hydrogen bonds between the amide carbonyl and the methylene CH adjacent to the ester carbonyl of neighboring molecules as well as between ester carbonyl and methylene group of the glycine moiety of adjacent molecules. Powder X-ray diffraction studies showed a linear dependence of the d-spacings on the acyl chain length, suggesting that all NAGEs adopt a structure similar to the packing exhibited in the crystal lattice of NMGME. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance.

PubMed

Meesapyodsuk, Dauenpen; Chen, Yan; Ng, Siew Hon; Chen, Jianan; Qiu, Xiao

2015-11-01

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance[S

PubMed Central

Meesapyodsuk, Dauenpen; Chen, Yan; Ng, Siew Hon; Chen, Jianan; Qiu, Xiao

2015-01-01

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a “push” (synthesis) and “pull” (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses. PMID:26323290

Production of acyl-homoserine lactone quorum-sensing signals is widespread in gram-negative Methylobacterium.

PubMed

Poonguzhali, Selvaraj; Madhaiyan, Munusamy; Sa, Tongmin

2007-02-01

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NT1 (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-DL-homoserine lactone (C8-HSL) and N-decanoyl-DL-homoserine lactone (C10-HSL).

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

Fatty acid desaturase gene variants, cardiovascular risk factors, and myocardial infarction in the costa rica study

USDA-ARS?s Scientific Manuscript database

Genetic variation in fatty acid desaturases (FADS) has previously been linked to long-chain polyunsaturated fatty acids (PUFAs) in adipose tissue and cardiovascular risk. The goal of our study was to test associations between six common FADS polymorphisms (rs174556, rs3834458, rs174570, rs2524299, r...

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

PubMed

Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

2013-10-15

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

PubMed

Zhang, Huidan; Lu, Dong; Li, Xin; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2018-05-02

Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L - 1 , which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Identification and characterization of a plastidial ω-3 fatty acid desaturase EgFAD8 from oil palm (Elaeis guineensis Jacq.) and its promoter response to light and low temperature

PubMed Central

Chen, Lizhi; Wang, Lei; Wang, Herong; Sun, Ruhao; You, Lili; Zheng, Yusheng; Yuan, Yijun

2018-01-01

In higher plants, ω-3 fatty acid desaturases are the key enzymes in the biosynthesis of alpha-linolenic acid (18:3), which plays key roles in plant metabolism as a structural component of both storage and membrane lipids. Here, the first ω-3 fatty acid desaturase gene was identified and characterized from oil palm. The bioinformatic analysis indicated it encodes a temperature-sensitive chloroplast ω-3 fatty acid desaturase, designated as EgFAD8. The expression analysis revealed that EgFAD8 is highly expressed in the oil palm leaves, when compared with the expression in the mesocarp. The heterologous expression of EgFAD8 in yeast resulted in the production of a novel fatty acid 18:3 (about 0.27%), when fed with 18:2 in the induction culture. Furthermore, to detect whether EgFAD8 could be induced by the environment stress, we detected the expression efficiency of the EgFAD8 promoter in transgenic Arabidopsis treated with low temperature and darkness, respectively. The results indicated that the promoter of EgFAD8 gene could be significantly induced by low temperature and slightly induced by darkness. These results reveal the function of EgFAD8 and the feature of its promoter from oil palm fruits, which will be useful for understanding the fuction and regulation of plastidial ω-3 fatty acid desaturases in higher plants. PMID:29698515

Changes in fatty acid composition in plant tissues expressing a mammalian delta9 desaturase.

PubMed

Moon, H; Hazebroek, J; Hildebrand, D F

2000-05-01

Plant tissues expressing a mammalian stearoyl-CoA delta9 desaturase were reported to accumulate delta9 hexadecenoic acid (16:1), normally very minor in most plant tissues. The transgenic plants were thoroughly analyzed for alterations of individual lipids in different subcellular sites. Western blot analysis indicated that the animal desaturase was targeted to the microsomes. The delta9 16:1 was incorporated into both the sn-1 and sn-2 positions of all the major membrane lipids tested, indicating that the endoplasmic reticulum acyltransferases do not exclude unsaturated C16 fatty acids from the sn-2 position. In addition to increases in monounsaturated and decreases in saturated fatty acids, accumulation of 16:1 was accompanied by a reduction in 18:3 in all the lipids tested except phosphatidylglycerol, and increases in 18:2 in phospholipids. Total C16 fatty acid content in the galactolipids of the transgenics was significantly higher than that in the control, but those in the phospholipids were unchanged. In transgenics, delta11 18:1 was detected in the sn-1 position of the lipids tested except phosphatidylinositol and phosphatidylserine. Introduction of the animal desaturase, controlled by a seed-specific phaseolin promoter, into soybean somatic embryo resulted in a significant reduction in saturated fatty acids. Such effects were greater in cotyledons than hypocotyl-radicles. This study demonstrated that the animal desaturase can be used to decrease the levels of saturated fatty acids in a crop plant.

Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation.

PubMed

Hsieh, S L; Kuo, C-M

2005-05-01

Desaturation of fatty acids is an important adaptation mechanism for fish to maintain membrane fluidity under thermal stress. To comprehend the temperature adaptation mechanism in fish, we investigated the difference in the changes of stearoyl-CoA desaturase expression and fatty acid composition between milkfish and grass carp under cold acclimation. We find that in both fish the proportions of unsaturated fatty acids at 15 degrees C are all higher than those at 25 degrees C. In milkfish Delta(9)-desaturation index (ratios of 16:1/16:0 and 18:1/18:0) increases significantly in the beginning of cold acclimation at 15 degrees C and decreases afterward, but in grass carp it increases slightly in the beginning of cold acclimation followed by a sustained dramatic increase. Similarly, activity of stearoyl-CoA desaturase in milkfish increases significantly in the beginning, peaks at day 4, and then decreases constantly, but in grass carp it increases gradually in the first week, rises dramatically afterward, and then maintains a very high level. The change of stearoyl-CoA desaturase activity is parallel to the change of Delta(9)-desaturation index in both milkfish and grass carp, but it is one day earlier than Delta(9)-desaturation index in milkfish. The difference of adaptation capability between milkfish and grass carp under cold stress is further evidenced by RT-PCR and Northern blot analysis of stearoyl-CoA desaturase gene expression.

Association of desaturase activity and C-reactive protein in European children.

PubMed

Wolters, Maike; Börnhorst, Claudia; Schwarz, Heike; Risé, Patrizia; Galli, Claudio; Moreno, Luis A; Pala, Valeria; Russo, Paola; Veidebaum, Toomas; Tornaritis, Michael; Fraterman, Arno; De Henauw, Stefaan; Eiben, Gabriele; Lissner, Lauren; Molnár, Dénes; Ahrens, Wolfgang

2017-01-01

Desaturase enzymes influence the fatty acid (FA) composition of body tissues and their activity affects the conversion rate of saturated to monounsaturated FA and of polyunsaturated FA (PUFA) to long-chain PUFA. Desaturase activity has further been shown to be associated with inflammation. We investigate the association between delta-9 (D9D), delta-6 (D6D) and delta-5 desaturase (D5D) activity and high-sensitive C-reactive protein (CRP) in young children. In the IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) cohort study children were examined at baseline (T0) and after 2 y (T1). D9D, D6D, and D5D activities were estimated from T0 product-precursor FA ratios. CRP was measured at T0 and T1. In a subsample of 1,943 children with available information on FA, CRP, and covariates, the cross-sectional and longitudinal associations of desaturase activity and CRP were analyzed. Cross-sectionally, a D9D increase of 0.01 units was associated with a 11% higher risk of having a serum CRP ≥ Percentile 75 (P75) (OR, 99% CI: 1.11 (1.01; 1.22)) whereas D6D and D5D were not associated with CRP. No significant associations were observed between baseline desaturase activity and CRP 2 y later. Cross-sectionally, our results indicate a positive association of D9D and CRP independent of weight status. High D9D activity may increase the risk of subclinical inflammation which is associated with metabolic disorders. As D9D expression increases with higher intake of saturated FA and carbohydrates, dietary changes may influence D9D activity and thus CRP. However, it remains to be investigated whether there is a causal relationship between D9D activity and CRP.

Alternation of plasma fatty acids composition and desaturase activities in children with liver steatosis

PubMed Central

Su, Hui-Min; Yao, Tsung-Chieh; Kuo, Ming-Ling; Lai, Ming-Wei; Tsai, Ming-Han; Huang, Jing-Long

2017-01-01

Objective The aim of this study was to investigate changes in plasma fatty acids proportions and estimated desaturase activities for variable grading of liver steatosis in children. Methods In total, 111 schoolchildren (aged 8–18 years) were included in the analysis from March 2015 to August 2016. Anthropometric evaluation, liver ultrasound examination and scoring for nonalcoholic fatty liver disease (NAFLD score = 0–6), and biochemical and plasma fatty acids analysis were performed. We compared the composition ratio of fatty acids between children with high-grade liver steatosis (NAFLD score = 4–6), low-grade liver steatosis (NAFLD score = 1–3), and healthy controls (NAFLD score = 0). In addition, correlation coefficients (r) between NAFLD score, metabolic variables, and estimated activity of desaturase indices (stearoyl-coenzyme A desaturase-1 (SCD1), delta-5 and delta-6 desaturase) were calculated. Results Compared with healthy controls, children with liver steatosis showed a higher proportion of monounsaturated fatty acids (21.16 ± 2.81% vs. 19.68 ± 2.71%, p = 0.024). In addition, children with high- grade liver steatosis exhibited higher proportions of palmitic acid (C16:0), palmitoleic acid (C16:1n-7), dihomo-γ-linolenic acid (C20:3n-6), adrenic acid (C22:4n-6), and docosapentaenoic acid (C22:5n-6); and lower proportions of eicosapentaenoic acid (C20:5n-3) (P< 0.05). In all subjects, the NAFLD score was positively correlated with body mass index (BMI) (kg/m2) (r = 0.696), homeostasis model of assessment ratio–index (HOMA-IR) (r = 0.510), SCD1(16) (r = 0.273), and the delta-6 index (r = 0.494); and inversely associated with the delta-5 index (r = -0.443). Conclusion Our current data suggested that children with liver steatosis was highly associated with obesity, and insulin resistance. In addition, increased endogenous lipogenesis through altered desaturase activity may contribute to the progression of liver steatosis in children. PMID:28759573

Analyses of barley spike mutant waxes identify alkenes, cyclopropanes and internally branched alkanes with dominating isomers at carbon 9.

PubMed

von Wettstein-Knowles, Penny

2007-01-01

About 15% of the epidermal wax on Hordeum vulgare cv. Bonus barley spikes is n-alkanes. Longer homologues are greatly reduced in the eceriferum mutants, cer-a(6), cer-e(8), cer-n(26), cer-n(53), cer-n(985), cer-x(60), cer-yc(135) and cer-yl(187). Simultaneously hydrocarbons accounting for only traces in the wild-type become prominent in the mutants, although their chain-length distributions remain unchanged. Accordingly several new hydrocarbon series were identified. The two major ones were C(23)-C(35)cis monoenoic alkenes (the major 9-ene isomer was part of a homologous series including 11, 13 and 15-enes), and the novel C(27)-C(31) cyclopropanes (the ring carbons of major isomers were 9,10 and 11,12 with lesser amounts of 13,14). Three minor series included 2- and 3-methylalkanes plus C(25)-C(33) internally branched alkanes (methyls on carbons 9, 11, 13, 15 or 17; shorter homologues dominated by the 9 isomer, longer homologues by 11, 13 or 15 isomers). Acyl chains destined for spike waxes are synthesized via acyl and polyketide elongase systems plus associated reductive and decarbonylative/decarboxylative enzyme systems. Both elongation systems are defective in synthesizing C(32) acyl chains in all nine mutants. The similarities in the position of the chemical groups (primarily on carbon 9, secondarily on carbon 11) of the alkenes, cyclopropanes and internally branched methyl alkanes imply an origin from a common, hitherto unrecognized associated pathway in barley, designated the enoic pathway. The elongation system leading to the enoic derived hydrocarbons differs from the known elongation systems by inclusion of a mechanism for introducing a double bond.

TEMPERATURE-SENSITIVE, POST-TRANSLATIONAL REGULATION OF PLANT OMEGA-3 FATTY ACID DESATURASES IS MEDIATED BY THE ER-ASSOCIATED DEGRADATION PATHWAY

USDA-ARS?s Scientific Manuscript database

In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty acid desaturases (Fad3s) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not upregulated during this adaptive response. Here, we expressed two closely related ...

Associations between plasma fatty acids, desaturase and elongase, and insulin resistance in children

USDA-ARS?s Scientific Manuscript database

Background and Objectives - Fatty acid profiles, desaturase (SCD-16, SCD018, D5D, D6D) and elongase (ELOVL6) enzyme activity have been associated with adiposity and metabolic disease. While this has been studied in adults, few studies have evaluated children. The objective of this study was to evalu...

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

PubMed Central

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

2001-01-01

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

Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

PubMed Central

Hao, Guixia; Burr, Thomas J.

2006-01-01

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N-acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI, ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI. The addition of synthetic AHLs (C16:1 and 3-O-C16:1) complemented grape necrosis in the avsR, avsI, ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR, two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI::lacZ fusion construct. PMID:16513747

Discovery of Potent Benzocycloalkane Derived Diapophytoene Desaturase Inhibitors with an Enhanced Safety Profile for the Treatment of MRSA, VISA, and LRSA Infections.

PubMed

Li, Baoli; Ni, Shuaishuai; Chen, Feifei; Mao, Fei; Wei, Hanwen; Liu, Yifu; Zhu, Jin; Lan, Lefu; Li, Jian

2018-03-09

Blocking the biosynthesis process of staphyloxanthin has emerged as a promising antivirulence strategy. Our previous research revealed that diapophytoene desaturase was an attractive and druggable target against infections caused by pigmented Staphylococcus aureus. Benzocycloalkane-derived compounds were effective inhibitors of diapophytoene desaturase but limited by high hERG (human Ether-a-go-go Related Gene) inhibition activity. Here, we identified a new type of benzo-hepta-containing cycloalkane derivative as diapophytoene desaturase inhibitors. Among the fifty-eight analogues, 48 (hERG inhibition activity, half maximal inhibitory concentration, IC 50 , of 16.1 μM) and 51 (hERG inhibition activity, IC 50 > 40 μM) were distinguished for effectively inhibiting the pigment production of Staphylococcus aureus Newman and three methicillin-resistant Staphylococcus aureus strains, and the four strains were highly sensitize to hydrogen peroxide killing without a bactericidal growth effect. In an in vivo assay, 48 and 51 displayed a comparable effect with linezolid and vancomycin in livers and hearts in mice against Staphylococcus aureus Newman and a more considerable effect against Mu50 and NRS271 with normal administration.

Evidence that norflurazon affects chloroplast lipid unsaturation in soybean leaves (Glycine max L.).

PubMed

Abrous-Belbachir, Ouzna; De Paepe, Rosine; Trémolières, Antoine; Mathieu, Chantal; Ad, Fatiha; Benhassaine-Kesri, Ghouziel

2009-12-09

Norflurazon is a bleaching herbicide known to block carotenoid biosynthesis by inhibiting phytoene desaturase activity. Soybean plants were treated with norflurazon, and we examined the effects on the desaturation of lipid molecular species in leaves using ammonium [1-(14)C] oleate labeling. In monogalactosyldiacylglycerol (MGDG), the main chloroplast lipid, a decrease in 18:3/18:3 molecular species and an increase in its precursors 18:2/18:3 and 18:2/18:2 were observed suggesting that the omega(3) FAD7 desaturase activity in planta was inhibited by norflurazon. The in vitro activity of MGDG synthase was also inhibited by 69%. In contrast, the amount of 18:3/18:3 molecular species of phosphatidylcholine (PC) in the extraplastid compartment increased. The observed increase in in vitro lysoPC-acyltransferase activity and activation of desaturation of [1-(14)C] oleate suggest that extraplastid omega(3)FAD3 desaturase was activated. Analysis of the expression of omega(3) FAD3 and omega(3) FAD7 genes in norflurazon treated plants indicate that omega(3) FAD7 and omega(3) FAD3 desaturases are controlled at the post-transcriptional level.

β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

DOE PAGES

McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; ...

2016-01-28

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

β-Ketoacyl-acyl Carrier Protein Synthase I (KASI) Plays Crucial Roles in the Plant Growth and Fatty Acids Synthesis in Tobacco

PubMed Central

Yang, Tianquan; Xu, Ronghua; Chen, Jianghua; Liu, Aizhong

2016-01-01

Fatty acids serve many functions in plants, but the effects of some key genes involved in fatty acids biosynthesis on plants growth and development are not well understood yet. To understand the functions of 3-ketoacyl-acyl-carrier protein synthase I (KASI) in tobacco, we isolated two KASI homologs, which we have designated NtKASI-1 and NtKASI-2. Expression analysis showed that these two KASI genes were transcribed constitutively in all tissues examined. Over-expression of NtKASI-1 in tobacco changed the fatty acid content in leaves, whereas over-expressed lines of NtKASI-2 exhibited distinct phenotypic features such as slightly variegated leaves and reduction of the fatty acid content in leaves, similar to the silencing plants of NtKASI-1 gene. Interestingly, the silencing of NtKASI-2 gene had no discernibly altered phenotypes compared to wild type. The double silencing plants of these two genes enhanced the phenotypic changes during vegetative and reproductive growth compared to wild type. These results uncovered that these two KASI genes had the partially functional redundancy, and that the KASI genes played a key role in regulating fatty acids synthesis and in mediating plant growth and development in tobacco. PMID:27509494

Catalytic properties of an expressed and purified higher plant type zeta-carotene desaturase from Capsicum annuum.

PubMed

Breitenbach, J; Kuntz, M; Takaichi, S; Sandmann, G

1999-10-01

The zeta-carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate zeta-carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7',9,9'-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the zeta-carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.

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

PubMed

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

2009-11-01

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

[Screening cold-acclimation differential expression candidate genes in the brain of common carp (Cyprinus carpio)].

PubMed

Xu, Li-Hua; Chang, Yu-Mei; Liu, Chun-Lei; Liang, Li-Qun; Liu, Jin-Liang; Chi, Bing-Jie

2011-03-01

In this study, 26 candidate genes were quantified and normalized in the brain cDNA of common carp (Cyprinus carpio) at 23°C and 6°C using double-standard curve method of real-time quantitative PCR. The results showed that five candidates up-regulated in the samples at 6°C (P<0.01) and quantified 2.11, 13.9, 2.52, 7.38, and 1.83 times more than in the samples at 23°C, respectively. Gene function searching indicated that the protein products of these five candidates were elongation of very long chain fatty acids protein, Acyl-CoA desaturase, Transcription initiation factor IIB, Myo-inositol- 1-phosphate synthase, and Blood-brain barrier HT7 antigen individually. Moreover, seven down-regulated candidates were also identified in the same samples at 6°C (P>0.05), and their expression levels were decreased by 21.8%, 25.9%, 16.6%, 23.7%, 15.8%, 16.3%, and 42.5%, respectively, in comparison with the samples at 23°C. These seven down-regulated candidates mainly participated in the inhibition of glycolysis, improvement of cell apoptosis, and intervention of synapse remodeling based on the results of function searching. The five cold-induced genes identified in this study will be used as important elements for fish with cold sensitive through transgenic technology in future.

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

PubMed

Wan, Huafang; Cui, Yixin; Ding, Yijuan; Mei, Jiaqin; Dong, Hongli; Zhang, Wenxin; Wu, Shiqi; Liang, Ying; Zhang, Chunyu; Li, Jiana; Xiong, Qing; Qian, Wei

2016-01-01

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola ( Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

Engineering Ashbya gossypii strains for de novo lipid production using industrial by-products.

PubMed

Lozano-Martínez, Patricia; Buey, Rubén M; Ledesma-Amaro, Rodrigo; Jiménez, Alberto; Revuelta, José Luis

2017-03-01

Ashbya gossypii is a filamentous fungus that naturally overproduces riboflavin, and it is currently exploited for the industrial production of this vitamin. The utilization of A. gossypii for biotechnological applications presents important advantages such as the utilization of low-cost culture media, inexpensive downstream processing and a wide range of molecular tools for genetic manipulation, thus making A. gossypii a valuable biotechnological chassis for metabolic engineering. A. gossypii has been shown to accumulate high levels of lipids in oil-based culture media; however, the lipid biosynthesis capacity is rather limited when grown in sugar-based culture media. In this study, by altering the fatty acyl-CoA pool and manipulating the regulation of the main ∆9 desaturase gene, we have obtained A. gossypii strains with significantly increased (up to fourfold) de novo lipid biosynthesis using glucose as the only carbon source in the fermentation broth. Moreover, these strains were efficient biocatalysts for the conversion of carbohydrates from sugarcane molasses to biolipids, able to accumulate lipids up to 25% of its cell dry weight. Our results represent a proof of principle showing the promising potential of A. gossypii as a competitive microorganism for industrial biolipid production using cost-effective feed stocks. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Identification and Expression Profiles of Sex Pheromone Biosynthesis and Transport Related Genes in Spodoptera litura

PubMed Central

Zhang, Ya-Nan; Zhu, Xiu-Yun; Fang, Li-Ping; He, Peng; Wang, Zhi-Qiang; Chen, Geng; Sun, Liang; Ye, Zhan-Feng; Deng, Dao-Gui; Li, Jin-Bu

2015-01-01

Although the general pathway of sex pheromone synthesis in moth species has been established, the molecular mechanisms remain poorly understood. The common cutworm Spodoptera litura is an important agricultural pest worldwide and causes huge economic losses annually. The female sex pheromone of S. litura comprises Z9,E11-14:OAc, Z9,E12-14:OAc, Z9-14:OAc, and E11-14:OAc. By sequencing and analyzing the transcriptomic data of the sex pheromone glands, we identified 94 candidate genes related to pheromone biosynthesis (55 genes) or chemoreception (39 genes). Gene expression patterns and phylogenetic analysis revealed that two desaturase genes (SlitDes5 and SlitDes11) and one fatty acyl reductase gene (SlitFAR3) showed pheromone gland (PG) biased or specific expression, and clustered with genes known to be involved in pheromone synthesis in other moth species. Furthermore, 4 chemoreception related genes (SlitOBP6, SlitOBP11, SlitCSP3, and SlitCSP14) also showed higher expression in the PG, and could be additional candidate genes involved in sex pheromone transport. This study provides the first solid background information that should facilitate further elucidation of sex pheromone biosynthesis and transport, and indicates potential targets to disrupt sexual communication in S. litura for a novel pest management strategy. PMID:26445454

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

PubMed

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

2016-08-01

Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n-3/18:2n-6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish. Copyright © 2016 Elsevier Inc. All rights reserved.

A LuxR homolog in a cottonwood tree endophyte that activates gene expression in response to a plant signal or specific peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schaefer, Amy L.; Oda, Yasuhiro; Coutinho, Bruna Goncalves

Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities.more » A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. As a result, the identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins.« less

A LuxR homolog in a cottonwood tree endophyte that activates gene expression in response to a plant signal or specific peptides

DOE PAGES

Schaefer, Amy L.; Oda, Yasuhiro; Coutinho, Bruna Goncalves; ...

2016-08-02

Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities.more » A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. As a result, the identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins.« less

Prevalence of LuxR- and LuxI-type quorum sensing circuits in members of the Populus deltoides microbiome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schaefer, Amy L; Lappala, Colin; Morlen, Ryan

2013-01-01

We are interested in the root microbiome of the fast-growing Eastern cottonwood tree, Populus 25 deltoides. There is a large bank of bacterial isolates from P. deltoides and there are 44 draft 26 genomes of bacterial endophyte and rhizosphere isolates. As a first step in efforts to understand 27 the roles of bacterial communication and plant-bacterial signaling in P. deltoides we focused on 28 the prevalence of acyl-homoserine lactone (AHL) quorum sensing signal production and 29 reception in members of the P. deltoides microbiome. We screened 129 bacterial isolates for 30 AHL production using a broad-spectrum bioassay that responds tomore » many but not all AHLs, and 31 we queried the available genome sequences of microbiome isolates for homologs of AHL 32 synthase and receptor genes. AHL signal production was detected in 40% of 129 strains tested. 33 Positive isolates included -, - and -Proteobacteria. Members of the luxI family of AHL 34 synthases were identified in 18 of 39 Proteobacteria genomes including genomes of some 35 isolates that tested negative in the bioassay. Members of the luxR family of transcription factors, 36 that include AHL-responsive factors, were more abundant than luxI homologs. There were 72 in 37 the 39 Proteobacteria genomes. Some of the luxR homologs appear to be members of a 38 subfamily of LuxRs that respond to as yet unknown plant signals rather than bacterial AHLs. 39 Apparently, there is a substantial capacity for AHL cell-to-cell communication in Proteobacteria 40 of the P. deltoides microbiota and there are also Proteobacteria with LuxR homologs of the type 41 hypothesized to respond to plant signals or cues.« less

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid

PubMed Central

2012-01-01

Background Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission. Results The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants. Conclusion In this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil. PMID:22377043

Temperature and metal exposure affect membrane fatty acid composition and transcription of desaturases and elongases in fathead minnow muscle and brain.

PubMed

Fadhlaoui, Mariem; Pierron, Fabien; Couture, Patrice

2018-02-01

In this study, we tested the hypothesis that metal exposure affected the normal thermal response of cell membrane FA composition and of elongase and desaturase gene transcription levels. To this end, muscle and brain membrane FA composition and FA desaturase (fads2, degs2 and scd2) and elongase (elovl2, elovl5 and elovl6) gene transcription levels were analyzed in fathead minnows (Pimephales promelas) acclimated for eight weeks to 15, 25 or 30°C exposed or not to cadmium (Cd, 6μg/l) or nickel (Ni, 450 6μg/l). The response of membrane FA composition to temperature variations or metal exposure differed between muscle and brain. In muscle, an increase of temperature induced a decrease of polyunsaturated FA (PUFA) and an increase of saturated FA (SFA) in agreement with the current paradigm. Although a similar response was observed in brain between 15 and 25°C, at 30°C, brain membrane unsaturation was higher than predicted. In both tissues, metal exposure affected the normal thermal response of membrane FA composition. The transcription of desaturases and elongases was higher in the brain and varied with acclimation temperature and metal exposure but these variations did not generally reflect changes in membrane FA composition. The mismatch between gene transcription and membrane composition highlights that several levels of control other than gene transcription are involved in adjusting membrane FA composition, including post-transcriptional regulation of elongases and desaturases and de novo phospholipid biosynthesis. Our study also reveals that metal exposure affects the mechanisms involved in adjusting cell membrane FA composition in ectotherms. Copyright © 2017 Elsevier Inc. All rights reserved.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

PubMed Central

Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

2012-01-01

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

PubMed Central

Hackett, Jeremiah D.; Wisecaver, Jennifer H.; Brosnahan, Michael L.; Kulis, David M.; Anderson, Donald M.; Bhattacharya, Debashish; Plumley, F. Gerald; Erdner, Deana L.

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand. PMID:22628533

Evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates.

PubMed

Hackett, Jeremiah D; Wisecaver, Jennifer H; Brosnahan, Michael L; Kulis, David M; Anderson, Donald M; Bhattacharya, Debashish; Plumley, F Gerald; Erdner, Deana L

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

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

PubMed

Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

2011-08-10

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. 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. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

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

Stabilization of the yeast desaturase system by low levels of oxygen

NASA Technical Reports Server (NTRS)

Volkmann, C. M.; Klein, H. P.

1983-01-01

The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen. The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells. These results suggest the evolutinary appearance of this system at a time when atmospheric oxygen was at a low level.

Oil biosynthesis in a basal angiosperm: transcriptome analysis of Persea Americana mesocarp.

PubMed

Kilaru, Aruna; Cao, Xia; Dabbs, Parker B; Sung, Ha-Jung; Rahman, Md Mahbubur; Thrower, Nicholas; Zynda, Greg; Podicheti, Ram; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Mockaitis, Keithanne; Ohlrogge, John B

2015-08-16

The mechanism by which plants synthesize and store high amounts of triacylglycerols (TAG) in tissues other than seeds is not well understood. The comprehension of controls for carbon partitioning and oil accumulation in nonseed tissues is essential to generate oil-rich biomass in perennial bioenergy crops. Persea americana (avocado), a basal angiosperm with unique features that are ancestral to most flowering plants, stores ~ 70 % TAG per dry weight in its mesocarp, a nonseed tissue. Transcriptome analyses of select pathways, from generation of pyruvate and leading up to TAG accumulation, in mesocarp tissues of avocado was conducted and compared with that of oil-rich monocot (oil palm) and dicot (rapeseed and castor) tissues to identify tissue- and species-specific regulation and biosynthesis of TAG in plants. RNA-Seq analyses of select lipid metabolic pathways of avocado mesocarp revealed patterns similar to that of other oil-rich species. However, only some predominant orthologs of the fatty acid biosynthetic pathway genes in this basal angiosperm were similar to those of monocots and dicots. The accumulation of TAG, rich in oleic acid, was associated with higher transcript levels for a putative stearoyl-ACP desaturase and endoplasmic reticulum (ER)-associated acyl-CoA synthetases, during fruit development. Gene expression levels for enzymes involved in terminal steps to TAG biosynthesis in the ER further indicated that both acyl-CoA-dependent and -independent mechanisms might play a role in TAG assembly, depending on the developmental stage of the fruit. Furthermore, in addition to the expression of an ortholog of WRINKLED1 (WRI1), a regulator of fatty acid biosynthesis, high transcript levels for WRI2-like and WRI3-like suggest a role for additional transcription factors in nonseed oil accumulation. Plastid pyruvate necessary for fatty acid synthesis is likely driven by the upregulation of genes involved in glycolysis and transport of its intermediates. Together, a comparative transcriptome analyses for storage oil biosynthesis in diverse plants and tissues suggested that several distinct and conserved features in this basal angiosperm species might contribute towards its rich TAG content. Our work represents a comprehensive transcriptome resource for a basal angiosperm species and provides insight into their lipid metabolism in mesocarp tissues. Furthermore, comparison of the transcriptome of oil-rich mesocarp of avocado, with oil-rich seed and nonseed tissues of monocot and dicot species, revealed lipid gene orthologs that are highly conserved during evolution. The orthologs that are distinctively expressed in oil-rich mesocarp tissues of this basal angiosperm, such as WRI2, ER-associated acyl-CoA synthetases, and lipid-droplet associated proteins were also identified. This study provides a foundation for future investigations to increase oil-content and has implications for metabolic engineering to enhance storage oil content in nonseed tissues of diverse species.

Rapid, transient, and highly localized induction of plastidial ω-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum

PubMed Central

Kirsch, Christoph; Takamiya-Wik, Monica; Reinold, Susanne; Hahlbrock, Klaus; Somssich, Imre E.

1997-01-01

Parsley (Petroselinum crispum) plants and suspension-cultured cells have been used extensively for studies of non-host-resistance mechanisms in plant/pathogen interactions. We now show that treatment of cultured parsley cells with a defined peptide elicitor of fungal origin causes rapid and large changes in the levels of various unsaturated fatty acids. While linoleic acid decreased and linolenic acid increased steadily for several hours, comparatively sharp increases in oleic acid followed a biphasic time course. In contrast, the overall level of stearic acid remained unaffected. Using a PCR-based approach, a parsley cDNA was isolated sharing high sequence similarity with ω-3 fatty acid desaturases. Subsequent isolation and characterization of a full-length cDNA enabled its functional identification as a plastid-localized ω-3 fatty acid desaturase by complementation of the Arabidopsis thaliana fad7/8 double mutant which is low in trienoic fatty acids. ω-3 Fatty acid desaturase mRNA accumulated rapidly and transiently in elicitor-treated cultured parsley cells, protoplasts, and leaves, as well as highly localized around fungal infection sites in parsley leaf buds. These results indicate that unsaturated fatty acid metabolism is yet another component of the highly complex, transcriptionally regulated pathogen defense response in plants. PMID:9050908

Novel Genes Encoding Hexadecanoic Acid Δ6-Desaturase Activity in a Rhodococcus sp.

PubMed

Araki, Hiroyuki; Hagihara, Hiroshi; Takigawa, Hirofumi; Tsujino, Yukiharu; Ozaki, Katsuya

2016-11-01

cis-6-Hexadecenoic acid, a major component of human sebaceous lipids, is involved in the defense mechanism against Staphylococcus aureus infection in healthy skin and closely related to atopic dermatitis. Previously, Koike et al. (Biosci Biotechnol Biochem 64:1064-1066, 2000) reported that a mutant strain of Rhodococcus sp. produced cis-6-hexadecenoate derivatives from palmitate alkyl esters. From the mutant Rhodococcus strain, we identified and sequenced two open reading frames present in an amplified 5.7-kb region; these open reading frames encoded tandemly repeated Δ6-desaturase-like genes, Rdes1 and Rdes2. A phylogenetic tree indicated that Rdes1 and Rdes2 were different from previously known Δ6-desaturase genes, and that they formed a new cluster. Rdes1 and Rdes2 were each introduced into vectors and then expressed separately in Escherichia coli, and the fatty acid composition of the transformed cells was analyzed by gas chromatography and mass spectrometry. The amount of cis-6-hexadecenoic acid was significantly higher in Rdes1- or Rdes2-transformed E. coli cells (twofold and threefold, respectively) than in vector-only control cells. These results showed that cis-6-hexadecenoic acid was produced in E. coli cells by the rhodococcal Δ6-desaturase-like proteins.

Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation.

PubMed

Kim, Hyun-Soo; Kim, Na-Rae; Choi, Wonja

2011-03-01

The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (∆9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (∆12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.

Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

NASA Astrophysics Data System (ADS)

Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Pan, Kehou; Pan, Jin; Yang, Guanpin

2011-03-01

A gene ( NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae). The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids (LCPUFAs), including eicosapentaenoic acid (20:5n-3, EPA). The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NANOC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid (LA) and α-linolenic acid (ALA).

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

PubMed

Sugimoto, H; Yamashita, S

1999-05-18

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.

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

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

PubMed

Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

2015-07-15

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity

PubMed Central

2015-01-01

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain. PMID:26120870

Parallel synthesis of a series of potentially brain penetrant aminoalkyl benzoimidazoles.

PubMed

Micco, Iolanda; Nencini, Arianna; Quinn, Joanna; Bothmann, Hendrick; Ghiron, Chiara; Padova, Alessandro; Papini, Silvia

2008-03-01

Alpha7 agonists were identified via GOLD (CCDC) docking in the putative agonist binding site of an alpha7 homology model and a series of aminoalkyl benzoimidazoles was synthesised to obtain potentially brain penetrant drugs. The array was prepared starting from the reaction of ortho-fluoronitrobenzenes with a selection of diamines, followed by reduction of the nitro group to obtain a series of monoalkylated phenylene diamines. N,N'-Carbonyldiimidazole (CDI) mediated acylation, followed by a parallel automated work-up procedure, afforded the monoacylated phenylenediamines which were cyclised under acidic conditions. Parallel work-up and purification afforded the array products in good yields and purities with a robust parallel methodology which will be useful for other libraries. Screening for alpha7 activity revealed compounds with agonist activity for the receptor.

Serum Fatty Acids, Desaturase Activities and Abdominal Obesity – A Population-Based Study of 60-Year Old Men and Women

PubMed Central

Alsharari, Zayed D.; Risérus, Ulf; Leander, Karin; Sjögren, Per; Carlsson, Axel C.; Vikström, Max; Laguzzi, Federica; Gigante, Bruna; Cederholm, Tommy; De Faire, Ulf; Hellénius, Mai-Lis

2017-01-01

Abdominal obesity is a key contributor of metabolic disease. Recent trials suggest that dietary fat quality affects abdominal fat content, where palmitic acid and linoleic acid influence abdominal obesity differently, while effects of n-3 polyunsaturated fatty acids are less studied. Also, fatty acid desaturation may be altered in abdominal obesity. We aimed to investigate cross-sectional associations of serum fatty acids and desaturases with abdominal obesity prevalence in a population-based cohort study. Serum cholesteryl ester fatty acids composition was measured by gas chromatography in 60-year old men (n = 1883) and women (n = 2015). Cross-sectional associations of fatty acids with abdominal obesity prevalence and anthropometric measures (e.g., sagittal abdominal diameter) were evaluated in multivariable-adjusted logistic and linear regression models, respectively. Similar models were employed to investigate relations between desaturase activities (estimated by fatty acid ratios) and abdominal obesity. In logistic regression analyses, palmitic acid, stearoyl-CoA-desaturase and Δ6-desaturase indices were associated with abdominal obesity; multivariable-adjusted odds ratios (95% confidence intervals) for highest versus lowest quartiles were 1.45 (1.19–1.76), 4.06 (3.27–5.05), and 3.07 (2.51–3.75), respectively. Linoleic acid, α-linolenic acid, docohexaenoic acid, and Δ5-desaturase were inversely associated with abdominal obesity; multivariable-adjusted odds ratios (95% confidence intervals): 0.39 (0.32–0.48), 0.74 (0.61–0.89), 0.76 (0.62–0.93), and 0.40 (0.33–0.49), respectively. Eicosapentaenoic acid was not associated with abdominal obesity. Similar results were obtained from linear regression models evaluating associations with different anthropometric measures. Sex-specific and linear associations were mainly observed for n3-polyunsaturated fatty acids, while associations of the other exposures were generally non-linear and similar across sexes. In accordance with findings from short-term trials, abdominal obesity was more common among individuals with relatively high proportions of palmitic acid, whilst the contrary was true for linoleic acid. Further trials should examine the potential role of linoleic acid and its main dietary source, vegetable oils, in abdominal obesity prevention. PMID:28125662

Hydroxytyrosol prevents reduction in liver activity of Δ-5 and Δ-6 desaturases, oxidative stress, and depletion in long chain polyunsaturated fatty acid content in different tissues of high-fat diet fed mice.

PubMed

Valenzuela, Rodrigo; Echeverria, Francisca; Ortiz, Macarena; Rincón-Cervera, Miguel Ángel; Espinosa, Alejandra; Hernandez-Rodas, María Catalina; Illesca, Paola; Valenzuela, Alfonso; Videla, Luis A

2017-04-11

Eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) are long-chain polyunsaturated fatty acids (LCPUFAs) with relevant roles in the organism. EPA and DHA are synthesized from the precursor alpha-linolenic acid (ALA, C18:3n-3), whereas AA is produced from linoleic acid (LA, C18:2n-6) through the action of Δ5 and Δ6-desaturases. High-fat diet (HFD) decreases the activity of both desaturases and LCPUFA accretion in liver and other tissues. Hydroxytyrosol (HT), a natural antioxidant, has an important cytoprotective effects in different cells and tissues. Male mice C57BL/6 J were fed a control diet (CD) (10% fat, 20% protein, 70% carbohydrates) or a HFD (60% fat, 20% protein, 20% carbohydrates) for 12 weeks. Animals were daily supplemented with saline (CD) or 5 mg HT (HFD), and blood and the studied tissues were analyzed after the HT intervention. Parameters studied included liver histology (optical microscopy), activity of hepatic desaturases 5 and 6 (gas-liquid chromatography of methyl esters derivatives) and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase by spectrophotometry), oxidative stress indicators (glutathione, thiobarbituric acid reactants, and the antioxidant capacity of plasma), gene expression assays for sterol regulatory element-binding protein 1c (SREBP-1c) (qPCR and ELISA), and LCPUFA profiles in liver, erythrocyte, brain, heart, and testicle (gas-liquid chromatography). HFD led to insulin resistance and liver steatosis associated with SREBP-1c upregulation, with enhancement in plasma and liver oxidative stress status and diminution in the synthesis and storage of n-6 and n-3 LCPUFAs in the studied tissues, compared to animals given control diet. HT supplementation significantly reduced fat accumulation in liver and plasma as well as tissue metabolic alterations induced by HFD. Furthermore, a normalization of desaturase activities, oxidative stress-related parameters, and tissue n-3 LCPUFA content was observed in HT-treated rats over control animals. HT supplementation prevents metabolic alterations in desaturase activities, oxidative stress status, and n-3 LCPUFA content in the liver and extrahepatic tissues of mice fed HFD.

Global and Phylogenetic Distribution of Quorum Sensing Signals, Acyl Homoserine Lactones, in the Family of Vibrionaceae

PubMed Central

Barker Rasmussen, Bastian; Fog Nielsen, Kristian; Machado, Henrique; Melchiorsen, Jette; Gram, Lone; Sonnenschein, Eva C.

2014-01-01

Bacterial quorum sensing (QS) and the corresponding signals, acyl homoserine lactones (AHLs), were first described for a luminescent Vibrio species. Since then, detailed knowledge has been gained on the functional level of QS; however, the abundance of AHLs in the family of Vibrionaceae in the environment has remained unclear. Three hundred and one Vibrionaceae strains were collected on a global research cruise and the prevalence and profile of AHL signals in this global collection were determined. AHLs were detected in 32 of the 301 strains using Agrobacterium tumefaciens and Chromobacterium violaceum reporter strains. Ethyl acetate extracts of the cultures were analysed by ultra-high performance liquid chromatography-high resolution mass spectrometry (MS) with automated tandem MS confirmation for AHLs. N-(3-hydroxy-hexanoyl) (OH-C6) and N-(3-hydroxy-decanoyl) (OH-C10) homoserine lactones were the most common AHLs found in 17 and 12 strains, respectively. Several strains produced a diversity of different AHLs, including N-heptanoyl (C7) HL. AHL-producing Vibrionaceae were found in polar, temperate and tropical waters. The AHL profiles correlated with strain phylogeny based on gene sequence homology, however not with geographical location. In conclusion, a wide range of AHL signals are produced by a number of clades in the Vibrionaceae family and these results will allow future investigations of inter- and intra-species interactions within this cosmopolitan family of marine bacteria. PMID:25419995

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

PubMed

Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

2000-03-01

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

PubMed Central

Lassner, M W; Lardizabal, K; Metz, J G

1996-01-01

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils. PMID:8742713

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

PubMed

Lassner, M W; Lardizabal, K; Metz, J G

1996-02-01

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.

Lipase regio- and stereoselectivities toward three enantiomeric pairs of didecanoyl-deoxyamino-O methyl glycerol: a kinetic study by the monomolecular film technique.

PubMed

Douchet, Isabelle; De Haas, Gerard; Verger, Robert

2003-03-01

A kinetic study was carried out on the regio- and stereoselectivities of 12 lipases of animal and microbial origin. For this purpose, monomolecular films consisting of three pairs of enantiomers (didecanoyl-deoxyamino-O methyl glycerol, DDG) containing a single hydrolyzable decanoyl ester bond and two lipase-resistant groups were spread at the air-water interface. Each of the lipases tested displayed a particular type of behavior, on the basis of which they were classified in two groups, depending on their ability to hydrolyze the sn-2 position. From the qualitative point of view, the sn-2 preference measured on triacylglycerides and DDG were in good agreement. The inductive chemical effect might explain why a greater level of hydrolytic activity was observed with the diglycerides than with DDG. With most of the lipases tested, it was observed that the enantiomeric pair having two distal acyl chains was more clearly differentiated stereochemically than the two homologous pairs with two adjacent acyl chains. This finding is consistent with the hypothesis that during the chiral recognition process two of the three attachment points may be the external (distal) hydrophobic chains, which is in line with the hypothesis of a tuning fork conformation of a triglyceride in the lipase active site. Copyright 2003 Wiley-Liss, Inc.

Gas Phase Dissociation Behavior of Acyl-Arginine Peptides.

PubMed

McGee, William M; McLuckey, Scott A

2013-11-15

The gas phase dissociation behavior of peptides containing acyl-arginine residues is investigated. These acylations are generated via a combination of ion/ion reactions between arginine-containing peptides and N -hydroxysuccinimide (NHS) esters and subsequent tandem mass spectrometry (MS/MS). Three main dissociation pathways of acylated arginine, labeled Paths 1-3, have been identified and are dependent on the acyl groups. Path 1 involves the acyl-arginine undergoing deguanidination, resulting in the loss of the acyl group and dissociation of the guanidine to generate an ornithine residue. This pathway generates selective cleavage sites based on the recently discussed "ornithine effect". Path 2 involves the coordinated losses of H 2 O and NH 3 from the acyl-arginine side chain while maintaining the acylation. We propose that Path 2 is initiated via cyclization of the δ-nitrogen of arginine and the C-terminal carbonyl carbon, resulting in rapid rearrangement from the acyl-arginine side chain and the neutral losses. Path 3 occurs when the acyl group contains α-hydrogens and is observed as a rearrangement to regenerate unmodified arginine while the acylation is lost as a ketene.

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

PubMed

Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

2016-01-01

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Why is golden rice golden (yellow) instead of red?

PubMed

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-05-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.

Why Is Golden Rice Golden (Yellow) Instead of Red?1[w

PubMed Central

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-01-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, ζ-carotene desaturase, carotene cis-trans-isomerase, β-lycopene cyclase, and β-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that β-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, α/β-lycopene cyclase, β-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or ζ-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the β-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed. PMID:15821145

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

Global analysis of gene expression profiles in developing physic nut (Jatropha curcas L.) seeds.

PubMed

Jiang, Huawu; Wu, Pingzhi; Zhang, Sheng; Song, Chi; Chen, Yaping; Li, Meiru; Jia, Yongxia; Fang, Xiaohua; Chen, Fan; Wu, Guojiang

2012-01-01

Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29-41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production.

[6]-gingerol dampens hepatic steatosis and inflammation in experimental nonalcoholic steatohepatitis.

PubMed

Tzeng, Thing-Fong; Liou, Shorong-Shii; Chang, Chia Ju; Liu, I-Min

2015-04-15

The aim of the study was to investigate the effects of [6]-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) in experimental models of non-alcoholic steatohepatitis. HepG2 cells were exposed to 500 µmol/l oleic acid (OA) for 24 h and preincubated for an additional 24 h with [6]-gingerol (25, 50 or 100 µmol/l). [6]-Gingerol (100 µmol/l) inhibited OA-induced triglyceride and inflammatory marker accumulation in HepG2 cells. After being fed a high-fat diet (HFD) for 2 weeks, male golden hamsters were dosed orally with [6]-gingerol (25, 50 or 100 mg/kg/day) once daily for 8 weeks while maintained on HFD. [6]-Gingerol (100 mg/kg/day) alleviated liver steatosis, inflammation, and reversed plasma markers of metabolic syndrome in HFD-fed hamsters. The expression of inflammatory cytokine genes and nuclear transcription factor-κB (NF-κB) were increased in the HFD group; these effects were attenuated by [6]-gingerol. The hepatic mRNA expression of lipogenic genes such as liver X receptor-α, sterol regulating element binding protein-1c and its target genes including acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and acyl-CoA:diacylglycerol acyltransferase 2 in HFD-fed hamsters was also blocked by [6]-gingerol. [6]-Gingerol may attenuate HFD-induced steatohepatitis by downregulating NF-κB-mediated inflammatory responses and reducing hepatic lipogenic gene expression. Copyright © 2015 Elsevier GmbH. All rights reserved.

The level of circulating octanoate does not predict ghrelin O-acyl transferase (GOAT)-mediated acylation of ghrelin during fasting.

PubMed

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

2015-01-01

Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Eight healthy young men (aged 18-28 years, body mass index range, 20.6-26.2 kg/m(2)) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14-C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity.

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…

Erythrocyte and plasma fatty acid patterns in dogs with atopic dermatitis and healthy dogs in the same household

PubMed Central

Zimmermann, Annett; Gück, Thomas; Oechtering, Gerhard

2006-01-01

Abstract Recent studies have indicated that dogs with canine atopic dermatitis (CAD) may have a disorder of fatty acid metabolism: possibly low or absent activity of Δ6-desaturase or Δ5-desaturase, or both. To clarify this possibility, we examined the erythrocyte and plasma fatty acid patterns of 8 dogs with CAD and their 8 healthy housemates. Atopic dermatitis was diagnosed according to the criteria proposed by Willemse; other causes of dermatitis were excluded clinically and by appropriate tests. Erythrocyte ghosts were prepared from blood samples. Membrane lipids were extracted and separated by thin-layer chromatography. From plasma and lipid fractions, fatty acid content was determined by gas chromatography. In erythrocytes, but not in plasma, we observed significant differences in the fatty acid pattern that suggested a reduction in the n6 fatty acid products of the Δ6- and Δ5-desaturases in dogs with atopic dermatitis when compared with healthy housemates. PMID:16850941

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides.

PubMed

Simkin, A J; Breitenbach, J; Kuntz, M; Sandmann, G

2000-10-01

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).

cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea.

PubMed

Zhu, Changfu; Yamamura, Saburo; Koiwa, Hiroyuki; Nishihara, Masashiro; Sandmann, Gerhard

2002-02-01

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

Cloning and expression analysis of phytoene desaturase and ζ-carotene desaturase genes in Carica papaya.

PubMed

Yan, P; Gao, X Z; Shen, W T; Zhou, P

2011-02-01

The fruit flesh color of papaya is an important nutritional quality trait and is due to the accumulation of carotenoid. To elucidate the carotenoid biosynthesis pathway in Carica papaya, the phytoene desaturase (PDS) and the ζ-carotene desaturase (ZDS) genes were isolated from papaya (named CpPDS and CpZDS) using the rapid amplification of cDNA ends (RACE) approach, and their expression levels were investigated in red- and yellow-fleshed papaya varieties. CpPDS contains a 1749 bp open reading frame coding for 583 amino acids, while CpZDS contains a 1716 bp open reading frame coding for 572 amino acids. The deduced CpPDS and CpZDS proteins contain a conserved dinucleotide-binding site at the N-terminus and a carotenoid-binding domain at the C-terminus. Papaya genome sequence analysis revealed that CpPDS and CpZDS are single copy; the CpPDS was mapped to papaya chromosome LG6, and the CpZDS was mapped to chromosome LG3. Quantitative PCR showed that both CpPDS and CpZDS were expressed in all tissues examined with the highest expression in maturing fruits, and that the expression of CpPDS and CpZDS were higher in red-fleshed fruits than in yellow-fleshed fruits. These results indicated that the differential accumulation of carotenoids in red- and yellow-fleshed papaya varieties might be partly explained by the transcriptional level of CpPDS and CpZDS.

Glucomannan- and glucomannan plus spirulina-enriched pork affect liver fatty acid profile, LDL receptor expression and antioxidant status in Zucker fa/fa rats fed atherogenic diets

PubMed Central

González-Torres, Laura; Matos, Cátia; Vázquez-Velasco, Miguel; Santos-López, Jorge A.; Sánchez-Martínez, Iria; García–Fernández, Camino; Bastida, Sara; Benedí, Juana; Sánchez-Muniz, Francisco J.

2017-01-01

ABSTRACT We evaluated the effects of glucomannan or glucomannan plus spirulina-restructured pork (RP) on liver fatty acid profile, desaturase/elongase enzyme activities and oxidative status of Zucker fa/fa rats for seven weeks. Control (C), glucomannan (G) and glucomannan/spirulina (GS)-RP; HC (cholesterol-enriched control), HG and HGS (cholesterol-enriched glucomannan and glucomannan/spirulina-RP) experimental diets were tested. Increased metabolic syndrome markers were found in C, G and GS rats. Cholesterol feeding increased liver size, fat, and cholesterol and reduced antioxidant enzyme levels and expressions. Cholesterolemia was lower in HG and HGS than in HC. GS vs. G showed higher stearic but lower oleic levels. SFA and PUFA decreased while MUFA increased by cholesterol feeding. The arachidonic/linoleic and docosahexaenoic/alpha-linolenic ratios were lower in HC, HG, and HGS vs. C, G, and GS, respectively, suggesting a delta-6-elongase-desaturase system inhibition. Moreover, cholesterol feeding, mainly in HGS, decreased low-density-lipoprotein receptor expression and the delta-5-desaturase activity and increased the delta-9-desaturase activity. In conclusion, the liver production of highly unsaturated fatty acids was limited to decrease their oxidation in presence of hypercholesterolaemia. Glucomannan or glucomannan/spirulina-RP has added new attributes to their functional properties in meat, partially arresting the negative effects induced by high-fat-high-cholesterol feeding on the liver fatty acid and antioxidant statuses. PMID:28325998

Skeletal muscle membrane lipid composition is related to adiposity and insulin action.

PubMed Central

Pan, D A; Lillioja, S; Milner, M R; Kriketos, A D; Baur, L A; Bogardus, C; Storlien, L H

1995-01-01

The cellular basis of insulin resistance is still unknown; however, relationships have been demonstrated between insulin action in muscle and the fatty acid profile of the major membrane structural lipid (phospholipid). The present study aimed to further investigate the hypothesis that insulin action and adiposity are associated with changes in the structural lipid composition of the cell. In 52 adult male Pima Indians, insulin action (euglycemic clamp), percentage body fat (pFAT; underwater weighing), and muscle phospholipid fatty acid composition (percutaneous biopsy of vastus lateralis) were determined. Insulin action (high-dose clamp; MZ) correlated with composite measures of membrane unsaturation (% C20-22 polyunsaturated fatty acids [r= 0.463, P < 0.001], unsaturation index [r= -0.369, P < 0.01]), a number of individual fatty acids and with delta5 desaturase activity (r= 0.451, P < 0.001). pFAT (range 14-53%) correlated with a number of individual fatty acids and delta5 desaturase activity (r= -0.610, P < 0.0001). Indices of elongase activity (r= -0.467, P < 0.001), and delta9 desaturase activity (r= 0.332, P < 0.05) were also related to pFAT but not insulin action. The results demonstrate that delta5 desaturase activity is independently related to both insulin resistance and obesity. While determining the mechanisms underlying this relationship is important for future investigations, strategies aimed at restoring "normal" enzyme activities, and membrane unsaturation, may have therapeutic importance in the "syndromes of insulin resistance." PMID:8675650

Stereochemistry of a bifunctional dihydroceramide delta 4-desaturase/hydroxylase from Candida albicans; a key enzyme of sphingolipid metabolism.

PubMed

Beckmann, Christoph; Rattke, Janine; Sperling, Petra; Heinz, Ernst; Boland, Wilhelm

2003-07-21

The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.

An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.

PubMed

Meesapyodsuk, Dauenpen; Qiu, Xiao

2008-07-01

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.

Transcriptional Activation of Two Delta-9 Palmitoyl-ACP Desaturase Genes by MYB115 and MYB118 Is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acids in the Endosperm of Arabidopsis Seeds

PubMed Central

Troncoso-Ponce, Manuel Adrián; Barthole, Guillaume; Tremblais, Geoffrey

2016-01-01

In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 mol% of total FAs, whereas these molecular species are 10-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase. PMID:27681170

Peroxisomal ATP-binding cassette transporters form mainly tetramers

PubMed Central

Geillon, Flore; Gondcaille, Catherine; Raas, Quentin; Dias, Alexandre M. M.; Pecqueur, Delphine; Truntzer, Caroline; Lucchi, Géraldine; Ducoroy, Patrick; Falson, Pierre; Savary, Stéphane; Trompier, Doriane

2017-01-01

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane. PMID:28258215

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

PubMed

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

2014-03-01

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.

Incorporation of Extracellular Fatty Acids by a Fatty Acid Kinase-Dependent Pathway in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Frank, Matthew W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Summary Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids. PMID:24673884

Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid.

PubMed

Wang, Hong-Lei; Zhao, Cheng-Hua; Wang, Chen-Zhu

2005-06-01

Two Helicoverpa species, H. armigera and H. assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as their sex attractant pheromone components but in opposite ratios. Since both female and male interspecific hybrids produced by female H. assulta and male H. armigera have been obtained in our laboratory, we can make a comparative study of sex pheromone composition and biosynthesis in the two species and their hybrid. With GC and GC-MS analyses using single gland extracts, the ratio of (Z)-9-hexadecenal to (Z)-11-hexadecenal was determined as 2.1:100 in H. armigera, and 1739:100 in H. assulta. The hybrid has a ratio of 4.0: 100, which is closer to that of H. armigera, but significantly different from H. armigera. We investigated pheromone biosynthesis with labeling experiments, using various fatty acid precursors in H. armigera, H. assulta and the hybrid. In H. armigera, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation; (Z)-9-hexadecenal results from delta11 desaturation of stearic acid, followed by one cycle of chain shortening, reduction and terminal oxidation. delta11 desaturase is the unique desaturase for the production of the two pheromone components. In our Chinese strain of H. assulta, palmitic acid is used as the substrate to form both the major pheromone component, (Z)-9-hexadecenal and the minor one, (Z)-11-hexadecenal. Our data suggest that delta9 desaturase is the major desaturase, and delta11 desaturase is responsible for the minor component in H. assulta, which is consistent with previous work. However, the weak chain shortening acting on (Z)-9 and (Z)-11-octadecenoic acid, which is present in the pheromone glands, does occur in this species to produce (Z)-7 and (Z)-9-hexadecenoic acid. In the hybrid, the major pheromone component, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation. The direct fatty acid precursor of the minor component, (Z)-9-hexadecenoic acid is mainly produced by delta9 desaturation of palmitic acid, but also by delta11 desaturation of stearic acid and one cycle of chain shortening. The greater relative amounts of (Z)-9-hexadecenal in the hybrid are due to the fact that both palmitic and stearic acids are used as substrates, whereas only stearic acid is used as substrate in H. armigera. The evolutionary relationships between the desaturases in several Helicoverpa species are also discussed in this paper.

Optimizing gelling parameters of gellan gum for fibrocartilage tissue engineering.

PubMed

Lee, Haeyeon; Fisher, Stephanie; Kallos, Michael S; Hunter, Christopher J

2011-08-01

Gellan gum is an attractive biomaterial for fibrocartilage tissue engineering applications because it is cell compatible, can be injected into a defect, and gels at body temperature. However, the gelling parameters of gellan gum have not yet been fully optimized. The aim of this study was to investigate the mechanics, degradation, gelling temperature, and viscosity of low acyl and low/high acyl gellan gum blends. Dynamic mechanical analysis showed that increased concentrations of low acyl gellan gum resulted in increased stiffness and the addition of high acyl gellan gum resulted in greatly decreased stiffness. Degradation studies showed that low acyl gellan gum was more stable than low/high acyl gellan gum blends. Gelling temperature studies showed that increased concentrations of low acyl gellan gum and CaCl₂ increased gelling temperature and low acyl gellan gum concentrations below 2% (w/v) would be most suitable for cell encapsulation. Gellan gum blends were generally found to have a higher gelling temperature than low acyl gellan gum. Viscosity studies showed that increased concentrations of low acyl gellan gum increased viscosity. Our results suggest that 2% (w/v) low acyl gellan gum would have the most appropriate mechanics, degradation, and gelling temperature for use in fibrocartilage tissue engineering applications. Copyright © 2011 Wiley Periodicals, Inc.

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

Genotype-specific responses in Atlantic salmon (Salmo salar) subject to dietary fish oil replacement by vegetable oil: a liver transcriptomic analysis.

PubMed

Morais, Sofia; Pratoomyot, Jarunan; Taggart, John B; Bron, James E; Guy, Derrick R; Bell, J Gordon; Tocher, Douglas R

2011-05-20

Expansion of aquaculture is seriously limited by reductions in fish oil (FO) supply for aquafeeds. Terrestrial alternatives such as vegetable oils (VO) have been investigated and recently a strategy combining genetic selection with changes in diet formulations has been proposed to meet growing demands for aquaculture products. This study investigates the influence of genotype on transcriptomic responses to sustainable feeds in Atlantic salmon. A microarray analysis was performed to investigate the liver transcriptome of two family groups selected according to their estimated breeding values (EBVs) for flesh lipid content, 'Lean' or 'Fat', fed diets containing either FO or a VO blend. Diet principally affected metabolism genes, mainly of lipid and carbohydrate, followed by immune response genes. Genotype had a much lower impact on metabolism-related genes and affected mostly signalling pathways. Replacement of dietary FO by VO caused an up-regulation of long-chain polyunsaturated fatty acid biosynthesis, but there was a clear genotype effect as fatty acyl elongase (elovl2) was only up-regulated and desaturases (Δ5 fad and Δ6 fad) showed a higher magnitude of response in Lean fish, which was reflected in liver fatty acid composition. Fatty acid synthase (FAS) was also up-regulated by VO and the effect was independent of genotype. Genetic background of the fish clearly affected regulation of lipid metabolism, as PPARα and PPARβ were down-regulated by the VO diet only in Lean fish, while in Fat salmon SREBP-1 expression was up-regulated by VO. In addition, all three genes had a lower expression in the Lean family group than in the Fat, when fed VO. Differences in muscle adiposity between family groups may have been caused by higher levels of hepatic fatty acid and glycerophospholipid synthesis in the Fat fish, as indicated by the expression of FAS, 1-acyl-sn-glycerol-3-phosphate acyltransferase and lipid phosphate phosphohydrolase 2. This study has identified metabolic pathways and key regulators that may respond differently to alternative plant-based feeds depending on genotype. Further studies are required but data suggest that it will be possible to identify families better adapted to alternative diet formulations that might be appropriate for future genetic selection programmes.

Genotype-specific responses in Atlantic salmon (Salmo salar) subject to dietary fish oil replacement by vegetable oil: a liver transcriptomic analysis

PubMed Central

2011-01-01

Background Expansion of aquaculture is seriously limited by reductions in fish oil (FO) supply for aquafeeds. Terrestrial alternatives such as vegetable oils (VO) have been investigated and recently a strategy combining genetic selection with changes in diet formulations has been proposed to meet growing demands for aquaculture products. This study investigates the influence of genotype on transcriptomic responses to sustainable feeds in Atlantic salmon. Results A microarray analysis was performed to investigate the liver transcriptome of two family groups selected according to their estimated breeding values (EBVs) for flesh lipid content, 'Lean' or 'Fat', fed diets containing either FO or a VO blend. Diet principally affected metabolism genes, mainly of lipid and carbohydrate, followed by immune response genes. Genotype had a much lower impact on metabolism-related genes and affected mostly signalling pathways. Replacement of dietary FO by VO caused an up-regulation of long-chain polyunsaturated fatty acid biosynthesis, but there was a clear genotype effect as fatty acyl elongase (elovl2) was only up-regulated and desaturases (Δ5 fad and Δ6 fad) showed a higher magnitude of response in Lean fish, which was reflected in liver fatty acid composition. Fatty acid synthase (FAS) was also up-regulated by VO and the effect was independent of genotype. Genetic background of the fish clearly affected regulation of lipid metabolism, as PPARα and PPARβ were down-regulated by the VO diet only in Lean fish, while in Fat salmon SREBP-1 expression was up-regulated by VO. In addition, all three genes had a lower expression in the Lean family group than in the Fat, when fed VO. Differences in muscle adiposity between family groups may have been caused by higher levels of hepatic fatty acid and glycerophospholipid synthesis in the Fat fish, as indicated by the expression of FAS, 1-acyl-sn-glycerol-3-phosphate acyltransferase and lipid phosphate phosphohydrolase 2. Conclusions This study has identified metabolic pathways and key regulators that may respond differently to alternative plant-based feeds depending on genotype. Further studies are required but data suggest that it will be possible to identify families better adapted to alternative diet formulations that might be appropriate for future genetic selection programmes. PMID:21599965

Efficient CRISPR/Cas9-based gene knockout in watermelon.

PubMed

Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

2017-03-01

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

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

PubMed

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

2017-01-25

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

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

PubMed Central

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

2017-01-01

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord. PMID:28120938

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

A Plastidial Lysophosphatidic Acid Acyltransferase from Oilseed Rape1

PubMed Central

Bourgis, Fabienne; Kader, Jean-Claude; Barret, Pierre; Renard, Michel; Robinson, David; Robinson, Colin; Delseny, Michel; Roscoe, Thomas J.

1999-01-01

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotic-type acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme. PMID:10398728

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

PubMed

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

2014-03-01

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis. © 2013 Scandinavian Plant Physiology Society.

Purification of a Jojoba Embryo Wax Synthase, Cloning of its cDNA, and Production of High Levels of Wax in Seeds of Transgenic Arabidopsis

PubMed Central

Lardizabal, Kathryn D.; Metz, James G.; Sakamoto, Tetsuo; Hutton, William C.; Pollard, Michael R.; Lassner, Michael W.

2000-01-01

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a β-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. 13C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds. PMID:10712527

Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation

PubMed Central

Raboune, Siham; Stuart, Jordyn M.; Leishman, Emma; Takacs, Sara M.; Rhodes, Brandon; Basnet, Arjun; Jameyfield, Evan; McHugh, Douglas; Widlanski, Theodore; Bradshaw, Heather B.

2014-01-01

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation. PMID:25136293

StearoylCoA Desaturase-5: A Novel Regulator of Neuronal Cell Proliferation and Differentiation

PubMed Central

Sinner, Debora I.; Kim, Gretchun J.; Henderson, Gregory C.; Igal, R. Ariel

2012-01-01

Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1), a Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5), a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls). De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFR)Akt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key lipogenic pathways and transduction signaling cascades, SCD5 participates in the regulation of neuronal cell growth and differentiation. PMID:22745828

Adding a purple corn extract in rats supplemented with chia oil decreases gene expression of SREBP-1c and retains Δ5 and Δ6 hepatic desaturase activity, unmodified the hepatic lipid profile.

PubMed

Reyna Gallegos, Sixto; Torres Arrunátegui, Génesis; Valenzuela, Rodrigo; Rincón-Cervera, Miguel Ángel; Villanueva Espinoza, María Elena

2018-05-01

Flavonoids upregulate gene expression of PPAR-α and underregulate the gene expression of SREBP-1c, and their intake increases the plasmatic concentration of n-3 LC-PUFAs. However, the biological mechanisms underlying these effects have not been elucidated. In this work, the effect of oral supplementation of ALA from chia (Salvia hispanica L.) seed oil and anthocyanins from a purple corn extract (PCE) on gene expression of SREBP-1c, PPAR-α and Δ5 and Δ6 desaturases (Δ5D and Δ6D), the activity of these enzymes in the liver as well as the hepatic lipid profile were evaluated in thirty-six female Sprague Dawley rats whose diet was supplemented with olive oil (OL), chia oil (CH), olive oil and PCE (OL + PCE) or chia oil and PCE (CH + PCE). Gene expression of PPAR-α was significantly higher when supplemented with CH and CH + PCE, SREBP-1c gene expression was higher when supplemented with chia oil. CH supplementation enhanced Δ5D expression whereas no significant differences between treatments were observed concerning Δ6D gene expression. Activities of both desaturases were increased by including olive oil (OL + PCE and OL), and they were found to be higher in CH + PCE respect to CH for both enzymes. The ALA and n-3 LCPUFAs hepatic content was higher with CH, decreasing the levels of AA and n-6 LCPUFAs. It is concluded that the joint action of flavonoids such as anthocyanins and ALA show an anti-adipogenic effect. Desaturase activity was inhibited by ALA and kept by the anthocyanins from PCE, thus anthocyanins would exert a protective effect on the desaturase activity but they would not affect on its gene expression, however, high doses of ALA increased the production of its metabolites, masking the effect of PCE. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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...

Perfluorodecanoic acid enhances the formation of oleic acid in rat liver.

PubMed Central

Yamamoto, A; Kawashima, Y

1997-01-01

The feeding of perfluorodecanoic acid (PFDA) to male rats at a dietary concentration of 0.005% (w/w) for 7 days resulted in a marked increase in the activity of microsomal stearoyl-CoA desaturation in the liver. This increase in the overall desaturation activity was due to the induction of terminal desaturase among the components comprising the desaturation system. In contrast, PFDA inhibited desaturation in vitro, seemingly due to interference with electron transport through the desaturation system. Accordingly, PFDA can be an inducer and also an inhibitor of delta9-desaturation. PFDA feeding enhanced the conversion of radioactive stearic acid into oleic acid in the liver in vivo, indicating that the induction of delta9-desaturase by PFDA functions in vivo. PFDA feeding increased the mass of octadecenoic acid (C18:1) in the liver and the proportion of C18:1 in microsomal lipid. A highly significant linear correlation existed between the microsomal desaturase activity and the proportion of C18:1 in microsomal lipid when compared using rats in five different physiological states: control, PFDA-fed, p-chlorophenoxyisobutyric acid (clofibric acid)-fed, starved and starved/refed. These results suggest that the increase in the hepatic level of C18:1 caused by feeding of PFDA to rats can be explained by the common concept of regulation, i.e. the hepatic level of C18:1 is under the control of delta9-desaturase. The dietary administration of PFDA also increased the content of cytochrome P-450 and the activity of 7-ethoxycoumarin O-de-ethylase in the liver. PMID:9230124

Improved eicosapentaenoic acid production in Pythium splendens RBB-5 based on metabolic regulation analysis.

PubMed

Ren, Liang; Zhou, Pengpeng; Zhu, Yuanmin; Zhang, Ruijiao; Yu, Longjiang

2017-05-01

Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid for human beings. At present, the production of commercially available long-chain polyunsaturated fatty acids, mainly from wild-caught ocean fish, is struggling to meet the increasing demand for EPA. Production of EPA by microorganisms may be an alternative, effective and economical method. The oleaginous fungus Pythium splendens RBB-5 is a potential source of EPA, and thanks to the simple culture conditions required, high yields can be achieved in a facile manner. In the study, lipid metabolomics was performed in an attempt to enhance EPA biosynthesis in Pythium splendens. Synthetic, metabolic regulation and gene expression analyses were conducted to clarify the mechanism of EPA biosynthesis, and guide optimization of EPA production. The results showed that the Δ 6 desaturase pathway is the main EPA biosynthetic route in this organism, and ∆ 6 , ∆ 12 and Δ 17 desaturases are the rate-limiting enzymes. All the three desaturase genes were separately introduced into the parent strain to increase the flow of fatty acids into the Δ 6 desaturase pathway. Enhanced expression of these key enzymes, in combination with improved regulation of metabolism, resulted in a maximum yield of 1.43 g/L in the D12 transgenic strain, which represents a tenfold increase over the parent strain before optimization. This is the higher EPA production yield yet reported for a microbial system. Our findings may allow the production of EPA at an industrial scale, and the strategy employed could be used to increase the production of EPA or other lipids in oleaginous microorganisms.

Genetic variation in polyunsaturated fatty acid metabolism and its potential relevance for human development and health.

PubMed

Glaser, Claudia; Lattka, Eva; Rzehak, Peter; Steer, Colin; Koletzko, Berthold

2011-04-01

Blood and tissue contents of polyunsaturated fatty acid (PUFA) and long-chain PUFA (LC-PUFA) are related to numerous health outcomes including cardiovascular health, allergies, mental health and cognitive development. Evidence has accumulated to show that in addition to diet, common polymorphisms in the fatty acid desaturase (FADS) gene cluster have very marked effects on human PUFA and LC-PUFA status. Recent results suggest that in addition to fatty acid desaturase 1 and fatty acid desaturase 2, the gene product of fatty acid desaturase 3 is associated with desaturating activity. New data have become available to show that FADS single nucleotide polymorphisms (SNPs) also modulate docosahexaenoic acid status in pregnancy as well as LC-PUFA levels in children and in human milk. There are indications that FADS SNPs modulate the risk for allergic disorders and eczema, and the effect of breastfeeding on later cognitive development. Mechanisms by which FADS SNPs modulate PUFA levels in blood, breast milk and tissues should be explored further. More studies are required to explore the effects of FADS gene variants in populations with different ethnic backgrounds, lifestyles and dietary habits, and to investigate in greater depth the interaction of gene variants, diet and clinical end points, including immune response and developmental outcomes. Analyses of FADS gene variants should be included into all sizeable cohort and intervention studies addressing biological effects of PUFA and LC-PUFA in order to consider these important confounders, and to enhance study sensitivity and precision. © 2011 Blackwell Publishing Ltd.

Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression.

PubMed

Ziesack, Marika; Rollins, Nathan; Shah, Aashna; Dusel, Brendon; Webster, Gordon; Silver, Pamela A; Way, Jeffrey C

2018-05-15

Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 ( Cp FatB1) (C 8 specificity, lower activity) and Cp FatB2 (C 14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C 8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify Cp FatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from Cp FatB1 into Cp FatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Similarly, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing. Copyright © 2018 American Society for Microbiology.

Effect of tanniniferous Terminalia chebula extract on rumen biohydrogenation, ∆(9)-desaturase activity, CLA content and fatty acid composition in longissimus dorsi muscle of kids.

PubMed

Rana, Madhu Suman; Tyagi, A; Hossain, Sk Asraf; Tyagi, A K

2012-03-01

Conjugated linoleic acid, a fatty acid found in milk fat and ruminant meat is one of the functional food components. Modifying fatty acid composition so as to increase CLA and other beneficial PUFA/MUFA level and reducing SFA levels might be a key to enhance the neutraceutical and therapeutic value of ruminant-derived food products. In the present experiment, the effect of supplementation of polyphenol rich Terminalia chebula plant extract at different concentrations (1.06g/kg and 3.18g/kg of body weight in T1 and T2 groups, respectively) was investigated on fatty acid composition of rumen fluid, plasma, intramuscular fat and Δ9-desaturase activity in longissimus dorsi muscle of crossbred kids. Total MUFA and PUFA content in muscle were enhanced by 25 and 35%, respectively, whereas SFA was reduced by 20% thereby improving the desaturation index. Δ9-desaturase activity also increased by 47% resulting in an enhancement of total CLA content (58.73%) in muscle. Copyright © 2011 Elsevier Ltd. All rights reserved.

Fatty acid utilization by young Wistar rats fed a cafeteria diet.

PubMed

Esteve, M; Rafecas, I; Fernández-López, J A; Remesar, X; Alemany, M

1992-12-02

The content and accretion of fatty acids in 30, 45 and 60-day old Wistar rats fed either reference chow or a cafeteria diet has been studied, together with their actual fatty acid intake during that period. Diet had a small overall effect on the pattern of deposition of fatty acids, but the deposition of fat was much higher in cafeteria rats. The fat-rich cafeteria diet allowed the direct incorporation of most fatty acids into lipid storage, whilst chow-feeding activated lipogenesis and the deposition of a shorter chain and more saturated type of fatty acids. During the second month of the rat's life, the elongation pathway as well as delta 9-desaturase became functional, thus helping to shape the pattern of fatty acids actually accrued. The 60-day rats showed a relative impairment in the operation of delta 5-desaturase, since their lipids had a higher C20:4/C20:3 ratio than those of the diet ingested. Cafeteria-diet feeding minimized this effect since the large supply of dietary polyunsaturated fatty acids made the operation of the elongation-desaturase pathways practically unnecessary.

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

Early life disruption to the ghrelin system with over-eating is resolved in adulthood in male rats.

PubMed

Sominsky, Luba; Ziko, Ilvana; Nguyen, Thai-Xinh; Andrews, Zane B; Spencer, Sarah J

2017-02-01

Early life overweight is a significant risk factor for developmental programming of adult obesity due to changes in the availability of metabolic factors crucial for the maturation of brain appetite-regulatory circuitry. The appetite-stimulating hormone, ghrelin, has been recently identified as a major regulator of the establishment of hypothalamic feeding pathways. Ghrelin exists in circulation in two major forms, as acylated and des-acylated ghrelin. While most research has focused on acyl ghrelin, the role of neonatal des-acyl ghrelin in metabolic programming is currently unknown. Here we assessed the influences of early life overfeeding on the ghrelin system, including acyl and des-acyl ghrelin's ability to access the hypothalamus in male rats. Our data show that early life overfeeding influences the ghrelin system short-term, leading to an acute reduction in circulating des-acyl ghrelin and increased expression of the growth hormone secretagogue receptor (GHSR) in the arcuate nucleus of the hypothalamus (ARC). These changes are associated with increased neuronal activation in response to exogenous acyl, but not des-acyl, ghrelin in the ARC and the paraventricular nucleus of the hypothalamus (PVN). Interestingly, while we observed no differences in the accessibility of the ARC to acyl or des-acyl ghrelin, less exogenous acyl ghrelin reaches the PVN in the neonatally overfed. Importantly, the influences of neonatal overfeeding on the ghrelin system were not maintained into adulthood. Therefore, while early life overfeeding results in excess body weight and stimulates acute changes in the brain's sensitivity to metabolic signals, this developmental mal-programming is at least partially alleviated in adulthood. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

PubMed

Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

2010-11-30

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

1998-12-01

Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated sialomucins in C9. Adenocarcinomas of the colloid type, thought to bear a poor prognosis, are the most heavily O-acylated.

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

PubMed Central

Wisner, Stephanie A; Chen, Xiao; Spiegelman, Nicole A; Linder, Maurine E

2017-01-01

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. PMID:29239724

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

Cancer.gov

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

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

PubMed Central

Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

2003-01-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments. PMID:14660416

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant.

PubMed

Van Dien, Stephen J; Marx, Christopher J; O'Brien, Brooke N; Lidstrom, Mary E

2003-12-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.

Investigation of acyl migration in mono- and dicaffeoylquinic acids under aqueous basic, aqueous acidic, and dry roasting conditions.

PubMed

Deshpande, Sagar; Jaiswal, Rakesh; Matei, Marius Febi; Kuhnert, Nikolai

2014-09-17

Acyl migration in chlorogenic acids describes the process of migration of cinnamoyl moieties from one quinic acid alcohol group to another, thus interconverting chlorogenic acid regioisomers. It therefore constitutes a special case of transesterification reaction. Acyl migration constitutes an important reaction pathway in both coffee roasting and brewing, altering the structure of chlorogenic acid initially present in the green coffee bean. In this contribution we describe detailed and comprehensive mechanistic studies comparing inter- and intramolecular acyl migration involving the seven most common chlorogenic acids in coffee. We employe aqueous acidic and basic conditions mimicking the brewing of coffee along with dry roasting conditions. We show that under aqueous basic conditions intramolecular acyl migration is fully reversible with basic hydrolysis competing with acyl migration. 3-Caffeoylquinic acid was shown to be most labile to basic hydrolysis. We additionally show that the acyl migration process is strongly pH dependent with increased transesterification taking place at basic pH. Under dry roasting conditions acyl migration competes with dehydration to form lactones. We argue that acyl migration precedes lactonization, with 3-caffeoylquinic acid lactone being the predominant product.

Quantitative visualization of passive transport across bilayer lipid membranes

PubMed Central

Grime, John M. A.; Edwards, Martin A.; Rudd, Nicola C.; Unwin, Patrick R.

2008-01-01

The ability to predict and interpret membrane permeation coefficients is of critical importance, particularly because passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. We present a method for the quantitative measurement of the permeation coefficients of protonophores by using laser confocal scanning microscopy coupled to microelectrochemistry, which is amenable to precise modeling with the finite element method. The technique delivers well defined and high mass transport rates and allows rapid visualization of the entire pH distribution on both the cis and trans side of model bilayer lipid membranes (BLMs). A homologous series of carboxylic acids was investigated as probe molecules for BLMs composed of soybean phosphatidylcholine. Significantly, the permeation coefficient decreased with acyl tail length contrary to previous work and to Overton's rule. The reasons for this difference are considered, and we suggest that the applicability of Overton's rule requires re-evaluation. PMID:18787114

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

PubMed

Pan, Jieru; Huang, Tianpei; Yao, Fan; Huang, Zhipeng; Powell, Charles A; Qiu, Sixin; Guan, Xiong

2008-01-01

AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.

Plasma levels of acylated ghrelin in patients with functional dyspepsia

PubMed Central

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

2012-01-01

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

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

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

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

PubMed

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

1995-03-01

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.

Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein.

PubMed

Dörmann, P; Spener, F; Ohlrogge, J B

1993-03-01

Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic a nd lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata.

Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

PubMed

Garg, Hari G; Mrabat, Hicham; Yu, Lunyin; Hales, Charles A; Li, Boyangzi; Moore, Casey N; Zhang, Fuming; Linhardt, Robert J

2011-08-01

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.

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

PubMed

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

2013-09-01

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

Global Analysis of Gene Expression Profiles in Developing Physic Nut (Jatropha curcas L.) Seeds

PubMed Central

Jiang, Huawu; Wu, Pingzhi; Zhang, Sheng; Song, Chi; Chen, Yaping; Li, Meiru; Jia, Yongxia; Fang, Xiaohua; Chen, Fan; Wu, Guojiang

2012-01-01

Background Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. Methodology/Principal Findings We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29–41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. Conclusions/Significance The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production. PMID:22574177

Hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia in sucrose-fed obese rats via two pathways.

PubMed

Uebanso, Takashi; Taketani, Yutaka; Fukaya, Makiko; Sato, Kazusa; Takei, Yuichiro; Sato, Tadatoshi; Sawada, Naoki; Amo, Kikuko; Harada, Nagakatsu; Arai, Hidekazu; Yamamoto, Hironori; Takeda, Eiji

2009-07-01

The mechanism by which replacement of some dietary carbohydrates with protein during weight loss favors lipid metabolism remains obscure. In this study, we investigated the effect of an energy-restricted, high-protein/low-carbohydrate diet on lipid metabolism in obese rats. High-sucrose-induced obese rats were assigned randomly to one of two energy-restricted dietary interventions: a carbohydrate-based control diet (CD) or a high-protein diet (HPD). Lean rats of the same age were assigned as normal control. There was significantly greater improvement in fatty liver and hypertriglyceridemia with the HPD diet relative to the CD diet. Expression of genes regulated by fibroblast growth factor-21 (FGF21) and involved in liver lipolysis and lipid utilitization, such as lipase and acyl-CoA oxidase, increased in obese rats fed the HPD. Furthermore, there was an inverse correlation between levels of FGF21 gene expression (regulated by glucagon/insulin balance) and increased triglyceride concentrations in liver from obese rats. Expression of hepatic stearoyl-CoA desaturase-1 (SCD1), regulated primarily by the dietary carbohydrate, was also markedly reduced in the HPD group (similar to plasma triglyceride levels in fasting animals) relative to the CD group. In conclusion, a hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia effectively relative to a carbohydrate diet. The two cellular pathways at work behind these benefits include stimulation of hepatic lipolysis and lipid utilization mediated by FGF21 and reduction of hepatic VLDL-TG production by SCD1 regulation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simpson, Jeffrey P.; Thrower, Nicholas; Ohlrogge, John B.

Bayberry (Myrica pensylvanica) fruits are covered with a remarkably thick layer of crystalline wax consisting of triacylglycerol (TAG) and diacylglycerol (DAG) esterified exclusively with saturated fatty acids. As the only plant known to accumulate soluble glycerolipids as a major component of surface waxes, Bayberry represents a novel system to investigate neutral lipid biosynthesis and lipid secretion by vegetative plant cells. The assembly of Bayberry wax is distinct from conventional TAG and other surface waxes, and instead proceeds through a pathway related to cutin synthesis (Simpson and Ohlrogge, 2016). In this study, microscopic examination revealed that the fruit tissue that producesmore » and secretes wax (Bayberry knobs) is fully developed before wax accumulates and that wax is secreted to the surface without cell disruption. Comparison of transcript expression to genetically related tissues (Bayberry leaves, M. rubra fruits), cutin-rich tomato and cherry fruit epidermis, and to oil-rich mesocarp and seeds, revealed exceptionally high expression of 13 transcripts for acyl-lipid metabolism together with down-regulation of fatty acid oxidases and desaturases. The predicted protein sequences of the most highly expressed lipid-related enzyme-encoding transcripts in Bayberry knobs are 100% identical to the sequences from Bayberry leaves,which do not produce surface DAG or TAG. Together, these results indicate that TAG biosynthesis and secretion in Bayberry is achieved by both up and down-regulation of a small subset of genes related to the biosynthesis of cutin and saturated fatty acids, and also implies that modifications in gene expression, rather than evolution of new gene functions, was the major mechanism by which Bayberry evolved its specialized lipid metabolism.« less

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content.

PubMed

Xu, Jingyu; Francis, Tammy; Mietkiewska, Elzbieta; Giblin, E Michael; Barton, Dennis L; Zhang, Yan; Zhang, Meng; Taylor, David C

2008-10-01

A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Pharmacological characterization of [trans-5'-(4-amino-7,7-dimethyl-2-trifluoromethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)-2',3'-dihydrospiro(cyclohexane-1,1'-inden)-4-yl]acetic acid monobenzenesulfonate (JTT-553), a novel acyl CoA:diacylglycerol transferase (DGAT) 1 inhibitor.

PubMed

Tomimoto, Daisuke; Okuma, Chihiro; Ishii, Yukihito; Akiyama, Yoshiyuki; Ohta, Takeshi; Kakutani, Makoto; Ohkuma, Yoshiaki; Ogawa, Nobuya

2015-01-01

Acyl CoA:diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the final step in triglyceride (TG) synthesis. This enzyme is considered to be a potential therapeutic target for obesity and diabetes. Here, results of an investigation of the pharmacological effects of JTT-553 [trans-5'-(4-amino-7,7-dimethyl-2-trifluoromethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)-2',3'-dihydrospiro(cyclohexane-1,1'-inden)-4-yl]acetic acid monobenzenesulfonate, a novel DGAT1 inhibitor, are reported. To measure the inhibitory activity of JTT-553 against DGAT1, TG synthesis using [(14)C]-labeled oleoyl-CoA was evaluated. Similarly, the inhibitory activity of JTT-553 against DGAT2, an isozyme of DGAT1, and acyl-CoA cholesterol acyltransferase (ACAT) 1, which is highly homologous to DGAT1, were evaluated. JTT-553 selectively inhibited human DGAT1 and showed comparable inhibitory effects on the activity of human, rat, and mouse DGAT. In vivo, JTT-553 suppressed plasma TG and chylomicron TG levels after olive oil loading in Sprague-Dawley (SD) rats. JTT-553 also inhibited TG synthesis in epididymal fat after [(14)C] oleic acid injection in C57BL/6J mice. Food intake was evaluated in SD rats fed 3.1%, 13%, or 35% (w/w) fat diets. In rats fed the 35% fat diet, JTT-553 reduced food intake. This reduction of food intake was observed 2 h after feeding, lasted for 24 h, and correlated with dietary fat content. Furthermore, JTT-553 reduced daily food intake and body weight gain in diet-induced obese rats after 4-week repeated administration. JTT-553 exerted multiple effects on intestinal fat absorption, adipose fat synthesis, and food intake, and consequently induced body weight reduction. Therefore, JTT-553 is expected to be an effective novel therapeutic agent for the treatment of obesity.

Altered fatty acid metabolism and reduced stearoyl-coenzyme a desaturase activity in asthma.

PubMed

Rodriguez-Perez, N; Schiavi, E; Frei, R; Ferstl, R; Wawrzyniak, P; Smolinska, S; Sokolowska, M; Sievi, N A; Kohler, M; Schmid-Grendelmeier, P; Michalovich, D; Simpson, K D; Hessel, E M; Jutel, M; Martin-Fontecha, M; Palomares, O; Akdis, C A; O'Mahony, L

2017-11-01

Fatty acids and lipid mediator signaling play an important role in the pathogenesis of asthma, yet this area remains largely underexplored. The aims of this study were (i) to examine fatty acid levels and their metabolism in obese and nonobese asthma patients and (ii) to determine the functional effects of altered fatty acid metabolism in experimental models. Medium- and long-chain fatty acid levels were quantified in serum from 161 human volunteers by LC/MS. Changes in stearoyl-coenzyme A desaturase (SCD) expression and activity were evaluated in the ovalbumin (OVA) and house dust mite (HDM) murine models. Primary human bronchial epithelial cells from asthma patients and controls were evaluated for SCD expression and activity. The serum desaturation index (an indirect measure of SCD) was significantly reduced in nonobese asthma patients and in the OVA murine model. SCD1 gene expression was significantly reduced within the lungs following OVA or HDM challenge. Inhibition of SCD in mice promoted airway hyper-responsiveness. SCD1 expression was suppressed in bronchial epithelial cells from asthma patients. IL-4 and IL-13 reduced epithelial cell SCD1 expression. Inhibition of SCD reduced surfactant protein C expression and suppressed rhinovirus-induced IP-10 secretion, which was associated with increased viral titers. This is the first study to demonstrate decreased fatty acid desaturase activity in humans with asthma. Experimental models in mice and human epithelial cells suggest that inhibition of desaturase activity leads to airway hyper-responsiveness and reduced antiviral defense. SCD may represent a new target for therapeutic intervention in asthma patients. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

[Desaturases of fatty acids (FADS) and their physiological and clinical implication].

PubMed

Žák, Aleš; Slabý, Adolf; Tvrzická, Eva; Jáchymová, Marie; Macášek, Jaroslav; Vecka, Marek; Zeman, Miroslav; Staňková, Barbora

States associated with insulin resistance, as overweight/obesity, type 2 diabetes mellitus (DM2), cardiovascular diseases (CVD), some cancers and neuropsychiatric diseases are characterized with a decrease of long-chain polyunsaturated fatty acids (LC-PUFA) levels. Amounts of LC-PUFA depend on the exogenous intake of their precursors [linoleic (LA) and α-linolenic acid (ALA)] and by rate of their metabolism, which is influenced by activities of enzymes, such as Δ6-desaturase (D6D, FADS2), D5D, FADS1, elongases (Elovl2, -5, 6).Altered activities of D5D/D6D were described in plenty of diseases, e.g. neuropsychiatric (depressive disorders, bipolar disorder, dementia), metabolic (obesity, metabolic syndrome, DM2) and cardiovascular diseases (arterial hypertension, coronary heart disease), inflammatory states and allergy (Crohns disease, atopic eczema) or some malignancies. Similar results were obtained in studies dealing with the associations between genotypes/haplotypes of FADS1/FADS2 and above mentioned diseases, or interactions of dietary intake of LA and ALA on one hand and of the polymorphisms of minor allels of FADS1/FADS2, usually characterized by lower activities, on the other hand.The decrease of the desaturases activities leads to decreased concentrations of products with concomitant increased concentrations of substrates. Associations of some SNP FADS with coronary heart disease, concentrations of plasma lipids, oxidative stress, glucose homeostasis, and inflammatory reaction, were described. Experimental studies on animal models and occurrence of rare diseases, associated with missing or with marked fall activities of D5D/D6D emphasized the significance of desaturases for healthy development of organism as well as for pathogenesis of some disease.

Induction of 1-acylglycerophosphocholine acyltransferase genes by fibrates in the liver of rats.

PubMed

Yamazaki, Tohru; Wakabayashi, Michiko; Ikeda, Erika; Tanaka, Shizuyo; Sakamoto, Takeshi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2012-01-01

The effect of fibrates (clofibric acid, bezafibrate and fenofibrate) on the gene expression and activity of 1-acylglycerophosphocholine acyltransferase (LPCAT) was investigated. The administration of 0.1% (w/w) clofibric acid, bezafibrate or fenofibrate in diet for 14 d to rats induced LPCAT activity in hepatic microsomes in the following order: fenofibrate>bezafibrate>clofibric acid. The LPCAT induced by fenofibrate preferred to arachidonoyl-CoA and linoleoyl-CoA to a greater extent than did LPCAT in control microsomes. The treatment with the fibrates resulted in upregulation of the relative expression of mRNAs encoding LPCAT3 and LPCAT4 in the following order: fenofibrate>bezafibrate>clofibric acid. The administration of fibrates did not change the expression of genes encoding either LPCAT1 or LPCAT2. The treatment with fibrates elevated relative levels of both mRNAs encoding Δ6 desaturase (Fads2) and Δ5 desaturase (Fads1) in the order of fenofibrate>bezafibrate>clofibric acid, and the extent of the increase in the level of Δ6 desaturase mRNA was greater than that of Δ5 desaturase. Fatty acid profile in hepatic phosphatidylcholine (PC) was significantly changed by the treatments with fibrates. These results suggest (i) that fibrates induce LPCAT activity in hepatic microsomes by elevating the expression of genes encoding LPCAT3 and LPCAT4, (ii) that the changes in fatty acid profile of hepatic PC are, in part, due to the elevated expression of two isoforms, LPCAT3 and LPCAT4, and (iii) that the ability of fibrates to induce these changes are in the order of fenofibrate>bezafibrate>clofibric acid.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

PubMed

Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Rowland, Gordon; Booker, Helen; You, Frank M; Cloutier, Sylvie

2013-10-01

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

Deviation of the neurosporaxanthin pathway towards beta-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene.

PubMed

Prado-Cabrero, Alfonso; Schaub, Patrick; Díaz-Sánchez, Violeta; Estrada, Alejandro F; Al-Babili, Salim; Avalos, Javier

2009-08-01

Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin-producing fungus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB-encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid-overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21-CarB to perform the fifth desaturation, as implied by the accumulation of gamma-carotene and beta-carotene, which arise through four-step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five-step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene-producing Escherichia coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate zeta-carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress▿

PubMed Central

Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca

2007-01-01

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ9 position. We expressed two sunflower (Helianthus annuus) oleate Δ12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp+ or Trp− strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains. PMID:17071783

Lipid accumulation, oxidative stress and immune-related molecules affected by tributyltin exposure in muscle tissues of rare minnow (Gobiocypris rarus).

PubMed

Zhang, Jiliang; Zhang, Chunnuan; Ma, Dongdong; Liu, Min; Huang, Shuntao

2017-12-01

Tributyltin (TBT) is reported to induce adipogenesis in fish, which might affect nutritional qualities and health status. Muscle tissues account for the majority of body mass, and have been described as a major site of fat deposition and an immunologically active organ. Therefore, the present study aims to evaluate whether chronic exposures of TBT, at environmental concentrations of 1, 10 and 100 ng/L, affects lipid accumulation, oxidative stress and immune status in muscle tissues of rare minnow (Gobiocypris rarus). After 60 d of exposure, TBT increased contents of total lipid, total cholesterol, triglyceride and fatty acids in muscle tissues. Interestingly, TBT exposure disrupted fatty acid composition and increased contents of unsaturated fatty acids (such as eicosapentaenoic acid and docosahexaenoic acid) in muscle tissues, which might be a response to preserve membrane functions from TBT exposure. Meanwhile, the concentrations of hepatic fatty acid desaturase 2 (Δ6-desaturase) and stearoyl-CoA desaturase (Δ9-desaturase) were increased after TBT exposure, which might contribute the increase of unsaturated fatty acids. Furthermore, TBT increased muscle lipid peroxidation products, antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), and the expression of immune-related molecules (tumor necrosis factor alpha, interleukin 1 beta and nuclear factor kappa B) in muscle tissues. The disruption of TBT on the lipid accumulation, oxidative stress and immune-toxic effects in muscle tissues of fish might reduce nutritional qualities, and affect growth and health status, which might pose a constant and serious threat to fish and result in economic loss in aquaculture. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

PubMed Central

Wagner, Gregory R.; Bhatt, Dhaval P.; O’Connell, Thomas M.; Thompson, J. Will; Dubois, Laura G.; Backos, Donald S.; Yang, Hao; Mitchell, Grant A.; Ilkayeva, Olga R.; Stevens, Robert D.; Grimsrud, Paul A.; Hirschey, Matthew D.

2017-01-01

SUMMARY The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism. PMID:28380375

Association of acylated ghrelin profiles with chronic inflammatory markers in overweight and obese postmenopausal women: a MONET study.

PubMed

St-Pierre, David H; Bastard, Jean-Philippe; Coderre, Lise; Brochu, Martin; Karelis, Antony D; Lavoie, Marie-Eve; Malita, Florin; Fontaine, Jonathan; Mignault, Diane; Cianflone, Katherine; Imbeault, Pascal; Doucet, Eric; Rabasa-Lhoret, Rémi

2007-10-01

Recent reports have suggested that the existence of associations between hormonal dysregulation and chronic upregulation of inflammatory markers, which may cause obesity-related disturbances. Thus, we examined whether acylated ghrelin (AcylG) and total ghrelin (TotG) levels could be associated with the following inflammatory markers: C-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), and soluble TNF receptor 1 (sTNF-R1). Cross-sectional study consisting of 50 overweight and obese postmenopausal women. AcylG and TotG levels were assessed at 0, 60, 160, 170, and 180 min of the euglycemic/hyperinsulinemic clamp (EHC). We evaluated insulin sensitivity, body composition, and blood lipid profiles as well as fasting concentrations of CRP, TNF-alpha, and sTNF-R1. In fasting conditions, sTNF-R1 was negatively correlated with AcylG (r = -0.48, P < 0.001) levels. In addition, AcylG/TotG was associated negatively with sTNF-R1 (r = -0.44, P = 0.002) and positively with TNF-alpha (r = 0.38, P = 0.009) values. During the EHC, TotG (at all time points) and AcylG (at 60 and 160 min) values were significantly decreased from fasting concentrations. AcylG maximal reduction and area under the curve (AUC) values were correlated to sTNF-R1 (r = -0.35, P = 0.02 and r = -0.34, P = 0.02, respectively). Meanwhile, the AcylG/TotG AUC ratio was associated negatively with sTNF-R1 (r = -0.29, P < 0.05) and positively with TNF-alpha (r = 0.36, P = 0.02). Following adjustments for total adiposity, sTNF-R1 remained correlated with fasting and maximal reduction AcylG values. Similarly, AcylG/TotG ratios remained significantly correlated with sTNF-R1 and TNF-alpha. Importantly, 23% of the variation in sTNF-R1 was independently predicted by fasting AcylG. These results are the first to suggest that both fasting and EHC-induced AcylG profiles are correlated with fasting values of sTNF-R1, a component of the TNF-alpha system. Thus, AcylG may act, at least in part, as one mediator of chronic inflammatory activity in human obesity.

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

PubMed

Turchetto-Zolet, Andreia C; Maraschin, Felipe S; de Morais, Guilherme L; Cagliari, Alexandro; Andrade, Cláudia M B; Margis-Pinheiro, Marcia; Margis, Rogerio

2011-09-20

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. 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. In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa. Overall, the data show that DGAT1 and DGAT2 are present in most eukaryotic organisms and belong to two different gene families. The phylogenetic and evolutionary analyses revealed that DGAT1 and DGAT2 evolved separately, with functional convergence, despite their wide molecular and structural divergence.

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. Overall, the data show that DGAT1 and DGAT2 are present in most eukaryotic organisms and belong to two different gene families. The phylogenetic and evolutionary analyses revealed that DGAT1 and DGAT2 evolved separately, with functional convergence, despite their wide molecular and structural divergence. PMID:21933415

Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope.

PubMed

Conde-Álvarez, Raquel; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Salvador-Bescós, Miriam; Bárcena-Varela, Marina; Aragón-Aranda, Beatriz; Martínez-Gómez, Estrella; Zúñiga-Ripa, Amaia; de Miguel, María J; Bartholomew, Toby Leigh; Hanniffy, Sean; Grilló, María-Jesús; Vences-Guzmán, Miguel Ángel; Bengoechea, José A; Arce-Gorvel, Vilma; Gorvel, Jean-Pierre; Moriyón, Ignacio; Iriarte, Maite

2017-01-01

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE , and lpxO , three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA , which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi . Free-lipid analysis revealed that lpxO corresponded to olsC , the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti , while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE , or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.

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

USDA-ARS?s Scientific Manuscript database

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...

40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

Code of Federal Regulations, 2013 CFR

2013-07-01

...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

Code of Federal Regulations, 2012 CFR

2012-07-01

...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

Code of Federal Regulations, 2014 CFR

2014-07-01

...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

Effects of two-months balanced diet in metabolically healthy obesity: lipid correlations with gender and BMI-related differences.

PubMed

Rondanelli, Mariangela; Klersy, Chaterine; Perna, Simone; Faliva, Milena Anna; Montorfano, Gigliola; Roderi, Paola; Colombo, Irma; Corsetto, Paola Antonia; Fioravanti, Marisa; Solerte, Sebastiano Bruno; Rizzo, Angela Maria

2015-10-29

Nowadays no researches has been performed on fatty acid profile (FA) and desaturase activity in metabolically healthy obesity (MHO). The aim of this study was to assessed gender and BMI-related difference in FA, estimated desaturase activities and the efficacy on metabolic changes produced by 2-months well-balance diet in MHO subjects. In 103 MHO subjects (30/73 M/F; age:42.2 ± 9.5) FA, estimated desaturase activity, body composition (by DXA), Body Mass Index (BMI), lipid profile, adipokines (leptin, adiponectin, grelin, glucagon-like peptide-1), insulin resistence (by Homestasis metabolic assessment), C-reactive proteine, Atherogenic index of plasma (AIP) and Body Shape Index (ABSI) have been assessed. Gender and BMI related difference have been evaluated and the efficacy produced by 2-months well-balance diet has been considered. At baseline, obese subjects, compared to overweight, show a significantly higher oleic (p <0.050), monounsaturated fatty acids (p <0.040), C18:0 delta-9 desaturase activity (D9D) (p <0.040) and lower linoleic acid (p <0.020), polyunsaturated fatty acids (p <0.020) and n-6 LCPUFA (p <0.010). Concerning gender-related difference, women show a significantly higher arachidonic acid (p <0.001), polyunsaturated fatty acids (p <0.001), n-6 LCPUFA (p <0.002), and lower monounsaturated fatty acids (p <0.001), D6D activity (p <0.030), C18:0 D9D (0.000) and C16:0 D9D (p <0.030). The 2-months diet was associated with a significantly increase in arachidonic acid (p = 0.007), eicosapentaenoic acid (p = 0.030), docosahexaenoic acid (p <0.001), long chain omega 3 polyunsaturated fatty acids (n-3 LCPUFA) (p <0.001), delta-5 desaturase activity (D5D) (p = 0.002), glucagon like peptide-1 (p <0.001) and a significant decrease in palmitoleic acid (p = <0.030), n-6/n-3 LCPUFA (p <0.001), insulin resistance (p = 0.006), leptin (p = 0.006), adiponectin (p <0.001), grelin (p = 0.030), CRP (p = 0.004), BMI (p <0.001) and android fat mass (p <0.001). The balanced diet intervention was effective in improving metabolic indices.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

PubMed

González-Mellado, Damián; von Wettstein-Knowles, Penny; Garcés, Rafael; Martínez-Force, Enrique

2010-05-01

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

Ghrelin enhancer, rikkunshito, improves postprandial gastric motor dysfunction in an experimental stress model.

PubMed

Harada, Y; Ro, S; Ochiai, M; Hayashi, K; Hosomi, E; Fujitsuka, N; Hattori, T; Yakabi, K

2015-08-01

Functional dyspepsia (FD) is one of the most common disorders of gastrointestinal (GI) diseases. However, no curable treatment is available for FD because the detailed mechanism of GI dysfunction in stressed conditions remains unclear. We aimed to clarify the association between endogenous acylated ghrelin signaling and gastric motor dysfunction and explore the possibility of a drug with ghrelin signal-enhancing action for FD treatment. Solid gastric emptying (GE) and plasma acylated ghrelin levels were evaluated in an urocortin1 (UCN1) -induced stress model. To clarify the role of acylated ghrelin on GI dysfunction in the model, exogenous acylated ghrelin, an endogenous ghrelin enhancer, rikkunshito, or an α2 -adrenergic receptor (AR) antagonist was administered. Postprandial motor function was investigated using a strain gauge force transducer in a free-moving condition. Exogenous acylated ghrelin supplementation restored UCN1-induced delayed GE. Alpha2 -AR antagonist and rikkunshito inhibited the reduction in plasma acylated ghrelin and GE in the stress model. The action of rikkunshito on delayed GE was blocked by co-administration of the ghrelin receptor antagonist. UCN1 decreased the amplitude of contraction in the antrum while increasing it in the duodenum. The motility index of the antrum but not the duodenum was significantly reduced by UCN1 treatment, which was improved by acylated ghrelin or rikkunshito. The UCN1-induced gastric motility dysfunction was mediated by abnormal acylated ghrelin dynamics. Supplementation of exogenous acylated ghrelin or enhancement of endogenous acylated ghrelin secretion by rikkunshito may be effective in treating functional GI disorders. © 2015 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.

Progress toward Understanding Protein S-acylation: Prospective in Plants

PubMed Central

Li, Yaxiao; Qi, Baoxiu

2017-01-01

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

Kinetic studies of the acylation of pig muscle–d-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism

PubMed Central

Harrigan, P. J.; Trentham, D. R.

1973-01-01

In the presence of NAD+ the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD+ and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H+ indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5–8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results. PMID:4360248

The Ghrelin/GOAT System Regulates Obesity-Induced Inflammation in Male Mice.

PubMed

Harvey, Rebecca E; Howard, Victor G; Lemus, Moyra B; Jois, Tara; Andrews, Zane B; Sleeman, Mark W

2017-07-01

Ghrelin plays a key role in appetite, energy homeostasis, and glucose regulation. Recent evidence suggests ghrelin suppresses inflammation in obesity; however, whether this is modulated by the acylated and/or des-acylated peptide is unclear. We used mice deficient in acylated ghrelin [ghrelin octanoyl-acyltransferase (GOAT) knockout (KO) mice], wild-type (WT) littermates, and C57BL/6 mice to examine the endogenous and exogenous effects of acyl and des-acyl ghrelin on inflammatory profiles under nonobese and obese conditions. We demonstrate that in the spleen, both ghrelin and GOAT are localized primarily in the red pulp. Importantly, in the thymus, ghrelin was predominantly localized to the medulla, whereas GOAT was found in the cortex, implying differing roles in T cell development. Acute exogenous treatment with acyl/des-acyl ghrelin suppressed macrophage numbers in spleen and thymus in obese mice, whereas only acyl ghrelin increased CD3+ T cells in the thymus in mice fed both chow and a high-fat-diet (HFD). Consistent with this result, macrophages were increased in the spleen of KO mice on a HFD. Whereas there was no difference in CD3+ T cells in the plasma, spleen, or thymus of WT vs KO mice, KO chow and HFD-fed mice displayed decreased leukocytes. Our results suggest that the acylation status affects the anti-inflammatory properties of ghrelin under chow and HFD conditions. Copyright © 2017 Endocrine Society.

Highly efficient and regioselective synthesis of dihydromyricetin esters by immobilized lipase.

PubMed

Li, Wei; Wu, Huan; Liu, Benguo; Hou, Xuedan; Wan, Duanji; Lou, Wenyong; Zhao, Jian

2015-04-10

Dihydromyricetin is the principle component of the Chinese herbal tea Teng-cha and a promising ingredient for functional food and nutraceuticals, but its low solubility limits its application potentials. This study explored enzymatic acylation of dihydromyricetin to improve its solubility in lipid systems. Acylation was achieved with several lipases with the synthesis of a major (>86%) product and a minor product. Isolation and purification of the products by preparative HPLC followed by LC-MS, (13)C NMR, (1)H NMR and 2 D-HSQC NMR analyses showed that the major product was a dihydromyricetin monoester with the acylation site at the 3-OH group of C ring. Quantum chemical calculations revealed that the 3-OH had the lowest antioxidant activity, and therefore acylation at this site was expected to have minimum impact on the antioxidant activity. Several factors, including solvent, acyl donor, enzyme origin, molar ratio of substrates and reaction temperature and time, exhibited significant effects on the initial rate, conversion yield and regioselectivity of the reaction. Acylation occurred only with vinyl acetate as the acyl donor, and highest conversion yields were achieved with immobilized Penicillium expansum lipase and Novozyme 435 with DMSO and acetonitrile being the best solvents. In general, the acylation results were found to be superior to previous reports on acylation of aglycone flavonoids with respects to conversion yield and regioselectivity. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2013-03-01

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

40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

Code of Federal Regulations, 2014 CFR

2014-07-01

...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

Code of Federal Regulations, 2013 CFR

2013-07-01

...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

Code of Federal Regulations, 2012 CFR

2012-07-01

...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

PubMed

Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

2013-01-01

Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

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

PubMed

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

2016-02-26

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

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

PubMed

Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11.

PubMed

Lagrange, Brice; Benaoudia, Sacha; Wallet, Pierre; Magnotti, Flora; Provost, Angelina; Michal, Fanny; Martin, Amandine; Di Lorenzo, Flaviana; Py, Bénédicte F; Molinaro, Antonio; Henry, Thomas

2018-01-16

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

PubMed

Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

1977-02-01

The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

PubMed

Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

2016-06-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. © 2016 American Society of Plant Biologists. All Rights Reserved.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

PubMed Central

Mei, Yu; Kernodle, Bliss M.; Hill, John H.

2016-01-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2014 CFR

2014-07-01

...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2013 CFR

2013-07-01

...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2012 CFR

2012-07-01

...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

40 CFR 721.10193 - 1-Butanaminium, N-(3-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts.

Code of Federal Regulations, 2011 CFR

2011-07-01

...-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts. 721.10193 Section 721.10193 Protection of...-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts. (a) Chemical substance and...-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts (PMN P-06-263, Chemical B; CAS No...

40 CFR 721.10174 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts.

Code of Federal Regulations, 2011 CFR

2011-07-01

...-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. 721.10174 Section 721.10174 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. (a) Chemical substance...-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts (PMN P-04-139...

Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: relationship with cholinesterase.

PubMed

Tham, Elaine; Liu, Jianhua; Innis, Sheila; Thompson, David; Gaylinn, Bruce D; Bogarin, Roberto; Haim, Alon; Thorner, Michael O; Chanoine, Jean-Pierre

2009-05-01

Acylated (octanoylated) ghrelin stimulates food intake and growth hormone secretion and is deacylated into desacyl ghrelin by butyrylcholinesterase. Acylated and desacyl ghrelin both promote adipogenesis. Ghrelin concentrations decrease with hyperglycemia and hyperinsulinism. We hypothesized that 1) acylated ghrelin increases during pregnancy, contributing positively to energy balance, but is lower in women with gestational diabetes and 2) butyrylcholinesterase activity is inversely correlated with acylated ghrelin concentrations. In a first group of subjects, using two-site sandwich ghrelin assays that specifically detect full-length forms, we investigated women with and without gestational diabetes (n = 14/group) during pregnancy and after delivery. We examined whether changes in ghrelin during a test meal were correlated with changes in pituitary growth hormone [assessed through calculation of the area under the curve (AUC) during the test meal]. In postpartum subjects, the percent of total ghrelin that is acylated was four to five times higher than previously observed using single antibody assays. During pregnancy, acylated ghrelin concentrations (mean +/- SE) were lower compared with the postpartum period throughout the meal (AUC 1.2 +/- 0.2 vs. 10.2 +/- 1.9 ng.ml(-1).90 min(-1), P < 0.001). In the postpartum, acylated ghrelin and growth hormone were positively correlated (r = 0.50, P = 0.007). Desacyl (but not acylated) ghrelin was increased in subjects with gestational diabetes during and after pregnancy (AUC 15.4 +/- 1.9 vs. 8.6 +/- 1.2 ng.ml(-1).90 min(-1), P = 0.005). In a second group of subjects (n = 13), acylated ghrelin was similarly suppressed during pregnancy. Circulating octanoate concentrations (3.1 +/- 0.5 vs. 4.5 +/- 0.6 microg/ml, P = 0.029) and cholinesterase activity (705 +/- 33 vs. 1,013 +/- 56 U/ml, P < 0.001) were lower during pregnancy compared with the postpartum period. In conclusion, acylated ghrelin markedly decreases during pregnancy, likely because of a decrease in the acylation process. Desacyl ghrelin increases in gestational diabetes, possibly reflecting resistance to the inhibitory effect of insulin on ghrelin secretion.

Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: relationship with cholinesterase

PubMed Central

Tham, Elaine; Liu, Jianhua; Innis, Sheila; Thompson, David; Gaylinn, Bruce D.; Bogarin, Roberto; Haim, Alon; Thorner, Michael O.; Chanoine, Jean-Pierre

2009-01-01

Acylated (octanoylated) ghrelin stimulates food intake and growth hormone secretion and is deacylated into desacyl ghrelin by butyrylcholinesterase. Acylated and desacyl ghrelin both promote adipogenesis. Ghrelin concentrations decrease with hyperglycemia and hyperinsulinism. We hypothesized that 1) acylated ghrelin increases during pregnancy, contributing positively to energy balance, but is lower in women with gestational diabetes and 2) butyrylcholinesterase activity is inversely correlated with acylated ghrelin concentrations. In a first group of subjects, using two-site sandwich ghrelin assays that specifically detect full-length forms, we investigated women with and without gestational diabetes (n = 14/group) during pregnancy and after delivery. We examined whether changes in ghrelin during a test meal were correlated with changes in pituitary growth hormone [assessed through calculation of the area under the curve (AUC) during the test meal]. In postpartum subjects, the percent of total ghrelin that is acylated was four to five times higher than previously observed using single antibody assays. During pregnancy, acylated ghrelin concentrations (mean ± SE) were lower compared with the postpartum period throughout the meal (AUC 1.2 ± 0.2 vs. 10.2 ± 1.9 ng·ml−1·90 min−1, P < 0.001). In the postpartum, acylated ghrelin and growth hormone were positively correlated (r = 0.50, P = 0.007). Desacyl (but not acylated) ghrelin was increased in subjects with gestational diabetes during and after pregnancy (AUC 15.4 ± 1.9 vs. 8.6 ± 1.2 ng·ml−1·90 min−1, P = 0.005). In a second group of subjects (n = 13), acylated ghrelin was similarly suppressed during pregnancy. Circulating octanoate concentrations (3.1 ± 0.5 vs. 4.5 ± 0.6 μg/ml, P = 0.029) and cholinesterase activity (705 ± 33 vs. 1,013 ± 56 U/ml, P < 0.001) were lower during pregnancy compared with the postpartum period. In conclusion, acylated ghrelin markedly decreases during pregnancy, likely because of a decrease in the acylation process. Desacyl ghrelin increases in gestational diabetes, possibly reflecting resistance to the inhibitory effect of insulin on ghrelin secretion. PMID:19240252

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

PubMed

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

2018-06-01

Overfeeding is a strategy for evaluating the effects of excess energy intake. In this secondary analysis we tested the possibility that different levels of dietary protein might differentially modify the response of fatty acyl-carnitines to overfeeding. Twenty-three healthy adult men and women were overfed by 40% for 8 weeks while in-patients with diets containing 5% (LPD), 15% (NPD) or 25% (HPD) protein. Plasma fatty acyl-carnitines were measured by gas chromatography/mass spectrometry (GC/MS) at baseline and after 8 weeks of overfeeding. Measurements included: body composition by DXA, energy expenditure by ventilated hood and doubly-labeled water, fat cell size from subcutaneous fat biopsies, and fat distribution by CT scan. Analysis was done on 5 groups of fatty acyl-carnitines identified by principal components analysis and 6 individual short-chain fatty acyl carnitines. Higher protein intake was associated with significantly lower 8 week levels of medium chain fatty acids and C2, C4-OH and C 6:1, but higher values of C3 and C5:1 acyl-carnitines derived from essential amino acids. In contrast energy and fat intake were only weakly related to changes in fatty acyl-carnitines. A decease or smaller rise in 8 week medium chain acyl-carnitines was associated with an increase in sleeping energy expenditure (P = 0.0004), and fat free mass (P < 0.0001) and a decrease in free fatty acid concentrations (FFA) (P = 0.0067). In contrast changes in short-chain fatty acyl-carnitines were related to changes in resting energy expenditure (P = 0.0026), and fat free mass (P = 0.0007), and C4-OH was positively related to FFA (P = 0006). Protein intake was the major factor influencing changes in fatty acyl carnitines during overfeeding with higher values of most acyl-fatty acids on the low protein diet. The association of dietary protein and fat intake may explain the changes in energy expenditure and metabolic variables resulting in the observed patterns of fatty acyl carnitines. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-02-01

Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels. The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups. Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst. Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P < .005) and acyl-ghrelin (14.7 ± 2.3 vs 27.8 ± 3.9 pg/mL, P < .05) levels were significantly lower in older adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P < .01). The ghrelin/GH association was more than 3-fold lower in the older group compared with the young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P < .001). These results provide further evidence of an age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.

PubMed

Hara, Yoshinori; Seki, Masahide; Matsuoka, Satoshi; Hara, Hiroshi; Yamashita, Atsushi; Matsumoto, Kouji

2008-12-01

The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis1

PubMed Central

Jasieniecka-Gazarkiewicz, Katarzyna; Lager, Ida; Carlsson, Anders S.; Gutbrod, Katharina; Peisker, Helga; Dörmann, Peter; Stymne, Sten; Banaś, Antoni

2017-01-01

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE. The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC. The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells. PMID:28408542

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.

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

PubMed

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

2013-01-17

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

Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides

PubMed Central

2015-01-01

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis. PMID:24617596

Total and acylated ghrelin in liver cirrhosis: correlation with clinical and nutritional status.

PubMed

El-Shehaby, Amal M; Obaia, Eman M; Alwakil, Sahar S; Hiekal, Ahmed A

2010-07-01

The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. The aim of the present study was to compare acylated and total ghrelin concentration in patients with liver cirrhosis and to investigate the possible relationship between ghrelin and clinical and nutritional parameters. Sixty patients with viral liver cirrhosis who did not have hepatocellular carcinoma or acute infections were studied. Twenty healthy volunteers were recruited after matching for age, gender, and body mass index with the patients and served as controls. Fasting levels of total, acylated ghrelin, leptin, TNF-alpha and insulin were measured in all subjects, in addition, clinical and nutrition parameters were assessed. In cirrhotic patients, plasma levels of both acylated and total ghrelin were significantly higher than those in the controls. The mean plasma acylated ghrelin levels were significantly higher in Child C cirrhosis compared to Child A and B. Ghrelin (total and acylated) were negatively correlated with leptin in cirrhotic patients confirming the fact that leptin acts as a physiological counterpart of ghrelin. Nutritional and metabolic abnormalities in cirrhotic patients may be dependent on the changes in the ghrelin/leptin systems, mainly the acylated form of ghrelin.

Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2[C][W

PubMed Central

Sanjaya; Miller, Rachel; Durrett, Timothy P.; Kosma, Dylan K.; Lydic, Todd A.; Muthan, Bagyalakshmi; Koo, Abraham J.K.; Bukhman, Yury V.; Reid, Gavin E.; Howe, Gregg A.; Ohlrogge, John; Benning, Christoph

2013-01-01

Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves. PMID:23417035

Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

PubMed

Kite, Geoffrey C; Larsson, Sonny; Veitch, Nigel C; Porter, Elaine A; Ding, Ning; Simmonds, Monique S J

2013-04-10

LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Miho; Nakahara, Keiko; Goto, Shintaro

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

Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.

PubMed

Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung

2015-06-01

N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.

Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

PubMed

Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J

2018-02-28

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

Administration of exogenous acylated ghrelin or rikkunshito, an endogenous ghrelin enhancer, improves the decrease in postprandial gastric motility in an acute restraint stress mouse model

PubMed Central

Nahata, M; Saegusa, Y; Sadakane, C; Yamada, C; Nakagawa, K; Okubo, N; Ohnishi, S; Hattori, T; Sakamoto, N; Takeda, H

2014-01-01

Background Physical or psychological stress causes functional disorders in the upper gastrointestinal tract. This study aims to elucidate the ameliorating effect of exogenous acylated ghrelin or rikkunshito, a Kampo medicine which acts as a ghrelin enhancer, on gastric dysfunction during acute restraint stress in mice. Methods Fasted and postprandial motor function of the gastric antrum was wirelessly measured using a strain gauge force transducer and solid gastric emptying was detected in mice exposed to restraint stress. Plasma corticosterone and ghrelin levels were also measured. To clarify the role of ghrelin on gastrointestinal dysfunction in mice exposed to stress, exogenous acylated ghrelin or rikkunshito was administered, then the mice were subjected to restraint stress. Key Results Mice exposed to restraint stress for 60 min exhibited delayed gastric emptying and increased plasma corticosterone levels. Gastric motility was decreased in mice exposed to restraint stress in both fasting and postprandial states. Restraint stress did not cause any change in plasma acylated ghrelin levels, but it significantly increased the plasma des-acyl ghrelin levels. Administration of acylated ghrelin or rikkunshito improved the restraint stress-induced delayed gastric emptying and decreased antral motility. Ameliorating effects of rikkunshito on stress-induced gastric dysfunction were abolished by simultaneous administration of a ghrelin receptor antagonist. Conclusions & Inferences Plasma acylated/des-acyl ghrelin imbalance was observed in acute restraint stress. Supplementation of exogenous acylated ghrelin or enhancement of endogenous ghrelin signaling may be useful in the treatment of decreased gastric function caused by stress. PMID:24684160

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko

2014-03-07

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

Stearoyl-CoA Desaturase and its Relation to High-Carbohydrate Diets and Obesity

PubMed Central

Flowers, Matthew T.; Ntambi, James M.

2009-01-01

Obesity is currently a worldwide epidemic and public health burden that increases the risk for developing insulin resistance and several chronic diseases such as diabetes, cardiovascular diseases and non-alcoholic fatty liver disease. The multifactorial causes of obesity include several genetic, dietary and lifestyle variables that together result in an imbalance between energy intake and energy expenditure. Dietary approaches to limit fat intake are commonly prescribed to achieve the hypocaloric conditions necessary for weight loss. But dietary fat restriction is often accompanied by increased carbohydrate intake, which can dramatically increase endogenous fatty acid synthesis depending upon carbohydrate composition. Since both dietary and endogenously synthesized fatty acids contribute to the whole-body fatty acid pool, obesity can therefore result from excessive fat or carbohydrate consumption. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a delta-9 fatty acid desaturase that converts saturated fatty acids into monounsaturated fatty acids (MUFA) and this activity is elevated by dietary carbohydrate. Mice lacking Scd1 are protected from obesity and insulin resistance and are characterized by decreased fatty acid synthesis and increased fatty acid oxidation. In this review, we address the association of high-carbohydrate diets with increased SCD activity and summarize the current literature on the subject of SCD1 and body weight regulation. PMID:19166967

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cripps, C.

De novo biosynthesis of linoleic acid was demonstrated in vivo in 8 of 32 insect species examined, including both holometabolous and hemimetabolous species. The incorporation of (1-{sup 14}C) acetate into linoleic acid was demonstrated by radio-gas-liquid chromatography (radio-GLC), and in selected species by radio-high-performance liquid chromatography, silver nitrate thin-layer chromatography, radio-GLC and GLC linked to mass spectrometry of ozonolysis products. Analysis of the ozonolysis products clearly demonstrated that the entire molecule was labeled and that synthesis of linoleate was de novo from acetate. The in vivo incorporation of (1-{sup 14}C)acetate into lipid was monitored during the final three stadia ofmore » both male and female house crickets, Acheta domesticus. Characterization of the {Delta}{sup 12}-desaturase showed that, in the house cricket, this enzyme is microsomal and requires a reduced pyridine dinucleotide as a cofactor, with NADPH the preferred electron donor. The optimal substrate concentration for desaturation is about 40 uM. Addition of the microsomal supernatant, MgCl{sub 2} or ATP did not enhance activity. The form of the substrate for the desaturase, oleic acid, was determined and appears to be a CoA derivative, as is true for most animal desaturases, rather than a complex lipid, as it is in plants.« less

cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo.

PubMed

Hsieh, S L; Liu, R W; Wu, C H; Cheng, W T; Kuo, Ching-Ming

2003-12-01

A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development. Copyright 2003 Wiley-Liss, Inc.

On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase

PubMed Central

Gemmecker, Sandra; Poussin-Courmontagne, Pierre; Mailliot, Justine; McEwen, Alastair G.; Ghisla, Sandro; Al-Babili, Salim; Cavarelli, Jean; Beyer, Peter

2012-01-01

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C40 hydrocarbon substrate. PMID:22745782

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

PubMed Central

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

1995-01-01

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

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

PubMed

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

1995-01-01

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

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

PubMed

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

2011-04-01

The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar range of non-acylated flavonoids. Copyright © 2011 Elsevier Ltd. All rights reserved.

SMART – Sunflower Mutant population And Reverse genetic Tool for crop improvement

PubMed Central

2013-01-01

Background Sunflower (Helianthus annuus L.) is an important oilseed crop grown widely in various areas of the world. Classical genetic studies have been extensively undertaken for the improvement of this particular oilseed crop. Pertaining to this endeavor, we developed a “chemically induced mutated genetic resource for detecting SNP by TILLING” in sunflower to create new traits. Results To optimize the EMS mutagenesis, we first conducted a “kill curve” analysis with a range of EMS dose from 0.5% to 3%. Based on the observed germination rate, a 50% survival rate i.e. LD50, treatment with 0.6% EMS for 8 hours was chosen to generate 5,000 M2 populations, out of which, 4,763 M3 plants with fertile seed set. Phenotypic characterization of the 5,000 M2 mutagenised lines were undertaken to assess the mutagenesis quality and to identify traits of interest. In the M2 population, about 1.1% of the plants showed phenotypic variations. The sunflower TILLING platform was setup using Endo-1-nuclease as mismatch detection system coupled with an eight fold DNA pooling strategy. As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations. Conclusion Based on the TILLING of FatA and SAD genes, we calculated the overall mutation rate to one mutation every 480 kb, similar to other report for this crop so far. As sunflower is a plant model for seed oil biosynthesis, we anticipate that the developed genetic resource will be a useful tool to identify novel traits for sunflower crop improvement. PMID:23496999

Identification of candidate genes from the SAD gene family in cotton for determination of cottonseed oil composition.

PubMed

Shang, Xiaoguang; Cheng, Chaoze; Ding, Jian; Guo, Wangzhen

2017-02-01

Cotton is an economically important crop grown for natural fiber and seed oil production. Cottonseed oil ranks third after soybean oil and colza oil in terms of edible oilseed tonnage worldwide. The fatty acid composition of cottonseed oil determines its industrial application and nutritional values. However, little progress has been made in understanding cottonseed oil biogenesis. Stearoyl-acyl carrier protein desaturase (SAD), the only known enzyme to convert saturated fatty acids into unsaturated fatty acids in plants, plays key roles in determining the fatty acid composition of cottonseed oil. In this study, we identified 9, 9, 18 and 19 SAD genes in the genomes of four sequenced cotton species: diploid Gossypium raimondii (D 5 ), G. arboreum (A 2 ), tetraploid G. hirsutum acc. TM-1 (AD 1 ) and G. barbadense cv. Xinhai21 (AD 2 ), respectively. Bioinformatic and phylogenetic analyses revealed that cotton SADs can be classified into two classes. Expression patterns showed developmental and spatial regulation of SADs in cotton. GhSAD2 and GhSAD4 were preferentially expressed in developing ovules 20-35 days post-anthesis, and significantly different expression patterns were found between high-oil and low-oil cotton cultivars, implying these two genes could be involved in cottonseed oil biogenesis. Association analysis further confirmed that GhSAD4-At expression was closely related to the oleic acid (O) content, linoleic acid (L) content and O/L value in cottonseed, implying GhSAD4 plays an important role in cottonseed oil composition. This study brings new perspectives for integrated genome-wide identification of SADs in cotton and provides references for the genetic improvement of cottonseed oil.

The Natural Diyne-Furan Fatty Acid EV-086 Is an Inhibitor of Fungal Delta-9 Fatty Acid Desaturation with Efficacy in a Model of Skin Dermatophytosis

PubMed Central

Diefenbacher, Melanie; Greve, Katrine B. V.; Brianza, Federico; Folly, Christophe; Heider, Harald; Lone, Museer A.; Long, Lisa; Meyer, Jean-Philippe; Roussel, Patrick; Ghannoum, Mahmoud A.; Schneiter, Roger; Sorensen, Alexandra S.

2014-01-01

Human fungal infections represent a therapeutic challenge. Although effective strategies for treatment are available, resistance is spreading, and many therapies have unacceptable side effects. A clear need for novel antifungal targets and molecules is thus emerging. Here, we present the identification and characterization of the plant-derived diyne-furan fatty acid EV-086 as a novel antifungal compound. EV-086 has potent and broad-spectrum activity in vitro against Candida, Aspergillus, and Trichophyton spp., whereas activities against bacteria and human cell lines are very low. Chemical-genetic profiling of Saccharomyces cerevisiae deletion mutants identified lipid metabolic processes and organelle organization and biogenesis as targets of EV-086. Pathway modeling suggested that EV-086 inhibits delta-9 fatty acid desaturation, an essential process in S. cerevisiae, depending on the delta-9 fatty acid desaturase OLE1. Delta-9 unsaturated fatty acids—but not saturated fatty acids—antagonized the EV-086-mediated growth inhibition, and transcription of the OLE1 gene was strongly upregulated in the presence of EV-086. EV-086 increased the ratio of saturated to unsaturated free fatty acids and phosphatidylethanolamine fatty acyl chains, respectively. Furthermore, EV-086 was rapidly taken up into the lipid fraction of the cell and incorporated into phospholipids. Together, these findings demonstrate that EV-086 is an inhibitor of delta-9 fatty acid desaturation and that the mechanism of inhibition might involve an EV-086–phospholipid. Finally, EV-086 showed efficacy in a guinea pig skin dermatophytosis model of topical Trichophyton infection, which demonstrates that delta-9 fatty acid desaturation is a valid antifungal target, at least for dermatophytoses. PMID:24189258

The natural diyne-furan fatty acid EV-086 is an inhibitor of fungal delta-9 fatty acid desaturation with efficacy in a model of skin dermatophytosis.

PubMed

Knechtle, Philipp; Diefenbacher, Melanie; Greve, Katrine B V; Brianza, Federico; Folly, Christophe; Heider, Harald; Lone, Museer A; Long, Lisa; Meyer, Jean-Philippe; Roussel, Patrick; Ghannoum, Mahmoud A; Schneiter, Roger; Sorensen, Alexandra S

2014-01-01

Human fungal infections represent a therapeutic challenge. Although effective strategies for treatment are available, resistance is spreading, and many therapies have unacceptable side effects. A clear need for novel antifungal targets and molecules is thus emerging. Here, we present the identification and characterization of the plant-derived diyne-furan fatty acid EV-086 as a novel antifungal compound. EV-086 has potent and broad-spectrum activity in vitro against Candida, Aspergillus, and Trichophyton spp., whereas activities against bacteria and human cell lines are very low. Chemical-genetic profiling of Saccharomyces cerevisiae deletion mutants identified lipid metabolic processes and organelle organization and biogenesis as targets of EV-086. Pathway modeling suggested that EV-086 inhibits delta-9 fatty acid desaturation, an essential process in S. cerevisiae, depending on the delta-9 fatty acid desaturase OLE1. Delta-9 unsaturated fatty acids-but not saturated fatty acids-antagonized the EV-086-mediated growth inhibition, and transcription of the OLE1 gene was strongly upregulated in the presence of EV-086. EV-086 increased the ratio of saturated to unsaturated free fatty acids and phosphatidylethanolamine fatty acyl chains, respectively. Furthermore, EV-086 was rapidly taken up into the lipid fraction of the cell and incorporated into phospholipids. Together, these findings demonstrate that EV-086 is an inhibitor of delta-9 fatty acid desaturation and that the mechanism of inhibition might involve an EV-086-phospholipid. Finally, EV-086 showed efficacy in a guinea pig skin dermatophytosis model of topical Trichophyton infection, which demonstrates that delta-9 fatty acid desaturation is a valid antifungal target, at least for dermatophytoses.

Reducing Isozyme Competition Increases Target Fatty Acid Accumulation in Seed Triacylglycerols of Transgenic Arabidopsis1[OPEN

PubMed Central

van Erp, Harrie; Shockey, Jay; Zhang, Meng; Adhikari, Neil D.; Browse, John

2015-01-01

One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies. PMID:25739701

Two Pathways of Sphingolipid Biosynthesis Are Separated in the Yeast Pichia pastoris*

PubMed Central

Ternes, Philipp; Wobbe, Tobias; Schwarz, Marnie; Albrecht, Sandra; Feussner, Kirstin; Riezman, Isabelle; Cregg, James M.; Heinz, Ernst; Riezman, Howard; Feussner, Ivo; Warnecke, Dirk

2011-01-01

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C16/C18 acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis. PMID:21303904

Cognitive function in adolescence: testing for interactions between breast-feeding and FADS2 polymorphisms.

PubMed

Martin, Nicolas W; Benyamin, Beben; Hansell, Narelle K; Montgomery, Grant W; Martin, Nicholas G; Wright, Margaret J; Bates, Timothy C

2011-01-01

Breast-fed C-allele carriers of the rs174575 single nucleotide polymorphism in the fatty acyl desaturase 2 (FADS2) gene have been reported to show a 6.4 to 7 IQ point advantage over formula-fed C-allele carriers, with no effect of breast-feeding in GG carriers. An Australian sample was examined to determine if an interaction between breast-feeding and the rs174575 single nucleotide polymorphism had any effect on IQ. This hypothesis was tested in more than 700 families of adolescent twins assessed for IQ and breast-feeding, birth weight, and FADS2 polymorphisms, and parental socioeconomic status and education, and maternal FADS2 status. No significant evidence for a moderating effect on IQ of rs174575 C-carrier status and breast-feeding was found, and there no effects of maternal FADS2 status on offspring IQ. In addition, no main effects of any FADS2 polymorphisms on IQ were found when the genotype was kept as two-homozygote and one-heterozygote categories and indeed no evidence for effects of breast-feeding on IQ scores after controlling for parental socioeconomic status and education. The investigation was extended to two additional FADS2 polymorphisms (rs1535 and rs174583), but again, although these polymorphisms code alleles affecting fatty acid metabolism, no main or interaction effects were found on IQ. These results support the view that apparent effects of breast-feeding on IQ reflect differential likelihood of breast-feeding as a function of parental education and did not support the predicted interaction effect of FADS2 and breast-feeding on IQ. Copyright © 2011 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.

Milk fatty acid unsaturation: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1).

PubMed

Schennink, A; Heck, J M L; Bovenhuis, H; Visker, M H P W; van Valenberg, H J F; van Arendonk, J A M

2008-05-01

With regard to human health aspects of milk fat, increasing the amount of unsaturated fatty acids in milk is an important selection objective. The cow's diet has an influence on the degree of unsaturation, but literature suggests that genetics also plays a role. To estimate genetic variation in milk fatty acid unsaturation indices, milk fatty acid composition of 1,933 Dutch Holstein Friesian heifers was measured and unsaturation indices were calculated. An unsaturation index represents the concentration of the unsaturated product proportional to the sum of the unsaturated product and the saturated substrate. Intraherd heritabilities were moderate, ranging from 0.23 +/- 0.07 for conjugated linoleic acid (CLA) index to 0.46 +/- 0.09 for C16 index. We genotyped the cows for the SCD1 A293V and DGAT1 K232A polymorphisms, which are known to alter milk fatty acid composition. Both genes explain part of the genetic variation in unsaturation indices. The SCD1 V allele is associated with lower C10, C12, and C14 indices, and with higher C16, C18, and CLA indices in comparison to the SCD1 A allele, with no differences in total unsaturation index. In comparison to the DGAT1 K allele, the DGAT1 A allele is associated with lower C10, C12, C14, and C16 indices and with higher C18, CLA, and total indices. We conclude that selective breeding can contribute to higher unsaturation indices, and that selective breeding can capitalize on genotypic information of both the SCD1 A293V and the DGAT1 K232A polymorphism.

Transcriptional Differences between Diapausing and Non-Diapausing D. montana Females Reared under the Same Photoperiod and Temperature

PubMed Central

Kankare, Maaria; Parker, Darren J.; Merisalo, Mikko; Salminen, Tiina S.; Hoikkala, Anneli

2016-01-01

Background A wide range of insects living at higher latitudes enter diapause at the end of the warm season, which increases their chances of survival through harsh winter conditions. In this study we used RNA sequencing to identify genes involved in adult reproductive diapause in a northern fly species, Drosophila montana. Both diapausing and non-diapausing flies were reared under a critical day length and temperature, where about half of the emerging females enter diapause enabling us to eliminate the effects of varying environmental conditions on gene expression patterns of the two types of female flies. Results RNA sequencing revealed large differences between gene expression patterns of diapausing and non-diapausing females, especially in genes involved with metabolism, fatty acid biosynthesis, and metal and nucleotide binding. Differently expressed genes included several gene groups, including myosin, actin and cytochromeP450 genes, which have been previously associated with diapause. This study also identified new candidate genes, including some involved in cuticular hydrocarbon synthesis or regulation (desat1 and desat2), and acyl-CoA Δ11-desaturase activity (CG9747), and few odorant-binding protein genes (e.g. Obp44A). Also, several transposable elements (TEs) showed differential expression between the two female groups motivating future research on their roles in diapause. Conclusions Our results demonstrate that the adult reproductive diapause in D. montana involves changes in the expression level of a variety of genes involved in key processes (e.g. metabolism and fatty acid biosynthesis) which help diapausing females to cope with overwintering. This is consistent with the view that diapause is a complex adaptive phenotype where not only sexual maturation is arrested, but also changes in adult physiology are required in order to survive over the winter. PMID:27571415

Dietary DHA/EPA ratio affected tissue fatty acid profiles, antioxidant capacity, hematological characteristics and expression of lipid-related genes but not growth in juvenile black seabream (Acanthopagrus schlegelii)

PubMed Central

Monroig, Óscar; Lu, You; Yuan, Ye; Li, Yi; Ding, Liyun; Tocher, Douglas R.; Zhou, Qicun

2017-01-01

An 8-week feeding trial was conducted to investigate the effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth performance, fatty acid profiles, antioxidant capacity, hematological characteristics and expression of some lipid metabolism related genes of juvenile black seabream (Acanthopagrus schlegelii) of initial weight 9.47 ± 0.03 g. Five isonitrogenous and isolipidic diets (45% crude protein and 14% crude lipid) were formulated to contain graded DHA/EPA ratios of 0.65, 1.16, 1.60, 2.03 and 2.67. There were no differences in growth performance and feed utilization among treatments. Fish fed higher DHA/EPA ratios had higher malondialdehyde (MDA) contents in serum than lower ratios. Serum triacylglycerol (TAG) content was significantly higher in fish fed the lowest DHA/EPA ratio. Tissue fatty acid profiles reflected the diets despite down-regulation of LC-PUFA biosynthesis genes, fatty acyl desaturase 2 (fads2) and elongase of very long-chain fatty acids 5 (elovl5), by high DHA/EPA ratios. Expression of acetyl-CoA carboxylase alpha (accα) and carnitine palmitoyl transferase 1A (cpt1a) were up-regulated by high DHA/EPA ratio, whereas sterol regulatory element-binding protein-1 (srebp-1) and hormone-sensitive lipase (hsl) were down-regulated. Fatty acid synthase (fas), 6-phosphogluconate dehydrogenase (6pgd) and peroxisome proliferator-activated receptor alpha (pparα) showed highest expression in fish fed intermediate (1.16) DHA/EPA ratio. Overall, this study indicated that dietary DHA/EPA ratio affected fatty acid profiles and significantly influenced lipid metabolism including LC-PUFA biosynthesis and other anabolic and catabolic pathways, and also had impacts on antioxidant capacity and hematological characteristics. PMID:28430821

Dietary DHA/EPA ratio affected tissue fatty acid profiles, antioxidant capacity, hematological characteristics and expression of lipid-related genes but not growth in juvenile black seabream (Acanthopagrus schlegelii).

PubMed

Jin, Min; Monroig, Óscar; Lu, You; Yuan, Ye; Li, Yi; Ding, Liyun; Tocher, Douglas R; Zhou, Qicun

2017-01-01

An 8-week feeding trial was conducted to investigate the effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth performance, fatty acid profiles, antioxidant capacity, hematological characteristics and expression of some lipid metabolism related genes of juvenile black seabream (Acanthopagrus schlegelii) of initial weight 9.47 ± 0.03 g. Five isonitrogenous and isolipidic diets (45% crude protein and 14% crude lipid) were formulated to contain graded DHA/EPA ratios of 0.65, 1.16, 1.60, 2.03 and 2.67. There were no differences in growth performance and feed utilization among treatments. Fish fed higher DHA/EPA ratios had higher malondialdehyde (MDA) contents in serum than lower ratios. Serum triacylglycerol (TAG) content was significantly higher in fish fed the lowest DHA/EPA ratio. Tissue fatty acid profiles reflected the diets despite down-regulation of LC-PUFA biosynthesis genes, fatty acyl desaturase 2 (fads2) and elongase of very long-chain fatty acids 5 (elovl5), by high DHA/EPA ratios. Expression of acetyl-CoA carboxylase alpha (accα) and carnitine palmitoyl transferase 1A (cpt1a) were up-regulated by high DHA/EPA ratio, whereas sterol regulatory element-binding protein-1 (srebp-1) and hormone-sensitive lipase (hsl) were down-regulated. Fatty acid synthase (fas), 6-phosphogluconate dehydrogenase (6pgd) and peroxisome proliferator-activated receptor alpha (pparα) showed highest expression in fish fed intermediate (1.16) DHA/EPA ratio. Overall, this study indicated that dietary DHA/EPA ratio affected fatty acid profiles and significantly influenced lipid metabolism including LC-PUFA biosynthesis and other anabolic and catabolic pathways, and also had impacts on antioxidant capacity and hematological characteristics.

Modeling the two-locus architecture of divergent pollinator adaptation: how variation in SAD paralogs affects fitness and evolutionary divergence in sexually deceptive orchids.

PubMed

Xu, Shuqing; Schlüter, Philipp M

2015-01-01

Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n-alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species-specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl-acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator-mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two-locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator-mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator-driven ecological speciation.

Occurrence and biological activity of palmitoleic acid isomers in phagocytic cells.

PubMed

Astudillo, Alma M; Meana, Clara; Guijas, Carlos; Pereira, Laura; Lebrero, Patricia; Balboa, María A; Balsinde, Jesús

2018-02-01

Recent studies have highlighted the role of palmitoleic acid [16:1 n-7 ( cis -9-hexadecenoic acid)] as a lipid hormone that coordinates cross-talk between liver and adipose tissue and exerts anti-inflammatory protective effects on hepatic steatosis and insulin signaling in murine models of metabolic disease. More recently, a 16:1 n-7 isomer, cis -7-hexadecenoic acid (16:1 n-9 ), that also possesses marked anti-inflammatory effects, has been described in human circulating monocytes and monocyte-derived macrophages. By using gas chromatographic/mass spectrometric analyses of dimethyl disulfide derivatives of fatty acyl methyl esters, we describe in this study the presence of a third 16:1 isomer, sapienic acid [16:1 n-10 (6- cis -hexadecenoic acid)], in phagocytic cells. Cellular levels of 16:1 n-10 appear to depend not only on the cellular content of linoleic acid, but also on the expression level of fatty acid desaturase 2, thus revealing a complex regulation both at the enzyme level, via fatty acid substrate competition, and directly at the gene level. However, unlike 16:1 n-7 and 16:1 n-9 , 16:1 n-10 levels are not regulated by the activation state of the cell. Moreover, while 16:1 n-7 and 16:1 n-9 manifest strong anti-inflammatory activity when added to the cells at low concentrations (10 μM), notably higher concentrations of 16:1 n-10 are required to observe a comparable effect. Collectively, these results suggest the presence in phagocytic cells of an unexpected variety of 16:1 isomers, which can be distinguished on the basis of their biological activity and cellular regulation. Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing.

PubMed

Singh, Monika; Bhoge, Rajesh K; Randhawa, Gurinderjit

2018-04-20

Background : Confirming the integrity of seed samples in powdered form is important priorto conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. Objective : A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase ( Sad1 ) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). Methods : The assays were performed at a constant temperature of 63°C for 30 min for visual LAMP and 62ºC for 40 min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR ® Green or detected as real-time amplification curves. Results : Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. Conclusions : The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. Highlights : LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

Cloning and Characterization of Lxr and Srebp1, and Their Potential Roles in Regulation of LC-PUFA Biosynthesis in Rabbitfish Siganus canaliculatus.

PubMed

Zhang, Qinghao; You, Cuihong; Liu, Fang; Zhu, Wendi; Wang, Shuqi; Xie, Dizhi; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

2016-09-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability to biosynthesize C20-22 long-chain polyunsaturated fatty acid (LC-PUFA) from C18 PUFA precursors, which is generally absent or low in marine teleosts. Thus, understanding the molecular basis of LC-PUFA biosynthesis in rabbitfish will contribute to efforts aimed at optimizing LC-PUFA biosynthesis in teleosts, especially marine species. In the present study, the importance of the transcription factors liver X receptor (Lxr) and sterol regulatory element-binding protein 1 (Srebp1) in regulation of LC-PUFA biosynthesis in rabbitfish was investigated. First, full-length cDNA of Lxr and Srebp1 were cloned and characterized. The Lxr mRNA displayed a ubiquitous tissue expression pattern while Srebp1 was highly expressed in eyes, brain and intestine. In rabbitfish primary hepatocytes treated with Lxr agonist T0901317, the expression of Lxr and Srebp1 was activated, accompanied by elevated mRNA levels of Δ4 and Δ6/Δ5 fatty acyl desaturase (Fad), key enzymes of LC-PUFA biosynthesis, as well as peroxisome proliferator-activated receptor γ (PPARγ). In addition, Srebp1 displayed higher expression levels in liver of rabbitfish fed a vegetable oil diet or reared at 10 ppt salinity, which were conditions reported to increase the liver expression of Δ4 and Δ6/Δ5 Fad and LC-PUFA biosynthetic ability, than fish fed a fish oil diet or reared at 32 ppt, respectively. These results suggested that Lxr and Srebp1 are involved in regulation of LC-PUFA biosynthesis probably by promoting the expression of two Fad in rabbitfish liver, which, to our knowledge, is the first report in marine teleosts.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.

PubMed

Diomandé, Sara Esther; Nguyen-the, Christophe; Abee, Tjakko; Tempelaars, Marcel H; Broussolle, Véronique; Brillard, Julien

2015-11-20

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available. Copyright © 2015 Elsevier B.V. All rights reserved.

Genotyping-by-Sequencing-Based Investigation of the Genetic Architecture Responsible for a ∼Sevenfold Increase in Soybean Seed Stearic Acid.

PubMed

Heim, Crystal B; Gillman, Jason D

2017-01-05

Soybean oil is highly unsaturated but oxidatively unstable, rendering it nonideal for food applications. Until recently, the majority of soybean oil underwent partial chemical hydrogenation, which produces trans fats as an unavoidable consequence. Dietary intake of trans fats and most saturated fats are conclusively linked to negative impacts on cholesterol levels and cardiovascular health. Two major soybean oil breeding targets are: (1) to reduce or eliminate the need for chemical hydrogenation, and (2) to replace the functional properties of partially hydrogenated soybean oil. One potential solution is the elevation of seed stearic acid, a saturated fat which has no negative impacts on cardiovascular health, from 3 to 4% in typical cultivars to > 20% of the seed oil. We performed QTL analysis of a population developed by crossing two mutant lines, one with a missense mutation affecting a stearoyl-acyl-carrier protein desaturase gene resulting in ∼11% seed stearic acid crossed to another mutant, A6, which has 24-28% seed stearic acid. Genotyping-by-sequencing (GBS)-based QTL mapping identified 21 minor and major effect QTL for six seed oil related traits and plant height. The inheritance of a large genomic deletion affecting chromosome 14 is the basis for largest effect QTL, resulting in ∼18% seed stearic acid. This deletion contains SACPD-C and another gene(s); loss of both genes boosts seed stearic acid levels to ≥ 18%. Unfortunately, this genomic deletion has been shown in previous studies to be inextricably correlated with reduced seed yield. Our results will help inform and guide ongoing breeding efforts to improve soybean oil oxidative stability. Copyright © 2017 Heim and Gillman.

Functional desaturase Fads1 (Δ5) and Fads2 (Δ6) orthologues evolved before the origin of jawed vertebrates.

PubMed

Castro, Luís Filipe Costa; Monroig, Óscar; Leaver, Michael J; Wilson, Jonathan; Cunha, Isabel; Tocher, Douglas R

2012-01-01

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

Induction of Lipid and Oleosin Biosynthesis by (+)-Abscisic Acid and Its Metabolites in Microspore-Derived Embryos of Brassica napus L.cv Reston (Biological Responses in the Presence of 8[prime]-Hydroxyabscisic Acid).

PubMed Central

Zou, J.; Abrams, G. D.; Barton, D. L.; Taylor, D. C.; Pomeroy, M. K.; Abrams, S. R.

1995-01-01

Microspore-derived (MD) embryos of Brassica napus L. cv Reston were used to test the effects of (+)-abscisic acid ([(+)-ABA]) and its metabolites, 8[prime]-hydroxyabscisic acid (8[prime]-OH ABA) and (-)-phaseic acid (PA), on the accumulation of very long-chain monounsaturated fatty acids (VLCMFAs) and induction of genes encoding a 19-kD oleosin protein and a [delta]15 desaturase during embryogenesis. Developing early to mid-cotyledonary MD embryos at 16 to 19 d in culture were treated with 10 [mu]M hormone/metabolite for 4 d. At various times during incubation, embryos and medium were analyzed to determine levels of hormone/metabolite, VLCMFAs, and oleosin or [delta]15 desaturase transcripts. The VLCMFAs, 20:1 and 22:1, primarily in triacylglycerols, increased by 200% after 72 h in the presence of (+)-ABA and 8[prime]-OH ABA relative to the control. In contrast, treatment with PA for 72 h had little effect (20% increase) on the level of VLCMFAs. The first 24 to 72 h of (+)-ABA treatment were critical in the induction of VLCMFA biosynthesis, with 8[prime]-OH ABA lagging slightly behind (+)-ABA in promoting this response. The accumulation of VLCMFAs was positively correlated with an increase in elongase activity. (+)-ABA and its 8[prime]-OH ABA metabolite induced the accumulation of a 19-kD oleosin transcript within 2 to 4 h in culture. In addition, both (+)-ABA and 8[prime]-OH ABA induced the same level of [delta]15 desaturase transcript by 8 h. PA had no effect on the induction of either oleosin or [delta]15 desaturase transcripts. To our knowledge, this is the first report of the biological activity of 8[prime]-OH ABA and of stimulatory effects of (+)-ABA and 8[prime]-OH ABA on lipid and oleosin biosynthesis. PMID:12228493

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

PubMed Central

Poliakov, Eugenia; Samuel, William; Duncan, Todd; Gutierrez, Danielle B.; Mata, Nathan L.; Redmond, T. Michael

2017-01-01

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide’s various therapeutic uses will begin to resolve the pleotropic nature of this compound. PMID:28448568

Variants in CPT1A, FADS1, and FADS2 are Associated with Higher Levels of Estimated Plasma and Erythrocyte Delta-5 Desaturases in Alaskan Eskimos.

PubMed

Voruganti, V Saroja; Higgins, Paul B; Ebbesson, Sven O E; Kennish, John; Göring, Harald H H; Haack, Karin; Laston, Sandra; Drigalenko, Eugene; Wenger, Charlotte R; Harris, William S; Fabsitz, Richard R; Devereux, Richard B; Maccluer, Jean W; Curran, Joanne E; Carless, Melanie A; Johnson, Matthew P; Moses, Eric K; Blangero, John; Umans, Jason G; Howard, Barbara V; Cole, Shelley A; Comuzzie, Anthony Gean

2012-01-01

The delta-5 and delta-6 desaturases (D5D and D6D), encoded by fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes, respectively, are rate-limiting enzymes in the metabolism of ω-3 and ω-6 fatty acids. The objective of this study was to identify genes influencing variation in estimated D5D and D6D activities in plasma and erythrocytes in Alaskan Eskimos (n = 761) participating in the genetics of coronary artery disease in Alaska Natives (GOCADAN) study. Desaturase activity was estimated by product: precursor ratio of polyunsaturated fatty acids. We found evidence of linkage for estimated erythrocyte D5D (eD5D) on chromosome 11q12-q13 (logarithm of odds score = 3.5). The confidence interval contains candidate genes FADS1, FADS2, 7-dehydrocholesterol reductase (DHCR7), and carnitine palmitoyl transferase 1A, liver (CPT1A). Measured genotype analysis found association between CPT1A, FADS1, and FADS2 single-nucleotide polymorphisms (SNPs) and estimated eD5D activity (p-values between 10(-28) and 10(-5)). A Bayesian quantitative trait nucleotide analysis showed that rs3019594 in CPT1A, rs174541 in FADS1, and rs174568 in FADS2 had posterior probabilities > 0.8, thereby demonstrating significant statistical support for a functional effect on eD5D activity. Highly significant associations of FADS1, FADS2, and CPT1A transcripts with their respective SNPs (p-values between 10(-75) and 10(-7)) in Mexican Americans of the San Antonio Family Heart Study corroborated our results. These findings strongly suggest a functional role for FADS1, FADS2, and CPT1A SNPs in the variation in eD5D activity.

Identification of Shared and Unique Serum Lipid Profiles in Diabetes Mellitus and Myocardial Infarction.

PubMed

Kjellqvist, Sanela; Klose, Christian; Surma, Michal A; Hindy, George; Mollet, Inês G; Johansson, Anna; Chavaux, Patrick; Gottfries, Johan; Simons, Kai; Melander, Olle; Fernandez, Céline

2016-11-29

Diabetes mellitus (DM) and cardiovascular disease are associated with dyslipidemia, but the detailed lipid molecular pattern in both diseases remains unknown. We used shotgun mass spectrometry to determine serum levels of 255 molecular lipids in 316 controls, 171 DM, and 99 myocardial infarction (MI) events from a cohort derived from the Malmö Diet and Cancer study. Orthogonal projections to latent structures analyses were conducted between the lipids and clinical parameters describing DM or MI. Fatty acid desaturases (FADS) and elongation of very long chain fatty acid protein 5 (ELOVL5) activities were estimated by calculating product to precursor ratios of polyunsaturated fatty acids in complex lipids. FADS genotypes encoding these desaturases were then tested for association with lipid levels and ratios. Differences in the levels of lipids belonging to the phosphatidylcholine and triacylglyceride (TAG) classes contributed the most to separating DM from controls. TAGs also played a dominating role in discriminating MI from controls. Levels of C18:2 fatty acids in complex lipids were lower both in DM and MI versus controls (DM, P=0.004; MI, P=6.0E-06) at least due to an acceleration in the metabolic flux from C18:2 to C20:4 (eg, increased estimated ELOVL5: DM, P=0.02; MI, P=0.04, and combined elongase-desaturase activities: DM, P=3.0E-06; MI, P=2.0E-06). Minor allele carriers of FADS genotypes were associated with increased levels of C18:2 (P≤0.007) and lower desaturase activity (P≤0.002). We demonstrate a possible relationship between decreased levels of C18:2 in complex lipids and DM or MI. We thereby highlight the importance of molecular lipids in the pathogenesis of both diseases. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

PubMed

Smith, Dennis A; Hammond, Timothy; Baillie, Thomas A

2018-06-01

While simple O - (ether-linked) and N -glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Structure and self-assembly properties of a new chitosan-based amphiphile.

PubMed

Huang, Yuping; Yu, Hailong; Guo, Liang; Huang, Qingrong

2010-06-17

A new chitosan-based amphiphile, octanoyl-chitosan-polyethylene glycol monomethyl ether (acylChitoMPEG), has been prepared using both hydrophobic octanoyl and hydrophilic polyethylene glycol monomethyl ether (MPEG) substitutions. The success of synthesis was confirmed by Fourier transform infrared (FT-IR) and (1)H NMR spectroscopy. The synthesized acylChitoMPEG exhibited good solubility in either aqueous solution or common organic solvents such as ethanol, acetone, and CHCl(3). The self-aggregation behavior of acylChitoMPEG in solutions was studied by a combination of pyrene fluorescence technique, dynamic light scattering, atomic force microscopy, and small-angle X-ray scattering (SAXS). The critical aggregation concentration (CAC) and hydrodynamic diameter were found to be 0.066 mg/mL and 24.4 nm, respectively. SAXS results suggested a coiled structure of the triple helical acylChitoMPEG backbone with the hydrophobic moieties hiding in the center of the backbone, and the hydrophilic MPEG chains surrounding the acylChitoMPEG backbone in a random Gaussian chain conformation. Cytotoxicity results showed that acylChitoMPEG exhibited negligible cytotoxicity even at concentrations as high as 1.0 mg/mL. All results implied that acylChitoMPEG has the potential to be used for biological or medical applications.

Understanding Acyl Chain and Glycerolipid Metabolism in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

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.

Oxonitriles: a grignard addition-acylation route to enamides.

PubMed

Fleming, Fraser F; Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W

2006-10-12

[reaction: see text] Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by an in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents or a trialkylzincate, effectively assembling highly substituted, cyclic enamides.

Oxonitriles: A Grignard Addition-Acylation Route to Enamides

PubMed Central

Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W.

2008-01-01

Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents, or a trialkyl zincate, effectively assembling highly substituted, cyclic enamides. PMID:17020332

Exogenous fatty acid metabolism in bacteria.

PubMed

Yao, Jiangwei; Rock, Charles O

2017-10-01

Bacterial type II fatty acid synthesis (FASII) is a target for novel antibiotic development. All bacteria encode for mechanisms to incorporate exogenous fatty acids, and some bacteria can use exogenous fatty acids to bypass FASII inhibition. Bacteria encode three different mechanisms for activating exogenous fatty acids for incorporation into phospholipid synthesis. Exogenous fatty acids are converted into acyl-CoA in Gammaproteobacteria such as E. coli. Acyl-CoA molecules constitute a separate pool from endogenously synthesized acyl-ACP. Acyl-CoA can be used for phospholipid synthesis or broken down by β-oxidation, but cannot be used for lipopolysaccharide synthesis. Exogenous fatty acids are converted into acyl-ACP in some Gram-negative bacteria. The resulting acyl-ACP undergoes the same fates as endogenously synthesized acyl-ACP. Exogenous fatty acids are converted into acyl-phosphates in Gram-positive bacteria, and can be used for phospholipid synthesis or become acyl-ACP. Only the order Lactobacillales can use exogenous fatty acids to bypass FASII inhibition. FASII shuts down completely in presence of exogenous fatty acids in Lactobacillales, allowing Lactobacillales to synthesize phospholipids entirely from exogenous fatty acids. Inhibition of FASII cannot be bypassed in other bacteria because FASII is only partially down-regulated in presence of exogenous fatty acid or FASII is required to synthesize essential metabolites such as β-hydroxyacyl-ACP. Certain selective pressures such as FASII inhibition or growth in biofilms can select for naturally occurring one step mutations that attenuate endogenous fatty acid synthesis. Although attempts have been made to estimate the natural prevalence of these mutants, culture-independent metagenomic methods would provide a better estimate. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis.

PubMed

Abbadi, A; Brummel, M; Spener, F

2000-10-01

3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e. inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP. This indicated a distinct attachment site for regulatory acyl-ACPs. Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short-chain acyl-ACPs in the case of C. lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.

Structure of the Bifunctional Acyltransferase/Decarboxylase LnmK from the Leinamycin Biosynthetic Pathway Revealing Novel Activity for a Double-Hot-Dog Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy R.; Bingman, Craig A.; George N. Phillips Jr.

The β-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900–6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of themore » monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. But, to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. Moreover, LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.« less

MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor.

PubMed

Matharu, A L; Cox, R J; Crosby, J; Byrom, K J; Simpson, T J

1998-12-01

It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis. We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

PubMed Central

Khosla, C; McDaniel, R; Ebert-Khosla, S; Torres, R; Sherman, D H; Bibb, M J; Hopwood, D A

1993-01-01

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases. Images PMID:8468280

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated microbubbles.

PubMed

Borden, Mark A; Pu, Gang; Runner, Gabriel J; Longo, Marjorie L

2004-06-01

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating-cooling followed by compression-expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.

Theoretical and structural analysis of the active site of the transcriptional regulators LasR and TraR, using molecular docking methodology for identifying potential analogues of acyl homoserine lactones (AHLs) with anti-quorum sensing activity.

PubMed

Ahumedo, Maicol; Díaz, Antonio; Vivas-Reyes, Ricardo

2010-02-01

In the present study the homology of transcriptional receptors LuxR type were evaluated using as point of reference the receptors TraR and LasR of the bacterial types Agrobacterium tumefaciens and Pseudomonas aureginosa respectively. A series of alignments were performed in order to demonstrate that the active site of the protein is conserved in wide range of gram negative bacteria. Moreover, some docking calculations were carried out for analogs of the acyl homoserin lactones (AHLs) and regulatory proteins LasR and TraR, to understand the complex microenvironment in which the ligands are exposed. The molecular alignments show clearly that there are preserved motifs in the residues (Y53, Y61, W57, D70, W85 to TraR, Y56, Y64, W60, D73, W88 to LasR) analyzed, which may serve as site-specific targets for the development of potential antagonists. In this study was found that the anti-quorum sensing activity of the AHLs molecular analogs appears to depend on; the structure of the lactone ring and on appropriate combination of absolute and relative stereochemistry of the carbonyl (C=O) and amide (NH(2)) groups of the side chain of these AHLs molecular analogs, in combination with the interactions with the conserved amino acids (D73, W60, Y56, S129 to LasR and D70, W57, Y53 to TraR) of the LuxR type protein family. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Molecular cloning and chromosomal localization of a pseudogene related to the human Acyl-CoA binding protein/diazepam binding inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gersuk, V.H.; Rose, T.M.; Todaro, G.J.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5{prime} ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-relatedmore » sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1. 33 refs., 3 figs., 1 tab.« less

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

PubMed

Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

2014-05-02

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Neurodegeneration in a Drosophila model of adrenoleukodystrophy: the roles of the Bubblegum and Double bubble acyl-CoA synthetases

PubMed Central

Sivachenko, Anna; Gordon, Hannah B.; Kimball, Suzanne S.; Gavin, Erin J.; Bonkowsky, Joshua L.; Letsou, Anthea

2016-01-01

ABSTRACT Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum (bgm) and double bubble (dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivo is causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6 (encoding a very-long-chain ACS), a human homolog of bgm and dbb. PMID:26893370

An Inserted α/β Subdomain Shapes the Catalytic Pocket of Lactobacillus johnsonii Cinnamoyl Esterase

PubMed Central

Vu, Clara; Xu, Xiaohui; Cui, Hong; Molloy, Sara; Savchenko, Alexei; Yakunin, Alexander; Gonzalez, Claudio F.

2011-01-01

Background Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. Methodology/Principal Findings We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser106, His225, and Asp197, while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. Conclusions The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases. PMID:21876742

An inserted α/β subdomain shapes the catalytic pocket of Lactobacillus johnsonii cinnamoyl esterase.

PubMed

Lai, Kin-Kwan; Stogios, Peter J; Vu, Clara; Xu, Xiaohui; Cui, Hong; Molloy, Sara; Savchenko, Alexei; Yakunin, Alexander; Gonzalez, Claudio F

2011-01-01

Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser(106), His(225), and Asp(197), while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases.

Lanthanum tricyanide-catalyzed acyl silane-ketone benzoin additions and kinetic resolution of resultant alpha-silyloxyketones.

PubMed

Tarr, James C; Johnson, Jeffrey S

2010-05-21

We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic alpha-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant alpha-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction.

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

PubMed

Nieto Penalver, Carlos G; Morin, Danièle; Cantet, Franck; Saurel, Olivier; Milon, Alain; Vorholt, Julia A

2006-01-23

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

Comparative Analyses of Cuticular Waxes on Various Organs of Potato (Solanum tuberosum L.).

PubMed

Guo, Yanjun; Jetter, Reinhard

2017-05-17

Complex mixtures of cuticular waxes coat plant surfaces to seal them against environmental stresses, with compositions greatly varying between species and possibly organs. This paper reports comprehensive analyses of the waxes on both above- and below-ground organs of potato, where total wax coverages varied between petals (2.6 μg/cm 2 ), leaves, stems, and tubers (1.8-1.9 μg/cm 2 ), and rhizomes (1.1 μg/cm 2 ). The wax mixtures on above-ground organs were dominated by alkanes, occurring in homologous series of isomeric C 25 -C 35 n-alkanes, C 25 -C 35 2-methylalkanes, and C 26 -C 34 3-methylalkanes. In contrast, below-ground organs had waxes rich in monoacylglycerols (C 22 -C 28 acyls) and C 18 -C 30 alkyl ferulates, together with fatty acids (rhizomes) or primary alcohols (tubers). The organ-specific wax coverages, compound class distribution, and chain length profiles suggest highly regulated activities of wax biosynthesis enzymes, likely related to organ-specific ecophysiological functions.

Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma.

PubMed

Bullen, Hayley E; Jia, Yonggen; Yamaryo-Botté, Yoshiki; Bisio, Hugo; Zhang, Ou; Jemelin, Natacha Klages; Marq, Jean-Baptiste; Carruthers, Vern; Botté, Cyrille Y; Soldati-Favre, Dominique

2016-03-09

The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Insights using the molecular model of Lipoxygenase from Finger millet (Eleusine coracana (L.)).

PubMed

Tiwari, Apoorv; Avashthi, Himanshu; Jha, Richa; Srivastava, Ambuj; Kumar Garg, Vijay; Wasudev Ramteke, Pramod; Kumar, Anil

2016-01-01

Lipoxygenase-1 (LOX-1) protein provides defense against pests and pathogens and its presence have been positively correlated with plant resistance against pathogens. Linoleate is a known substrate of lipoxygenase and it induces necrosis leading to the accumulation of isoflavonoid phytoalexins in plant leaves. Therefore, it is of interest to study the structural features of LOX-1 from Finger millet. However, the structure ofLOX-1 from Finger millet is not yet known. A homology model of LOX-1 from Finger millet is described. Domain architecture study suggested the presence of two domains namely PLAT (Phospho Lipid Acyl Transferase) and lipoxygenase. Molecular docking models of linoleate with lipoxygenase from finger millet, rice and sorghum are reported. The features of docked models showed that finger millet have higher pathogen resistance in comparison to other cereal crops. This data is useful for the molecular cloning of fulllength LOX-1 gene for validating its role in improving plant defense against pathogen infection and for various other biological processes.

Global analysis of the Burkholderia thailandensis quorum sensing-controlled regulon.

PubMed

Majerczyk, Charlotte; Brittnacher, Mitchell; Jacobs, Michael; Armour, Christopher D; Radey, Mathew; Schneider, Emily; Phattarasokul, Somsak; Bunt, Richard; Greenberg, E Peter

2014-04-01

Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei.

Chlorogenic acids and the acyl-quinic acids: discovery, biosynthesis, bioavailability and bioactivity.

PubMed

Clifford, Michael N; Jaganath, Indu B; Ludwig, Iziar A; Crozier, Alan

2017-12-13

Covering: 2000 up to late 2017This review is focussed upon the acyl-quinic acids, the most studied group within the ca. 400 chlorogenic acids so far reported. The acyl-quinic acids, the first of which was characterised in 1846, are a diverse group of plant-derived compounds produced principally through esterification of an hydroxycinnamic acid and 1l-(-)-quinic acid. Topics addressed in this review include the confusing nomenclature, quantification and characterisation by NMR and MS, biosynthesis and role in planta, and the occurrence of acyl-quinic acids in coffee, their transformation during roasting and delivery to the beverage. Coffee is the major human dietary source world-wide of acyl-quinic acids and consideration is given to their absorption and metabolism in the upper gastrointestinal tract, and the colon where the microbiota play a key role in the formation of catabolites. Evidence on the potential of the in vivo metabolites and catabolites of acyl-quinic acids to promote the consumer's health is evaluated.

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations.

PubMed

Patelski, J; Pioruńska-Stolzmann, M

1985-01-01

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.

Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense*

PubMed Central

Yang, Ming-Te; Chang, Cheng-Hsiang; Wang, Jiou Ming; Wu, Tung Kung; Wang, Yu-Kuo; Chang, Chin-Yuan; Li, TienHsiung Thomas

2011-01-01

The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent Km and kcat/Km values of 52.66 mm and 40.42 mm−1 min−1, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC50 values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of NSQAETQC-C-DNS and C-DNS-NQRC sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition. PMID:21193394

Bleaching Herbicide Flurtamone Interferes with Phytoene Desaturase

PubMed Central

Sandmann, Gerhard; Ward, Carl E.; Lo, William C.; Nagy, Jon O.; Böger, Peter

1990-01-01

The mode of action of the furanone herbicide flurtamone and derivatives was investigated with cress seedlings and with the unicellular cyanobacterium Anacystis. Either in the light or in the dark these compounds inhibited the formation of α- and β-carotene and all of the xanthophylls in the seedlings. Instead, phytoene, a precursor of colored carotenoids, was accumulated. In illuminated seedlings photooxidative destruction of chlorophyll was observed. The I50 value of flurtamone inhibition of carotenoid biosynthesis in intact Anacystis cells and the K1 value for interaction of flurtamone with phytoene desaturase with Anacystis thylakoids were 30 and 18 nanomoles, respectively. Concentrations of flurtamone which strongly inhibited carotenoid synthesis had no direct peroxidative activities and did not inhibit photosynthetic electron transport. PMID:16667736

Elevated Medium-Chain Acylcarnitines Are Associated With Gestational Diabetes Mellitus and Early Progression to Type 2 Diabetes and Induce Pancreatic β-Cell Dysfunction.

PubMed

Batchuluun, Battsetseg; Al Rijjal, Dana; Prentice, Kacey J; Eversley, Judith A; Burdett, Elena; Mohan, Haneesha; Bhattacharjee, Alpana; Gunderson, Erica P; Liu, Ying; Wheeler, Michael B

2018-05-01

Specific circulating metabolites have emerged as important risk factors for the development of diabetes. The acylcarnitines (acylCs) are a family of metabolites known to be elevated in type 2 diabetes (T2D) and linked to peripheral insulin resistance. However, the effect of acylCs on pancreatic β-cell function is not well understood. Here, we profiled circulating acylCs in two diabetes cohorts: 1 ) women with gestational diabetes mellitus (GDM) and 2 ) women with recent GDM who later developed impaired glucose tolerance (IGT), new-onset T2D, or returned to normoglycemia within a 2-year follow-up period. We observed a specific elevation in serum medium-chain (M)-acylCs, particularly hexanoyl- and octanoylcarnitine, among women with GDM and individuals with T2D without alteration in long-chain acylCs. Mice treated with M-acylCs exhibited glucose intolerance, attributed to impaired insulin secretion. Murine and human islets exposed to elevated levels of M-acylCs developed defects in glucose-stimulated insulin secretion and this was directly linked to reduced mitochondrial respiratory capacity and subsequent ability to couple glucose metabolism to insulin secretion. In conclusion, our study reveals that an elevation in circulating M-acylCs is associated with GDM and early stages of T2D onset and that this elevation directly impairs β-cell function. © 2018 by the American Diabetes Association.

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

PubMed Central

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

2015-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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...

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

DTIC Science & Technology

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

Diversion of a thioglycoligase for the synthesis of 1-O-acyl arabinofuranoses.

PubMed

Pavic, Quentin; Tranchimand, Sylvain; Lemiègre, Loïc; Legentil, Laurent

2018-05-15

An arabinofuranosylhydrolase from the GH51 family was transformed into an acyl transferase by mutation of the catalytic acid/base amino acid. The resulting enzyme was able to transfer carboxylic acid onto the anomeric position of arabinose with complete chemo- and stereoselectivity. A wide range of acyl α-l-arabinofuranoses was obtained with yields ranging from 25 to 83%. Using this method, ibuprofen and N-Boc phenylalanine were successfully transformed into their corresponding acyl conjugates, expanding the scope of the reaction to drugs and amino acids.

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

Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

PubMed

Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

2013-04-02

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Lanthanum Tricyanide-Catalyzed Acyl Silane-Ketone Benzoin Additions and Kinetic Resolution of Resultant α-Silyloxyketones

PubMed Central

Tarr, James C.

2010-01-01

We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic α-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant α-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction. PMID:20392127

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids.

PubMed

Ardhaoui, M; Falcimaigne, A; Ognier, S; Engasser, J M; Moussou, P; Pauly, G; Ghoul, M

2004-06-10

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and omega-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on omega-substituted fatty acid chain were unfavourable to these reactions. The 1H NMR and 13C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4"'-OH of the rhamnose residue of rutin. Copyright 2004 Elsevier B.V.

Synthesis and Characterization of AlCl3 Impregnated Molybdenum Oxide as Heterogeneous Nano-Catalyst for the Friedel-Crafts Acylation Reaction in Ambient Condition.

PubMed

Jadhav, Arvind H; Chinnappan, Amutha; Hiremath, Vishwanath; Seo, Jeong Gil

2015-10-01

Aluminum trichloride (AlCl3) impregnated molybdenum oxide heterogeneous nano-catalyst was prepared by using simple impregnation method. The prepared heterogeneous catalyst was characterized by powder X-ray diffraction, FT-IR spectroscopy, solid-state NMR spectroscopy, SEM imaging, and EDX mapping. The catalytic activity of this protocol was evaluated as heterogeneous catalyst for the Friedel-Crafts acylation reaction at room temperature. The impregnated MoO4(AlCl2)2 catalyst showed tremendous catalytic activity in Friedel-Crafts acylation reaction under solvent-free and mild reaction condition. As a result, 84.0% yield of acyl product with 100% consumption of reactants in 18 h reaction time at room temperature was achieved. The effects of different solvents system with MoO4(AlCl2)2 catalyst in acylation reaction was also investigated. By using optimized reaction condition various acylated derivatives were prepared. In addition, the catalyst was separated by simple filtration process after the reaction and reused several times. Therefore, heterogeneous MoO4(AlCl2)2 catalyst was found environmentally benign catalyst, very convenient, high yielding, and clean method for the Friedel-Crafts acylation reaction under solvent-free and ambient reaction condition.

Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat.

PubMed

Grillo, M P; Benet, L Z

2001-08-01

Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl-N-acyl-cysteine products rather than the S-acyl-NAC conjugate.

Hepatic structure-pharmacokinetic relationships: the hepatic disposition and metabolite kinetics of a homologous series of O-acyl derivatives of salicylic acid.

PubMed

Hung, D Y; Mellick, G D; Anissimov, Y G; Weiss, M; Roberts, M S

1998-08-01

1. The hepatic disposition and metabolite kinetics of a homologous series of O-acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single-pass, in-situ rat liver preparation. 2. The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non-parametric moments analysis was used to determine the area under the curve (AUC'), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3. Pregenerated SA ([14C]-salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4. The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5. The metabolite AUC' value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC' decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6. The two-compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUC', MTT and CV2 for the parent ester and metabolite. The moments values obtained using the two-compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non-parametric approach. 7. The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirin's selectivity as an anti-thrombotic agent has been postulated to be due to selective anti-platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti-thrombotic agents than aspirin.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

PubMed

Mellbye, Brett L; Spieck, Eva; Bottomley, Peter J; Sayavedra-Soto, Luis A

2017-11-15

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus , Nitrobacter , and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N -decanoyl-l-homoserine lactone (C 10 -HSL), N -3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C 14 -HSL), a monounsaturated AHL (C 10:1 -HSL), and N -octanoyl-l-homoserine lactone (C 8 -HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification. Copyright © 2017 American Society for Microbiology.

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.

PubMed

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

2014-06-01

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1 cisΔ11 ) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2 cisΔ9,12 ; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1 cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3 cisΔ9,12,15 ) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. © 2014 American Society of Plant Biologists. All Rights Reserved.

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets.

PubMed Central

Bakken, A M; Farstad, M

1992-01-01

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium. PMID:1471991

One-plasmid tunable coexpression for mycobacterial protein–protein interaction studies

PubMed Central

Chang, Yong; Mead, David; Dhodda, Vinay; Brumm, Phil; Fox, Brian G

2009-01-01

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA Δ9 desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications. PMID:19760663

Genetic variants in desaturase gene, erythrocyte fatty acids, and risk for type 2 diabetes in Chinese Hans.

PubMed

Huang, Tao; Sun, Jianqin; Chen, Yanqiu; Xie, Hua; Xu, Danfeng; Huang, Jinyan; Li, Duo

2014-01-01

The aim of this study was to examine the association of the genetic variants in the fatty acid desaturase (FADS) gene cluster with erythrocyte phospholipid fatty acids (PLFA), and their relation to risk for type 2 diabetes mellitus (T2DM) in Han Chinese. Seven hundred and fifty-eight patients with T2DM and 400 healthy individuals were recruited. The erythrocyte PLFA and single-nucleotide polymorphism were determined by standard method. Minor allele homozygotes and heterozygotes of rs174575 and rs174537 had lower PL 20:4 ω-6 levels in healthy individuals. Minor allele homozygotes and heterozygotes of rs174455 in FADS3 gene had lower levels of 22:5 ω-3, 20:4 ω-6, and Δ5desaturase activity in patients with T2DM. Erythrocyte membrane PL 18:3 ω-3 (P for trend = 0.002), 22:5 ω-3 (P for trend < 0.001), ω-3 polyunsaturated fatty acid (P for trend < 0.001), and ω-3:ω-6 (P for trend < 0.001) were significantly inversely associated with risk for T2DM. Genetic variants in the FADS gene cluster are associated with altered erythrocyte PLFAs. High levels of PL 18:3 ω-3, 22:5 ω-3, and total ω-3 polyunsaturated fatty acid were associated with low risk for T2DM. Copyright © 2014 Elsevier Inc. All rights reserved.

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

PubMed

Wen, Shijie; Liu, Hao; Li, Xingyu; Chen, Xiaoping; Hong, Yanbin; Li, Haifen; Lu, Qing; Liang, Xuanqiang

2018-05-01

A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

RAPID 2D NMR METHOD FOR DETERMINING P-COUMARATE AND FERULATE LEVELS IN CORN (AND OTHER GRASS) CELL WALLS

USDA-ARS?s Scientific Manuscript database

Grass cell wall components are acylated by the hydroxycinnamates p-coumarate and ferulate. p-Coumarates largely acylate lignin sidechains, exclusively at the gamma-position, whereas ferulates primarily acylate the arabinosyl C5-position of arabinoxylans. Such components can be quantified as the corr...

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…

Acyl hydrazides as acyl donors for the synthesis of diaryl and aryl alkyl ketones.

PubMed

Akhbar, Ahmed R; Chudasama, Vijay; Fitzmaurice, Richard J; Powell, Lyn; Caddick, Stephen

2014-01-21

In this communication we describe a novel strategy for the formation of valuable diaryl and aryl alkyl ketones from acyl hydrazides. A wide variety of ketones are prepared and the mild reaction conditions allow for the use of a range of functionalities, especially in the synthesis of diaryl ketones.

Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals

PubMed Central

Xu, Peng; Qiao, Kangjian; Ahn, Woo Suk; Stephanopoulos, Gregory

2016-01-01

Harnessing lipogenic pathways and rewiring acyl-CoA and acyl-ACP (acyl carrier protein) metabolism in Yarrowia lipolytica hold great potential for cost-efficient production of diesel, gasoline-like fuels, and oleochemicals. Here we assessed various pathway engineering strategies in Y. lipolytica toward developing a yeast biorefinery platform for sustainable production of fuel-like molecules and oleochemicals. Specifically, acyl-CoA/acyl-ACP processing enzymes were targeted to the cytoplasm, peroxisome, or endoplasmic reticulum to generate fatty acid ethyl esters and fatty alkanes with tailored chain length. Activation of endogenous free fatty acids and the subsequent reduction of fatty acyl-CoAs enabled the efficient synthesis of fatty alcohols. Engineering a hybrid fatty acid synthase shifted the free fatty acids to a medium chain-length scale. Manipulation of alternative cytosolic acetyl-CoA pathways partially decoupled lipogenesis from nitrogen starvation and unleashed the lipogenic potential of Y. lipolytica. Taken together, the strategies reported here represent promising steps to develop a yeast biorefinery platform that potentially upgrades low-value carbons to high-value fuels and oleochemicals in a sustainable and environmentally friendly manner. PMID:27621436

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

PubMed

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

2018-06-04

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

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

PubMed

Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

2004-03-31

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

The gut hormone ghrelin partially reverses energy substrate metabolic alterations in the failing heart.

PubMed

Mitacchione, Gianfranco; Powers, Jeffrey C; Grifoni, Gino; Woitek, Felix; Lam, Amy; Ly, Lien; Settanni, Fabio; Makarewich, Catherine A; McCormick, Ryan; Trovato, Letizia; Houser, Steven R; Granata, Riccarda; Recchia, Fabio A

2014-07-01

The gut-derived hormone ghrelin, especially its acylated form, plays a major role in the regulation of systemic metabolism and exerts also relevant cardioprotective effects; hence, it has been proposed for the treatment of heart failure (HF). We tested the hypothesis that ghrelin can directly modulate cardiac energy substrate metabolism. We used chronically instrumented dogs, 8 with pacing-induced HF and 6 normal controls. Human des-acyl ghrelin [1.2 nmol/kg per hour] was infused intravenously for 15 minutes, followed by washout (rebaseline) and infusion of acyl ghrelin at the same dose. (3)H-oleate and (14)C-glucose were coinfused and arterial and coronary sinus blood sampled to measure cardiac free fatty acid and glucose oxidation and lactate uptake. As expected, cardiac substrate metabolism was profoundly altered in HF because baseline oxidation levels of free fatty acids and glucose were, respectively, >70% lower and >160% higher compared with control. Neither des-acyl ghrelin nor acyl ghrelin significantly affected function and metabolism in normal hearts. However, in HF, des-acyl and acyl ghrelin enhanced myocardial oxygen consumption by 10.2±3.5% and 9.9±3.7%, respectively (P<0.05), and cardiac mechanical efficiency was not significantly altered. This was associated, respectively, with a 41.3±6.7% and 32.5±10.9% increase in free fatty acid oxidation and a 31.3±9.2% and 41.4±8.9% decrease in glucose oxidation (all P<0.05). Acute increases in des-acyl or acyl ghrelin do not interfere with cardiac metabolism in normal dogs, whereas they enhance free fatty acid oxidation and reduce glucose oxidation in HF dogs, thus partially correcting metabolic alterations in HF. This novel mechanism might contribute to the cardioprotective effects of ghrelin in HF. © 2014 American Heart Association, Inc.

α-melanocyte stimulating hormone modulates the central acyl ghrelin-induced stimulation of feeding, gastrointestinal motility, and colonic secretion.

PubMed

Huang, Hsien-Hao; Chen, Liang-Yu; Doong, Ming-Luen; Chang, Shi-Chuan; Chen, Chih-Yen

2017-01-01

Acyl ghrelin-induced intake depends on hypothalamic neuropeptide Y and agouti-related protein (AgRP) neurotransmitters. Intracerebroventricular (ICV) injection of AgRP increases feeding through competitive antagonism at melanocortin receptors. ICV administration of α-melanocyte stimulating hormone (α-MSH), a natural antagonist of AgRP, may modulate the acyl ghrelin-induced orexigenic effect. This study aimed to investigate the modulating effect of α-MSH on the central acyl ghrelin-induced food intake, gastrointestinal motility, and colonic secretion in rats. We examined the effects of α-MSH and acyl ghrelin on food intake, gastric emptying, small intestinal transit, colonic motility, and secretion in conscious rats with a chronic implant of ICV catheters. ICV injection of O - n -octanoylated ghrelin (0.1 nmol/rat) significantly increased the cumulative food intake up to 8 h ( P <0.01), enhanced non-nutrient semi-liquid gastric emptying ( P <0.001), increased the geometric center and running percentage of small intestinal transit ( P <0.001), accelerated colonic transit time ( P <0.05), and increased fecal pellet output ( P <0.01) and total fecal weight ( P <0.01). Pretreatment with ICV injection of α-MSH (1.0 and 2.0 nmol/rat) attenuated the acyl ghrelin-induced hyperphagic effect, fecal pellet output, and total fecal weight, while higher dose of α-MSH (2.0 nmol/rat) attenuated the increase in the geometric center of small intestinal transit ( P <0.01). However, neither dose of α-MSH altered acyl ghrelin-stimulated gastroprokinetic effect, increase in the running percentage of small intestinal transit, nor accelerated colonic transit time. α-MSH is involved in central acyl ghrelin-elicited feeding, small intestinal transit, fecal pellet output, and fecal weight. α-MSH does not affect central acyl ghrelin-induced acceleration of gastric emptying and colonic transit time in rats.

Characterization of N-palmitoylated human growth hormone by in situ liquid-liquid extraction and MALDI tandem mass spectrometry.

PubMed

Sachon, Emmanuelle; Nielsen, Per Franklin; Jensen, Ole Nørregaard

2007-06-01

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.

Utilization of acidic α-amino acids as acyl donors: an effective stereo-controllable synthesis of aryl-keto α-amino acids and their derivatives.

PubMed

Wang, Lei; Murai, Yuta; Yoshida, Takuma; Okamoto, Masashi; Tachrim, Zetryana Puteri; Hashidoko, Yasuyuki; Hashimoto, Makoto

2014-05-16

Aryl-keto-containing α-amino acids are of great importance in organic chemistry and biochemistry. They are valuable intermediates for the construction of hydroxyl α-amino acids, nonproteinogenic α-amino acids, as well as other biofunctional components. Friedel-Crafts acylation is an effective method to prepare aryl-keto derivatives. In this review, we summarize the preparation of aryl-keto containing α-amino acids by Friedel-Crafts acylation using acidic α-amino acids as acyl-donors and Lewis acids or Brönsted acids as catalysts.

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

PubMed

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

2013-06-01

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

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

PubMed

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

2013-11-15

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

Environmentally friendly surface modification of silk fiber: Chitosan grafting and dyeing

NASA Astrophysics Data System (ADS)

Davarpanah, Saideh; Mahmoodi, Niyaz Mohammad; Arami, Mokhtar; Bahrami, Hajir; Mazaheri, Firoozmehr

2009-01-01

In this paper, the surface modification of silk fiber using anhydrides to graft the polysaccharide chitosan and dyeing ability of the grafted silk were studied. Silk fiber was degummed and acylated with two anhydrides, succinic anhydride (SA) and phthalic anhydride (PA), in different solvents (dimethyl sulfoxide (DMSO) and N, N-dimethyl formamide (DMF)). The effects of anhydrides, solvents, anhydride concentration, liquor ratio (L:R) and reaction time on acylation of silk were studied. The polysaccharide chitosan was grafted to the acylated silk fiber and dyed by acid dye (Acid Black NB.B). The effects of pH, chitosan concentration, and reaction time on chitosan grafting of acylated silk were investigated. The physical properties show sensible changes regardless of weight gain. Scanning electron microscopy (SEM) analysis showed the presence of foreign materials firmly attached to the surface of silk. FTIR spectroscopy provided evidence that chitosan was grafted onto the acylated silk through the formation of new covalent bonds. The dyeing of the chitosan grafted-acylated silk fiber indicated the higher dye ability in comparison to the acylated and degummed silk samples. The mechanism of chitosan grafting over degummed silk through anhydride linkage was proposed. The findings of this research support the potential production of new environmentally friendly textile fibers. It is worthwhile to mention that the grafted samples have antibacterial potential due to the antibacterial property of chitosan molecules.

Density functional theory studies on the nano-scaled composites consisted of graphene and acyl hydrazone molecules

NASA Astrophysics Data System (ADS)

Ren, J. L.; Zhou, L.; Lv, Z. C.; Ding, C. H.; Wu, Y. H.; Bai, H. C.

2016-07-01

Graphene, which is the first obtained single atomic layer 2D materials, has drawn a great of concern in nano biotechnology due to the unique property. On one hand, acyl hydrazone compounds belonging to the Schif bases have aroused considerable attention in medicine, pharmacy, and analytical reagent. However, few understanding about the interaction between graphene and acyl hydrazone molecules is now available. And such investigations are much crucial for the applications of these new nano-scaled composites. The current work revealed theoretical investigations on the nano-scaled composites built by acyl hydrazone molecules loaded on the surface of graphene. The relative energy, electronic property and the interaction between the counterparts of graphene/acyl hydrazone composites are investigated based on the density functional theory calculations. According to the obtained adsorption energy, the formation of the nano-scaled composite from the isolated graphene and acyl hydrazone molecule is exothermic, and thus it is energetically favorable to form these nano composites in viewpoint of total energy change. The frontier molecular orbital for the nano composite is mainly distributed at the graphene part, leading to that the energy levels of the frontier molecular orbital of the nano composites are very close to that of isolated graphene. Moreover, the counterpart interaction for the graphene/acyl hydrazone composites is also explored based on the discussions of orbital hybridization, charge redistribution and Van der Waals interaction.

Inverse association of des-acyl ghrelin with worksite blood pressure in overweight/obese male workers.

PubMed

Narisada, Akihiko; Hasegawa, Tomomi; Nakahigashi, Maki; Hirobe, Takaaki; Ikemoto, Tatsunori; Ushida, Takahiro; Kobayashi, Fumio

2015-05-01

Job strain, defined as a combination of high job demands and low job control, has been reported to elevate blood pressure (BP) during work. Meanwhile, a recent experimental study showed that ghrelin blunted the BP response to such mental stress. In the present study, we examined the hypothesis that des-acyl ghrelin may have some beneficial effects on worksite BP through modulating the BP response to work-related mental stress, i.e., job strain. Subjects were 34 overweight/obese male day-shift workers (mean age 41.7 ± 6.7 years). No subjects had received any anti-hypertensive medication. A 24-h ambulatory BP monitoring was recorded every 30 min on a regular working day. The average BP was calculated for Work BP, Morning BP, and Home BP. Job strain was assessed using the short version of the Japanese Job Content Questionnaire. Des-acyl ghrelin showed significant inverse correlations with almost all BPs except Morning SBP, Morning DBP, and Home DBP. In multiple regression analysis, des-acyl ghrelin inversely correlated with Work SBP after adjusting for confounding factors. Des-acyl ghrelin was also negatively associated with BP changes from Sleep to Morning, Sleep to Work, and Sleep to Home. Des-acyl ghrelin was inversely associated with Worksite BP, suggesting a unique beneficial effect of des-acyl ghrelin on Worksite BP in overweight/obese male day-shift workers.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling | Center for Cancer Research

Cancer.gov

The cover image illuminates the non-enzymatic “ghost writers” of lysine acylation. Meier et al. detail the development of a chemoproteomic strategy that harnesses thioester reactivity to discover candidate cellular targets of non-enzymatic acylation. Application of this approach reveals that glycolytic enzymes can be strongly inhibited by reactive thioesters, including the

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

USDA-ARS?s Scientific Manuscript database

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 ...

Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils†

PubMed Central

Wang, Ya-Juan; Leadbetter, Jared Renton

2005-01-01

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, 14C-labeled acyl-HSLs were mineralized to 14CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol · h−1 · g of fresh weight soil−1. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2 but that the two processes were well coupled kinetically. A most probable number of 4.6 × 105 cells · g of turf soil−1 degraded physiologically relevant amounts of hexanoyl-[1-14C]HSL to 14CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems. PMID:15746331

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

PubMed

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

2016-08-01

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

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

PubMed

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

2018-01-01

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

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.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

PubMed

Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

2011-12-01

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

Gauging a Hydrocarbon Ruler by an Intrinsic Exciton Probe†

PubMed Central

Khan, M. Adil; Neale, Chris; Michaux, Catherine; Pomés, Régis; Privé, Gilbert G.; Woody, Robert W.; Bishop, Russell E.

2016-01-01

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of Gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid: lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane β-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26–Trp66 phenol–indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton’s expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler–exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment. PMID:17375935

Validation of a method for the quantitation of ghrelin and unacylated ghrelin by HPLC.

PubMed

Staes, Edith; Rozet, Eric; Ucakar, Bernard; Hubert, Philippe; Préat, Véronique

2010-02-05

An HPLC/UV method was first optimized for the separation and quantitation of human acylated and unacylated (or des-acyl) ghrelin from aqueous solutions. This method was validated by an original approach using accuracy profiles based on tolerance intervals for the total error measurement. The concentration range that achieved adequate accuracy extended from 1.85 to 59.30microM and 1.93 to 61.60microM for acylated and unacylated ghrelin, respectively. Then, optimal temperature, pH and buffer for sample storage were determined. Unacylated ghrelin was found to be stable in all conditions tested. At 37 degrees C acylated ghrelin was stable at pH 4 but unstable at pH 7.4, the main degradation product was unacylated ghrelin. Finally, this validated HPLC/UV method was used to evaluate the binding of acylated and unacylated ghrelin to liposomes.

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

PubMed Central

Gupta, Chhitar M.; Costello, Catherine E.; Khorana, H. Gobind

1979-01-01

Sonicated vesicles of 1-fatty acyl-2-ω-(2-diazo-3,3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphoryl-cholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C—H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms. Images PMID:16592675

Erythrocyte polyunsaturated fatty acid composition is associated with depression and FADS genotype in Caucasians.

PubMed

Cribb, Lachlan; Murphy, Jenifer; Froud, Amy; Oliver, Georgina; Bousman, Chad A; Ng, Chee H; Sarris, Jerome

2017-05-29

Polyunsaturated fatty acids (PUFAs) play an important role in the pathophysiology of major depressive disorder (MDD), related, in part, to their role in inflammatory systems. The enzymes δ-5 and δ-6 desaturase are the rate-limiting steps in the metabolism of PUFAs and are encoded in the genes fatty acid desaturase (FADS) 1 and 2, respectively. Single nucleotide polymorphisms (SNPs) and haplotypes within the FADS gene cluster have been shown to influence PUFA composition. The objective of this study was to determine whether key omega-3 (n-3) and omega-6 (n-6) fatty acids may be associated with depression, and to explore the role of FADS genotype in PUFA variation. Four erythrocyte long chain (LC) fatty acids (linoleic acid [LA], α-linolenic acid [ALA], arachidonic acid [AA] and Eicosapentaenoic acid [EPA]), as well as six SNPs (rs174537, rs174547, rs174570, rs174575, rs498793 and rs3834458) within the FADS gene cluster were measured in a sample of 207 participants (154 with MDD versus 53 non-depressed controls). The precursor LC-PUFAs LA and ALA appeared to be negatively associated with depression (P < 0.001 and P < 0.01, respectively), while AA:LA (surrogate measure of desaturase activity) was positively associated with depression (P < 0.01). No significant differences were noted in erythrocyte EPA, AA or AA:EPA between groups. Minor alleles of each SNP (excluding rs498793) were associated with variation in desaturase activity and LA. Both rs174537 and rs174547 were associated with ALA. No genotype was associated with EPA or AA. Minor alleles of rs174537 and rs174547 were significantly associated with lower odds of MDD (although significance was lost after correction for multiple comparisons). Precursor LC-PUFAs, LA and ALA, appear to be associated with MDD and potentially modulated by genetic variation in the FADS gene cluster. These results provide support for the consideration of PUFA composition, diet and FADS genetic variation in the pathophysiology of MDD.

Fatty acid desaturase 1 knockout mice are lean with improved glycemic control and decreased development of atheromatous plaque

PubMed Central

Powell, David R; Gay, Jason P; Smith, Melinda; Wilganowski, Nathaniel; Harris, Angela; Holland, Autumn; Reyes, Maricela; Kirkham, Laura; Kirkpatrick, Laura L; Zambrowicz, Brian; Hansen, Gwenn; Platt, Kenneth A; van Sligtenhorst, Isaac; Ding, Zhi-Ming; Desai, Urvi

2016-01-01

Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for ω3, ω6, and ω9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. PMID:27382320

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...

Acylation, Diastereoselective Alkylation, and Cleavage of an Oxazolidinone Chiral Auxiliary: A Multistep Asymmetric Synthesis Experiment for Advanced Undergraduates

ERIC Educational Resources Information Center

Smith, Thomas E.; Richardson, David P.; Truran, George A.; Belecki, Katherine; Onishi, Megumi

2008-01-01

An introduction to the concepts and experimental techniques of diastereoselective synthesis using a chiral auxiliary is described. The 4-benzyl-2-oxazolidinone chiral auxiliary developed by Evans is acylated with propionic anhydride under mild conditions using DMAP as an acyl transfer catalyst. Deprotonation with NaN(TMS)[subscript 2] at -78…

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...

40 CFR 721.10174 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. 721.10174 Section 721.10174 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. (a) Chemical substance...

N-Acylated and d Enantiomer Derivatives of a Nonamer Core Peptide of Lactoferricin B Showing Improved Antimicrobial Activity

PubMed Central

Wakabayashi, Hiroyuki; Matsumoto, Hiroshi; Hashimoto, Koichi; Teraguchi, Susumu; Takase, Mitsunori; Hayasawa, Hirotoshi

1999-01-01

N-acylated or d enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B. PMID:10223949

N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.

PubMed

Wakabayashi, H; Matsumoto, H; Hashimoto, K; Teraguchi, S; Takase, M; Hayasawa, H

1999-05-01

N-acylated or D enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

Shifting Native Chemical Ligation into Reverse through N→S Acyl Transfer

PubMed Central

Macmillan, Derek; Adams, Anna; Premdjee, Bhavesh

2011-01-01

Peptide thioester synthesis by N→S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straightforward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts towards the synthesis of peptide thioesters through N→S acyl transfer in native peptide sequences. We have found that several peptide thioesters can be readily prepared and, what’s more, there appears to be ample opportunity for further development and discovery. PMID:22347724

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

PubMed

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

2016-05-23

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

Synthesis of 2-acyl-1,4-diketones via the diacylation of {alpha},{beta}-unsaturated ketones

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, N.S.; Yu, S.; Kabalka, G.W.

1998-08-17

The first example of a diacylation of the carbon-carbon double bond in {alpha},{beta}-unsaturated ketones is described. The reaction of acylcyanocuprate reagents with {alpha},{beta}-unsaturated ketones, followed by C-acylation, produces 2-acyl-1,4-diketones in good yields (50--89%). The 1,4-addition of organocuprate reagents to conjugated enones, followed by trapping of the enolate intermediates with various electrophiles, is one of the most useful synthetic reactions. However, to the best of the authors` knowledge, 1,4-acylation followed by trapping of the enolate intermediates with acid chloride has not been reported.

Toward Green Acylation of (Hetero)arenes: Palladium-Catalyzed Carbonylation of Olefins to Ketones

PubMed Central

2017-01-01

Green Friedel–Crafts acylation reactions belong to the most desired transformations in organic chemistry. The resulting ketones constitute important intermediates, building blocks, and functional molecules in organic synthesis as well as for the chemical industry. Over the past 60 years, advances in this topic have focused on how to make this reaction more economically and environmentally friendly by using green acylating conditions, such as stoichiometric acylations and catalytic homogeneous and heterogeneous acylations. However, currently well-established methodologies for their synthesis either produce significant amounts of waste or proceed under harsh conditions, limiting applications. Here, we present a new protocol for the straightforward and selective introduction of acyl groups into (hetero)arenes without directing groups by using available olefins with inexpensive CO. In the presence of commercial palladium catalysts, inter- and intramolecular carbonylative C–H functionalizations take place with good regio- and chemoselectivity. Compared to classical Friedel–Crafts chemistry, this novel methodology proceeds under mild reaction conditions. The general applicability of this methodology is demonstrated by the direct carbonylation of industrial feedstocks (ethylene and diisobutene) as well as of natural products (eugenol and safrole). Furthermore, synthetic applications to drug molecules are showcased. PMID:29392174

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

PubMed

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

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

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

An annotated database of Arabidopsis mutants of acyl lipid metabolism

DOE PAGES

McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

2014-12-10

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism ( fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypesmore » of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

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.

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

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

PubMed

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

1995-03-01

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

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

PubMed Central

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

2016-01-01

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

Des-acyl ghrelin inhibits the capacity of macrophages to stimulate the expression of aromatase in breast adipose stromal cells.

PubMed

Au, CheukMan C; Docanto, Maria M; Zahid, Heba; Raffaelli, Francesca-Maria; Ferrero, Richard L; Furness, John B; Brown, Kristy A

2017-06-01

Des-acyl ghrelin is the unacylated form of the well-characterized appetite-stimulating hormone ghrelin. It affects a number of physiological processes, including increasing adipose lipid accumulation and inhibiting adipose tissue inflammation. Breast adipose tissue inflammation in obesity is associated with an increase in the expression of the estrogen biosynthetic enzyme, aromatase, and is hypothesized to create a hormonal milieu conducive to tumor growth. We previously reported that des-acyl ghrelin inhibits the expression and activity of aromatase in isolated human adipose stromal cells (ASCs), the main site of aromatase expression in the adipose tissue. The current study aimed to examine the effect of des-acyl ghrelin on the capacity of mouse macrophages (RAW264.7 cells) and human adipose tissue macrophages (ATMs) to stimulate aromatase expression in primary human breast ASCs. RAW264.7 cells were treated with 0, 10 and 100pM des-acyl ghrelin following activation with phorbol 12-myristate 13-acetate, and cells and conditioned media were collected after 6 and 24h. The effect of des-acyl ghrelin on macrophage polarization was examined by assessing mRNA expression of pro-inflammatory M1-specific marker Cd11c and anti-inflammatory M2-specific marker Cd206, as well as expression of Tnf and Ptgs2, known mediators of the macrophage-dependent stimulation of aromatase. TNF protein in conditioned media was assessed by ELISA. The effect of RAW264.7 and ATM-conditioned media on aromatase expression in ASCs was assessed after 6h. Results demonstrate des-acyl ghrelin significantly increases the expression of Cd206 and suppresses the expression of Cd11c, Tnf and Ptgs2 in activated RAW264.7 cells. Treatment of RAW264.7 and ATMs with des-acyl ghrelin also significantly reduces the capacity of these cells to stimulate aromatase transcript expression in human breast ASCs. Overall, these findings suggest that in addition to direct effects on aromatase in ASCs, des-acyl ghrelin also has the capacity to inhibit the macrophage-dependent induction of aromatase, and provides a novel mechanism for potential effects of des-acyl ghrelin to break the linkage between obesity and breast cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea

DOE PAGES

Mukai, Takahito; Crnković, Ana; Umehara, Takuya; ...

2017-05-09

ABSTRACT The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA Cysis first acylated with phosphoserine (Sep) byO-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA Cysby Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN,Euryarchaeota, TACK, and Asgard) andmore » two groups of bacteria (“CandidatusParcubacteria” andChloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNA Cysspecies with cysteinein vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS-SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea andChloroflexibacteria. We further discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.« less

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukai, Takahito; Crnković, Ana; Umehara, Takuya

ABSTRACT The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA Cysis first acylated with phosphoserine (Sep) byO-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA Cysby Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN,Euryarchaeota, TACK, and Asgard) andmore » two groups of bacteria (“CandidatusParcubacteria” andChloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNA Cysspecies with cysteinein vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS-SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea andChloroflexibacteria. We further discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.« less

Jejunal administration of glucose enhances acyl ghrelin suppression in obese humans

PubMed Central

Sidani, Reem M.; Garcia, Anna E.; Antoun, Joseph; Isbell, James M.; Abumrad, Naji N.

2016-01-01

Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (iv) infusions of glucose. These “isoglycemic clamps” enabled us to determine the contribution of the GI tract and postabsorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared with gastric glucose administration (P < 0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P ≤ 0.01). However, gastric and isoglycemic iv glucose infusions resulted in similar degrees of acyl ghrelin suppression (P > 0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion. PMID:27279247

Novel acyloxy derivatives of branched mono- and polyol esters of sal fat: multiviscosity grade lubricant base stocks.

PubMed

Kamalakar, Kotte; Sai Manoj, Gorantla N V T; Prasad, Rachapudi B N; Karuna, Mallampalli S L

2014-12-10

Sal fat, a nontraditional seed oil, was chemically modified to obtain base stocks with a wide range of specifications that can replace mineral oil base stocks. Sal fatty acids were enriched to 72.6% unsaturation using urea adduct method and reacted with branched mono alcohol, 2-ethylhexanol (2-EtH), and polyols namely neopentyl glycol (NPG) and trimethylolpropane (TMP) to obtain corresponding esters. The esters were hydroxylated and then acylated using propionic, butyric, and hexanoic anhydrides to obtain corresponding acylated derivatives. The acylated TMP esters exhibited very high viscosities (427.35-471.93 cSt at 40 °C) similar to those of BS 150 mineral oil base stock range, ISO VG 460, while the acylated NPG esters (268.81-318.84 cSt at 40 °C) and 2-EtH esters viscosities (20.94-24.44 cSt at 40 °C) exhibited viscosities in the range of ISO VG 320 and 22 respectively with good viscosity indices. Acylated NPG esters were found suitable for high temperature and acylated 2-ethylhexyl esters for low viscosity grade industrial applications. It was observed that the thermo-oxidative stabilities of all acylated products were found better compared to other vegetable oil based base stocks. Overall, all the sal fat based lubricant base stocks are promising candidates with a wide range of properties, which can replace most of the mineral oil base stocks with appropriate formulations.

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

PubMed Central

Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

2014-01-01

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757