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Sample records for acid biosynthesis fas

  1. Fatty acid biosynthesis in actinomycetes

    PubMed Central

    Gago, Gabriela; Diacovich, Lautaro; Arabolaza, Ana; Tsai, Shiou-Chuan; Gramajo, Hugo

    2011-01-01

    All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multi-enzyme FAS II system and Corynebacterium species exclusively FAS I. In this review we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with anti-mycobacterial properties. PMID:21204864

  2. Crystal structure of FAS thioesterase domain with polyunsaturated fatty acyl adduct and inhibition by dihomo-[gamma]-linolenic acid

    SciTech Connect

    Zhang, Wei; Chakravarty, Bornali; Zheng, Fei; Gu, Ziwei; Wu, Hongmei; Mao, Jianqiang; Wakil, Salih J.; Quiocho, Florante A.

    2012-05-29

    Human fatty acid synthase (hFAS) is a homodimeric multidomain enzyme that catalyzes a series of reactions leading to the de novo biosynthesis of long-chain fatty acids, mainly palmitate. The carboxy-terminal thioesterase (TE) domain determines the length of the fatty acyl chain and its ultimate release by hydrolysis. Because of the upregulation of hFAS in a variety of cancers, it is a target for antiproliferative agent development. Dietary long-chain polyunsaturated fatty acids (PUFAs) have been known to confer beneficial effects on many diseases and health conditions, including cancers, inflammations, diabetes, and heart diseases, but the precise molecular mechanisms involved have not been elucidated. We report the crystal structure of the hFAS TE domain covalently modified and inactivated by methyl {gamma}-linolenylfluorophosphonate. Whereas the structure confirmed the phosphorylation by the phosphonate head group of the active site serine, it also unexpectedly revealed the binding of the 18-carbon polyunsaturated {gamma}-linolenyl tail in a long groove-tunnel site, which itself is formed mainly by the emergence of an {alpha} helix (the 'helix flap'). We then found inhibition of the TE domain activity by the PUFA dihomo-{gamma}-linolenic acid; {gamma}- and {alpha}-linolenic acids, two popular dietary PUFAs, were less effective. Dihomo-{gamma}-linolenic acid also inhibited fatty acid biosynthesis in 3T3-L1 preadipocytes and selective human breast cancer cell lines, including SKBR3 and MDAMB231. In addition to revealing a novel mechanism for the molecular recognition of a polyunsaturated fatty acyl chain, our results offer a new framework for developing potent FAS inhibitors as therapeutics against cancers and other diseases.

  3. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration.

  4. Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel.

    PubMed

    Blatti, Jillian L; Michaud, Jennifer; Burkart, Michael D

    2013-06-01

    Microalgae are a promising feedstock for biodiesel and other liquid fuels due to their fast growth rate, high lipid yields, and ability to grow in a broad range of environments. However, many microalgae achieve maximal lipid yields only under stress conditions hindering growth and providing compositions not ideal for biofuel applications. Metabolic engineering of algal fatty acid biosynthesis promises to create strains capable of economically producing fungible and sustainable biofuels. The algal fatty acid biosynthetic pathway has been deduced by homology to bacterial and plant systems, and much of our understanding is gleaned from basic studies in these systems. However, successful engineering of lipid metabolism in algae will necessitate a thorough characterization of the algal fatty acid synthase (FAS) including protein-protein interactions and regulation. This review describes recent efforts to engineer fatty acid biosynthesis toward optimizing microalgae as a biodiesel feedstock.

  5. [Biosynthesis of adipic acid].

    PubMed

    Han, Li; Chen, Wujiu; Yuan, Fei; Zhang, Yuanyuan; Wang, Qinhong; Ma, Yanhe

    2013-10-01

    Adipic acid is a six-carbon dicarboxylic acid, mainly for the production of polymers such as nylon, chemical fiber and engineering plastics. Its annual demand is close to 3 million tons worldwide. Currently, the industrial production of adipic acid is based on the oxidation of aromatics from non-renewable petroleum resources by chemo-catalytic processes. It is heavily polluted and unsustainable, and the possible alternative method for adipic acid production should be developed. In the past years, with the development of synthetic biology and metabolic engineering, green and clean biotechnological methods for adipic acid production attracted more attention. In this study, the research advances of adipic acid and its precursor production are reviewed, followed by addressing the perspective of the possible new pathways for adipic acid production.

  6. Salicylic Acid Biosynthesis and Metabolism

    PubMed Central

    Dempsey, D'Maris Amick; Vlot, A. Corina; Wildermuth, Mary C.; Klessig, Daniel F.

    2011-01-01

    Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented. PMID:22303280

  7. Biosynthesis and metabolism of salicylic acid.

    PubMed Central

    Lee, H I; León, J; Raskin, I

    1995-01-01

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-beta-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. PMID:11607533

  8. Biosynthesis and metabolism of salicylic acid

    SciTech Connect

    Lee, H.; Leon, J.; Raskin, I.

    1995-05-09

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-{beta}-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. 62 refs., 1 fig.

  9. Phenol biosynthesis in higher plants. Gallic acid

    PubMed Central

    Dewick, P. M.; Haslam, E.

    1969-01-01

    The biosynthesis of gallic acid in a number of higher plants was investigated by using l-[U-14C]phenylalanine, (−)-[G-14C]shikimic acid, d-[1-14C]glucose and d-[6-14C]glucose as tracers. The results are compared with those obtained similarly for caffeic acid and are interpreted in terms of the dehydrogenation of 5-dehydroshikimic acid as a normal route of metabolism for gallic acid. PMID:5807212

  10. Cyclopiazonic acid biosynthesis by Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA) is an indole-tetramic acid mycotoxin produced by some strains of Aspergillus flavus. Characterization of the CPA biosynthesis gene cluster confirmed that formation of CPA is via a three-enzyme pathway. This review examines the structure and organization of the CPA genes, elu...

  11. Pantothenic acid biosynthesis in zymomonas

    SciTech Connect

    Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V.

    2014-07-01

    Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

  12. Amino Acid Biosynthesis Pathways in Diatoms

    PubMed Central

    Bromke, Mariusz A.

    2013-01-01

    Amino acids are not only building blocks for proteins but serve as precursors for the synthesis of many metabolites with multiple functions in growth and other biological processes of a living organism. The biosynthesis of amino acids is tightly connected with central carbon, nitrogen and sulfur metabolism. Recent publication of genome sequences for two diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum created an opportunity for extensive studies on the structure of these metabolic pathways. Based on sequence homology found in the analyzed diatomal genes, the biosynthesis of amino acids in diatoms seems to be similar to higher plants. However, one of the most striking differences between the pathways in plants and in diatomas is that the latter possess and utilize the urea cycle. It serves as an important anaplerotic pathway for carbon fixation into amino acids and other N-containing compounds, which are essential for diatom growth and contribute to their high productivity. PMID:24957993

  13. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis.

    PubMed

    Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

    2016-12-01

    Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS.

  14. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis

    PubMed Central

    Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

    2016-01-01

    Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS. PMID:27874013

  15. Abscisic acid: biosynthesis, inactivation, homoeostasis and signalling.

    PubMed

    Dong, Ting; Park, Youngmin; Hwang, Inhwan

    2015-01-01

    The phytohormone abscisic acid (ABA) plays crucial roles in numerous physiological processes during plant growth and abiotic stress responses. The endogenous ABA level is controlled by complex regulatory mechanisms involving biosynthesis, catabolism, transport and signal transduction pathways. This complex regulatory network may target multiple levels, including transcription, translation and post-translational regulation of genes involved in ABA responses. Most of the genes involved in ABA biosynthesis, catabolism and transport have been characterized. The local ABA concentration is critical for initiating ABA-mediated signalling during plant development and in response to environmental changes. In this chapter we discuss the mechanisms that regulate ABA biosynthesis, catabolism, transport and homoeostasis. We also present the findings of recent research on ABA perception by cellular receptors, and ABA signalling in response to cellular and environmental conditions.

  16. Teichoic acid biosynthesis as an antibiotic target.

    PubMed

    Pasquina, Lincoln W; Santa Maria, John P; Walker, Suzanne

    2013-10-01

    The relentless spread of antibiotic-resistant pathogens makes it imperative to develop new chemotherapeutic strategies to overcome infection. The bacterial cell wall has served as a rich source for both validated and unexploited pathways that are essential for virulence and survival. Lipoteichoic acids (LTAs) and wall teichoic acids (WTAs) are cell wall polymers that play fundamental roles in Gram-positive bacterial physiology and pathogenesis, and both have been proposed as novel antibacterial targets. Here we describe recent progress toward the discovery of teichoic acid biosynthesis inhibitors and their potential as antibiotics to combat Staphylococcus aureus infections.

  17. 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

  18. Fatty acid biosynthesis in pea root plastids

    SciTech Connect

    Stahl, R.J.; Sparace, S.A. )

    1989-04-01

    Fatty acid biosynthesis from (1-{sup 14}C)acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 {mu}M acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl{sub 2}, 1 mM each of the MnCl{sub 2} and glycerol-3-phosphate, 15 mM KHCO{sub 3}, and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 {mu}g/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO{sub 3}, divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg{sup 2+} and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor.

  19. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

  20. Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis.

    PubMed

    Tokuoka, Masafumi; Kikuchi, Tomoki; Shinohara, Yasutomo; Koyama, Akifumi; Iio, Shin-Ichiro; Kubota, Takaaki; Kobayashi, Jun'ichi; Koyama, Yasuji; Totsuka, Akira; Shindo, Hitoshi; Sato, Kazuo

    2015-01-01

    Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.

  1. Retinoic acid: its biosynthesis and metabolism.

    PubMed

    Napoli, J L

    1999-01-01

    This article presents a model that integrates the functions of retinoid-binding proteins with retinoid metabolism. One of these proteins, the widely expressed (throughout retinoid target tissues and in all vertebrates) and highly conserved cellular retinol-binding protein (CRBP), sequesters retinol in an internal binding pocket that segregates it from the intracellular milieu. The CRBP-retinol complex appears to be the quantitatively major form of retinol in vivo, and may protect the promiscuous substrate from nonenzymatic degradation and/or non-specific enzymes. For example, at least seven types of dehydrogenases catalyze retinal synthesis from unbound retinol in vitro (NAD+ vs. NADP+ dependent, cytosolic vs. microsomal, short-chain dehydrogenases/reductases vs. medium-chain alcohol dehydrogenases). But only a fraction of these (some of the short-chain de-hydrogenases/reductases) have the fascinating additional ability of catalyzing retinal synthesis from CRBP-bound retinol as well. Similarly, CRBP and/or other retinoid-binding proteins function in the synthesis of retinal esters, the reduction of retinal generated from intestinal beta-carotene metabolism, and retinoic acid metabolism. The discussion details the evidence supporting an integrated model of retinoid-binding protein/metabolism. Also addressed are retinoid-androgen interactions and evidence incompatible with ethanol causing fetal alcohol syndrome by competing directly with retinol dehydrogenation to impair retinoic acid biosynthesis.

  2. Biosynthesis of very long chain fatty acids in Trypanosoma cruzi.

    PubMed

    Livore, Verónica I; Uttaro, Antonio D

    2015-01-01

    Trypanosoma brucei and Trypanosoma cruzi showed similar fatty acid (FA) compositions, having a high proportion of unsaturated FAs, mainly 18:2Δ9,12 (23-39%) and 18:1Δ9 (11-17%). C22 polyunsaturated FAs are in significant amounts only in T. brucei (12-20%) but represent a mere 2% of total FAs in T. cruzi. Both species have also similar profiles of medium- and long-chain saturated FAs, from 14:0 to 20:0. Interestingly, procyclic and bloodstream forms of T. brucei lack very long chain FAs (VLCFAs), whereas epimastigotes and trypomastigotes of T. cruzi contain 22:0 (0.1-0.2%), 24:0 (1.5-2%), and 26:0 (0.1-0.2%). This is in agreement with the presence of an additional FA elongase gene (TcELO4) in T. cruzi. TcELO4 was expressed in a Saccharomyces cerevisiae mutant lacking the endogenous ScELO3, rescuing the synthesis of saturated and hydroxylated C26 FAs in the yeast. Expression of TcELO4 also rescued the synthetic lethality of a ScELO2, ScELO3 double mutation, and the VLCFA profile of the transformed yeast was similar to that found in T. cruzi. By identifying TcELO4 as the enzyme responsible for the elongation of FA from 16:0 and 18:0 up to 26:0, with 24:0 being the preferred product, this work completed the characterization of FA elongases in Trypanosoma spp.

  3. A novel cisplatin mediated apoptosis pathway is associated with acid sphingomyelinase and FAS proapoptotic protein activation in ovarian cancer.

    PubMed

    Maurmann, L; Belkacemi, L; Adams, N R; Majmudar, P M; Moghaddas, S; Bose, R N

    2015-07-01

    Platinum-based anticancer drugs, including cisplatin and carboplatin, have been cornerstones in the treatment of solid tumors. We report here that these DNA-damaging agents, particularly cisplatin, induce apoptosis through plasma membrane disruption, triggering FAS death receptor via mitochondrial (intrinsic) pathways. Our objectives were to: quantify the composition of membrane metabolites; and determine the potential involvement of acid sphingomyelinase (ASMase) in the FAS-mediated apoptosis in ovarian cancer after cisplatin treatment. The resulting analysis revealed enhanced apoptosis as measured by: increased phosphocholine, and glycerophosphocholine; elevated cellular energetics; and phosphocreatine and nucleoside triphosphate concentrations. The plasma membrane alterations were accompanied by increased ASMase activity, leading to the upregulation of FAS, FASL and related pro-apoptotic BAX and PUMA genes. Moreover FAS, FASL, BAX, PUMA, CASPASE-3 and -9 proteins were upregulated. Our findings implicate ASMase activity and the intrinsic pathways in cisplatin-mediated membrane demise, and contribute to our understanding of the mechanisms by which ovarian tumors may become resistant to cisplatin.

  4. Mechanisms of cancer chemoprevention by hop bitter acids (beer aroma) through induction of apoptosis mediated by Fas and caspase cascades.

    PubMed

    Chen, Wei-Jen; Lin, Jen-Kun

    2004-01-14

    The bitter acids of hops (Humulus lupulus L.) mainly consist of alpha-acids, beta-acids, and their oxidation products that contribute the unique aroma of the beer beverage. Hop bitter acids displayed a strong growth inhibitory effect against human leukemia HL-60 cells, with an estimated IC(50) value of 8.67 microg/mL, but were less effective against human histolytic lymphoma U937 cells. Induction of apoptosis was confirmed in HL-60 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by dissipation of mitochondrial membrane potential, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of PARP and DFF-45 were accompanied with activation of caspase-9 and -3 triggered by hop bitter acids in HL-60 cells. The change in the expression of Bcl-2, Bcl-X(L), and Bax in response to hop bitter acids was studied, and the Bcl-2 protein level slightly decreased; however, the Bcl-X(L) protein level was obviously decreased, whereas the Bax protein level was dramatically increased, indicating that the control of Bcl-2 family proteins by hop bitter acids might participate in the disruption of mitochondrial integrity. In addition, the results showed that hop bitter acids promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in hop bitter acids-induced cells. Taken together, these findings suggest that a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to cell death induced by hop bitter acids. The induction of apoptosis by hop bitter acids may offer a pivotal mechanism for their chemopreventive action.

  5. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines w...

  6. Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

    PubMed Central

    Norman, Shirley M.; Poling, Stephen M.; Maier, Vincent P.; Orme, Edward D.

    1983-01-01

    The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds. PMID:16662775

  7. Regulation of collagen biosynthesis by ascorbic acid: a review.

    PubMed Central

    Pinnell, S. R.

    1985-01-01

    L-ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L-ascorbic acid preferentially stimulates collagen synthesis in a manner which appears unrelated to the effect of L-ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L-ascorbic acid preferentially stimulates collagen-specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture. PMID:3008449

  8. Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?1[OPEN

    PubMed Central

    Nichols, David S.; Smith, Jason; Chourey, Prem S.; McAdam, Erin L.; Quittenden, Laura

    2016-01-01

    The biosynthesis of the main auxin in plants (indole-3-acetic acid [IAA]) has been elucidated recently and is thought to involve the sequential conversion of Trp to indole-3-pyruvic acid to IAA. However, the pathway leading to a less well studied auxin, phenylacetic acid (PAA), remains unclear. Here, we present evidence from metabolism experiments that PAA is synthesized from the amino acid Phe, via phenylpyruvate. In pea (Pisum sativum), the reverse reaction, phenylpyruvate to Phe, is also demonstrated. However, despite similarities between the pathways leading to IAA and PAA, evidence from mutants in pea and maize (Zea mays) indicate that IAA biosynthetic enzymes are not the main enzymes for PAA biosynthesis. Instead, we identified a putative aromatic aminotransferase (PsArAT) from pea that may function in the PAA synthesis pathway. PMID:27208245

  9. 2-Octadecynoic acid as a dual life stage inhibitor of Plasmodium infections and plasmodial FAS-II enzymes.

    PubMed

    Carballeira, Néstor M; Bwalya, Angela Gono; Itoe, Maurice Ayamba; Andricopulo, Adriano D; Cordero-Maldonado, María Lorena; Kaiser, Marcel; Mota, Maria M; Crawford, Alexander D; Guido, Rafael V C; Tasdemir, Deniz

    2014-09-01

    The malaria parasite Plasmodium goes through two life stages in the human host, a non-symptomatic liver stage (LS) followed by a blood stage with all clinical manifestation of the disease. In this study, we investigated a series of 2-alkynoic fatty acids (2-AFAs) with chain lengths between 14 and 18 carbon atoms for dual in vitro activity against both life stages. 2-Octadecynoic acid (2-ODA) was identified as the best inhibitor of Plasmodium berghei parasites with ten times higher potency (IC50=0.34 μg/ml) than the control drug. In target determination studies, the same compound inhibited three Plasmodium falciparum FAS-II (PfFAS-II) elongation enzymes PfFabI, PfFabZ, and PfFabG with the lowest IC50 values (0.28-0.80 μg/ml, respectively). Molecular modeling studies provided insights into the molecular aspects underlying the inhibitory activity of this series of 2-AFAs and a likely explanation for the considerably different inhibition potentials. Blood stages of P. falciparum followed a similar trend where 2-ODA emerged as the most active compound, with 20 times less potency. The general toxicity and hepatotoxicity of 2-AFAs were evaluated by in vitro and in vivo methods in mammalian cell lines and zebrafish models, respectively. This study identifies 2-ODA as the most promising antiparasitic 2-AFA, particularly towards P. berghei parasites.

  10. Amino Acid Biosynthesis in the Halophilic Archaeon Haloarcula hispanica

    PubMed Central

    Hochuli, Michel; Patzelt, Heiko; Oesterhelt, Dieter; Wüthrich, Kurt; Szyperski, Thomas

    1999-01-01

    Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya. PMID:10322026

  11. Inhibition of Fatty Acid Synthase (FAS): A New Drug for Breast Cancer Chemoprevention

    DTIC Science & Technology

    2005-04-01

    induced obesity. Proc Natl Acad Sci U S A 99: 9498-9502, 2002. 6. Hakkak, R., Holley, A. W., Macleod, S. L ., Simpson, P. M., Fuchs, G. J., Jo, C. H...transgenic mice Patricia M Alli1, Michael L Pinn1, Elizabeth M Jaffee2, Jill M McFadden3 and Francis P Kuhajda*,1,2,4 Department of Pathology, The Johns...dependent densation of acetyl -CoA 1989). Inhibition of FAS in breast and prostate cancer cells led to apoptosis both in vitro and in vivo (Pizer et al

  12. GENETIC ANALYSIS OF ABSCISIC ACID BIOSYNTHESIS

    SciTech Connect

    MCCARTY D R

    2012-01-10

    The carotenoid cleavage dioxygenases (CCD) catalyze synthesis of a variety of apo-carotenoid secondary metabolites in plants, animals and bacteria. In plants, the reaction catalyzed by the 11, 12, 9-cis-epoxy carotenoid dioxygenase (NCED) is the first committed and key regulated step in synthesis of the plant hormone, abscisic acid (ABA). ABA is a key regulator of plant stress responses and has critical functions in normal root and seed development. The molecular mechanisms responsible for developmental control of ABA synthesis in plant tissues are poorly understood. Five of the nine CCD genes present in the Arabidopsis genome encode NCED's involved in control of ABA synthesis in the plant. This project is focused on functional analysis of these five AtNCED genes as a key to understanding developmental regulation of ABA synthesis and dissecting the role of ABA in plant development. For this purpose, the project developed a comprehensive set of gene knockouts in the AtNCED genes that facilitate genetic dissection of ABA synthesis. These mutants were used in combination with key molecular tools to address the following specific objectives: (1) the role of ABA synthesis in root development; (2) developmental control of ABA synthesis in seeds; (3) analysis of ATNCED over-expressers; (4) preliminary crystallography of the maize VP14 protein.

  13. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  14. Plant amino acid-derived vitamins: biosynthesis and function.

    PubMed

    Miret, Javier A; Munné-Bosch, Sergi

    2014-04-01

    Vitamins are essential organic compounds for humans, having lost the ability to de novo synthesize them. Hence, they represent dietary requirements, which are covered by plants as the main dietary source of most vitamins (through food or livestock's feed). Most vitamins synthesized by plants present amino acids as precursors (B1, B2, B3, B5, B7, B9 and E) and are therefore linked to plant nitrogen metabolism. Amino acids play different roles in their biosynthesis and metabolism, either incorporated into the backbone of the vitamin or as amino, sulfur or one-carbon group donors. There is a high natural variation in vitamin contents in crops and its exploitation through breeding, metabolic engineering and agronomic practices can enhance their nutritional quality. While the underlying biochemical roles of vitamins as cosubstrates or cofactors are usually common for most eukaryotes, the impact of vitamins B and E in metabolism and physiology can be quite different on plants and animals. Here, we first aim at giving an overview of the biosynthesis of amino acid-derived vitamins in plants, with a particular focus on how this knowledge can be exploited to increase vitamin contents in crops. Second, we will focus on the functions of these vitamins in both plants and animals (and humans in particular), to unravel common and specific roles for vitamins in evolutionary distant organisms, in which these amino acid-derived vitamins play, however, an essential role.

  15. Kinetic Modeling of Sunflower Grain Filling and Fatty Acid Biosynthesis.

    PubMed

    Durruty, Ignacio; Aguirrezábal, Luis A N; Echarte, María M

    2016-01-01

    Grain growth and oil biosynthesis are complex processes that involve various enzymes placed in different sub-cellular compartments of the grain. In order to understand the mechanisms controlling grain weight and composition, we need mathematical models capable of simulating the dynamic behavior of the main components of the grain during the grain filling stage. In this paper, we present a non-structured mechanistic kinetic model developed for sunflower grains. The model was first calibrated for sunflower hybrid ACA855. The calibrated model was able to predict the theoretical amount of carbohydrate equivalents allocated to the grain, grain growth and the dynamics of the oil and non-oil fraction, while considering maintenance requirements and leaf senescence. Incorporating into the model the serial-parallel nature of fatty acid biosynthesis permitted a good representation of the kinetics of palmitic, stearic, oleic, and linoleic acids production. A sensitivity analysis showed that the relative influence of input parameters changed along grain development. Grain growth was mostly affected by the specific growth parameter (μ') while fatty acid composition strongly depended on their own maximum specific rate parameters. The model was successfully applied to two additional hybrids (MG2 and DK3820). The proposed model can be the first building block toward the development of a more sophisticated model, capable of predicting the effects of environmental conditions on grain weight and composition, in a comprehensive and quantitative way.

  16. Kinetic Modeling of Sunflower Grain Filling and Fatty Acid Biosynthesis

    PubMed Central

    Durruty, Ignacio; Aguirrezábal, Luis A. N.; Echarte, María M.

    2016-01-01

    Grain growth and oil biosynthesis are complex processes that involve various enzymes placed in different sub-cellular compartments of the grain. In order to understand the mechanisms controlling grain weight and composition, we need mathematical models capable of simulating the dynamic behavior of the main components of the grain during the grain filling stage. In this paper, we present a non-structured mechanistic kinetic model developed for sunflower grains. The model was first calibrated for sunflower hybrid ACA855. The calibrated model was able to predict the theoretical amount of carbohydrate equivalents allocated to the grain, grain growth and the dynamics of the oil and non-oil fraction, while considering maintenance requirements and leaf senescence. Incorporating into the model the serial-parallel nature of fatty acid biosynthesis permitted a good representation of the kinetics of palmitic, stearic, oleic, and linoleic acids production. A sensitivity analysis showed that the relative influence of input parameters changed along grain development. Grain growth was mostly affected by the specific growth parameter (μ′) while fatty acid composition strongly depended on their own maximum specific rate parameters. The model was successfully applied to two additional hybrids (MG2 and DK3820). The proposed model can be the first building block toward the development of a more sophisticated model, capable of predicting the effects of environmental conditions on grain weight and composition, in a comprehensive and quantitative way. PMID:27242809

  17. Biosynthesis of myristic acid in luminescent bacteria. [Vibrio harveyi

    SciTech Connect

    Byers, D.M.

    1987-05-01

    In vivo pulse-label studies have demonstrated that luminescent bacteria can provide myritic acid (14:0) required for the synthesis of the luciferase substrate myristyl aldehyde. Luminescent wild type Vibrio harveyi incubated with (/sup 14/C) acetate in a nutrient-depleted medium accumulated substantial tree (/sup 14/C)fatty acid (up to 20% of the total lipid label). Radio-gas chromatography revealed that > 75% of the labeled fatty acid is 14:0. No free fatty acid was detected in wild type cells labeled prior to the development of bioluminescence in the exponential growth phase, or in a dark mutant of V. harveyi (mutant M17) that requires exogenous 14:0 for light emission. The preferential accumulation of 14:0 was not observed when wild type cells were labeled with (/sup 14/C)acetate in regular growth medium. Moreover, all V. harveyi strains exhibited similar fatty acid mass compositions regardless of the state of bioluminescence. Since earlier work has shown that a luminescence-related acyltransferase (defective in the M17 mutant) can catalyze the deacylation of fatty acyl-acyl carrier protein in vitro, the present results are consistent with a model in which this enzyme diverts 14:0 to the luminescence system during fatty acid biosynthesis. Under normal conditions, the supply of 14:0 by this pathway is tightly regulated such that bioluminescence development does not significantly alter the total fatty acid composition.

  18. Effect of low temperature on highly unsaturated fatty acid biosynthesis in activated sludge.

    PubMed

    He, Su; Ding, Li-Li; Xu, Ke; Geng, Jin-Ju; Ren, Hong-Qiang

    2016-07-01

    Low temperature is a limiting factor for the microbial activity of activated sludge for sewage treatment plant in winter. Highly unsaturated fatty acid (UFA) biosynthesis, phospholipid fatty acid (PLFA) constituents and microbial structure in activated sludge at low temperature were investigated. Over 12 gigabases of metagenomic sequence data were generated with the Illumina HiSeq 2000 platform. The result showed 43.11% of phospholipid fatty acid (PLFA) in the activated sludge participated in UFA biosynthesis, and γ-Linolenic could be converted to Arachidonic acid at low temperature. The highly UFA biosynthesis in activated sludge was n-6 highly UFA biosynthesis, rather than n-3 highly UFA biosynthesis. The microbial community structures of activated sludge were analyzed by PLFA and high-throughput sequencing (HiSeq) simultaneously. Acidovorax, Pseudomonas, Flavobacterium and Polaromonas occupied higher percentage at 5°C, and genetic changes of highly UFA biosynthesis derived from microbial community structures change.

  19. A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism.

    PubMed

    Cabruja, Matías; Mondino, Sonia; Tsai, Yi Ting; Lara, Julia; Gramajo, Hugo; Gago, Gabriela

    2017-02-01

    Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

  20. Pseudomonas aeruginosa Directly Shunts β-Oxidation Degradation Intermediates into De Novo Fatty Acid Biosynthesis

    PubMed Central

    Yuan, Yanqiu; Leeds, Jennifer A.

    2012-01-01

    We identified the fatty acid synthesis (FAS) initiation enzyme in Pseudomonas aeruginosa as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the accompanying article (Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of fabY can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in P. aeruginosa PAO1 intercepts C8-CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in Escherichia coli likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed in vitro substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in P. aeruginosa to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed. PMID:22753057

  1. Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium.

    PubMed

    Creelman, R A; Gage, D A; Stults, J T; Zeevaart, J A

    1987-11-01

    RESEARCH ON THE BIOSYNTHESIS OF ABSCISIC ACID (ABA) HAS FOCUSED PRIMARILY ON TWO PATHWAYS: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in (18)O(2). It was found that in stressed leaves three atoms of (18)O from (18)O(2) are incorporated into the ABA molecule, and that the amount of (18)O incorporated increases with time. One (18)O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in (18)O(2) shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more (18)O into the tertiary hydroxyl group at C-1' after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, (18)O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied (14)C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional (18)O incorporated during 8'-hydroxylation of ABA to phaseic acid.

  2. Carnosic acid biosynthesis elucidated by a synthetic biology platform

    PubMed Central

    Ignea, Codruta; Athanasakoglou, Anastasia; Ioannou, Efstathia; Georgantea, Panagiota; Trikka, Fotini A.; Loupassaki, Sofia; Roussis, Vassilios; Makris, Antonios M.

    2016-01-01

    Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial “chassis” have provided access to important industrial compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis. Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies. PMID:26976595

  3. Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

    PubMed Central

    2010-01-01

    Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source. PMID:20184738

  4. Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters▿ ‡

    PubMed Central

    Geng, Haifeng; Bruhn, Jesper Bartholin; Nielsen, Kristian F.; Gram, Lone; Belas, Robert

    2008-01-01

    The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda−) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed. PMID:18192410

  5. Biosynthesis of Ascorbic Acid in Legume Root Nodules1

    PubMed Central

    Matamoros, Manuel A.; Loscos, Jorge; Coronado, Maria J.; Ramos, Javier; Sato, Shusei; Testillano, Pilar S.; Tabata, Satoshi; Becana, Manuel

    2006-01-01

    Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by l-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling. PMID:16766673

  6. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    PubMed Central

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  7. Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

    PubMed

    Lu, J Z; Muench, S P; Allary, M; Campbell, S; Roberts, C W; Mui, E; McLeod, R L; Rice, D W; Prigge, S T

    2007-12-01

    Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

  8. Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1)

    PubMed Central

    Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; Allen, Eric E.; Kalyuzhnaya, Marina G.

    2017-01-01

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph. PMID:28119683

  9. Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants

    SciTech Connect

    Voelker, T.A.; Worrell, A.C.; Anderson, L.; Bleibaum, J.; Fan, C.; Hawkins, D.J.; Radke, S.E.; Davies, H.M. )

    1992-07-03

    Medium-chain fatty acids (FAs), found in storage lipids of certain plants, are an important renewable resource. Seeds of undomesticated California bay accumulate laurate (12:0), and a 12:0-acyl-carrier protein thioesterase (BTE) has been purified from this tissue. Sequencing of BTE enabled the cloning of a complementary DNA coding for a plastid-targeted preprotein. Expression of the complementary DNA in the seeds of Arabidopsis thaliana resulted in BTE activity, and medium chains accumulated at the expense of long-chain ({ge}16) FAs. Laurate became the most abundant FA species and was deposited in the storage triacylglycerols. These results demonstrate a mechanism for medium-chain FA synthesis in plants.

  10. Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae Porphyridium purpureum.

    PubMed

    Su, Gaomin; Jiao, Kailin; Li, Zheng; Guo, Xiaoyi; Chang, Jingyu; Ndikubwimana, Theoneste; Sun, Yong; Zeng, Xianhai; Lu, Yinghua; Lin, Lu

    2016-07-01

    Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04-1.2 g/L), temperature (25, 30 and 33 °C) and phosphate (0.00-0.14 g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO3 dose of 0.8 g/L and moderate temperature of 30 °C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38 mg/L and ARA yield of 159.74 mg/L were obtained at a phosphate concentration of 0.035 g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ∆6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ∆17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.

  11. The Effect of TRANSPARENT TESTA2 on Seed Fatty Acid Biosynthesis and Tolerance to Environmental Stresses during Young Seedling Establishment in Arabidopsis1[W][OA

    PubMed Central

    Chen, Mingxun; Wang, Zhong; Zhu, Yana; Li, Zhilan; Hussain, Nazim; Xuan, Lijie; Guo, Wanli; Zhang, Guoping; Jiang, Lixi

    2012-01-01

    In plants, fatty acids (FAs) and FA-derived complex lipids are major carbon and energy reserves in seeds. They are essential components of cellular membranes and cellular signal or hormone molecules. Although TRANSPARENT TESTA2 (TT2) is well studied for its function in regulating proanthocyanidin biosynthesis in the seed coat, little attention has been given to its role in affecting seed FA accumulation and tolerance to environmental stresses. We demonstrate that the tt2 mutation remarkably increased the seed FA content, decreased seed weight, and altered the FA composition. The increase in FA content in the tt2 seeds was due to the relative decrease of seed coat proportion as well as the more efficient FA synthesis in the tt2 embryo. Microarray analysis revealed that tt2 mutation up-regulated a group of genes critical to FA biosynthesis and embryonic development. The mutation also altered the gene expressions that respond to stress. The microarray analysis discovered that the increase in FA accumulation of the tt2 seeds were accompanied by the significant up-regulation of FUSCA3, a transcriptional factor for embryonic development and FATTY ACID ELONGASE1, which catalyzes the elongation of FA chains. Moreover, lower seed protein accumulation during seed maturation also contributed to the increased seed FA accumulation in tt2 mutants. This study advances the understanding of the TT2 gene in seed FA accumulation and abiotic stresses during seed germination and seedling establishment. PMID:22879396

  12. Evolution of the biosynthesis of the branched-chain amino acids

    NASA Technical Reports Server (NTRS)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-01-01

    The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  13. Evolution of the biosynthesis of the branched-chain amino acids

    NASA Astrophysics Data System (ADS)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-06-01

    The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from α-ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  14. Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1)

    DOE PAGES

    Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; ...

    2017-01-10

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less

  15. Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis.

    PubMed Central

    Elkins, Jonathan M; Clifton, Ian J; Hernández, Helena; Doan, Linh X; Robinson, Carol V; Schofield, Christopher J; Hewitson, Kirsty S

    2002-01-01

    During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding. PMID:12020346

  16. Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although the biosynthesis of oxalic acid is known to occur in a number of bacteria, the mechanism(s) regulating its production remains largely unknown. To date, there is no report on the identification of an oxalic acid biosynthetic pathway gene from bacteria. In an attempt to identify such a gene...

  17. Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis

    SciTech Connect

    Leon, J.; Shulaev, V.; Yalpani, N.

    1995-10-24

    Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicyclic acid with {sup 18}O{sub 2} suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation of benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[{sup 35}S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes. 21 refs., 6 figs., 1 tab.

  18. Microbial biosynthesis and secretion of l-malic acid and its applications.

    PubMed

    Chi, Zhe; Wang, Zhi-Peng; Wang, Guang-Yuan; Khan, Ibrar; Chi, Zhen-Ming

    2016-01-01

    l-Malic acid has many uses in food, beverage, pharmaceutical, chemical and medical industries. It can be produced by one-step fermentation, enzymatic transformation of fumaric acid to l-malate and acid hydrolysis of polymalic acid. However, the process for one-step fermentation is preferred as it has many advantages over any other process. The pathways of l-malic acid biosynthesis in microorganisms are partially clear and three metabolic pathways including non-oxidative pathway, oxidative pathway and glyoxylate cycle for the production of l-malic acid from glucose have been identified. Usually, high levels of l-malate are produced under the nitrogen starvation conditions, l-malate, as a calcium salt, is secreted from microbial cells and CaCO3 can play an important role in calcium malate biosynthesis and regulation. However, it is still unclear how it is secreted into the medium. To enhance l-malate biosynthesis and secretion by microbial cells, it is very important to study the mechanisms of l-malic acid biosynthesis and secretion at enzymatic and molecular levels.

  19. Autoxidated linolenic acid inhibits aflatoxin biosynthesis in Aspergillus flavus via oxylipin species.

    PubMed

    Yan, Shijuan; Liang, Yating; Zhang, Jindan; Chen, Zhuang; Liu, Chun-Ming

    2015-08-01

    Aflatoxins produced by Aspergillus species are among the most toxic and carcinogenic compounds in nature. Although it has been known for a long time that seeds with high oil content are more susceptible to aflatoxin contamination, the role of fatty acids in aflatoxin biosynthesis remains controversial. Here we demonstrate in A. flavus that both the saturated stearic acid (C18:0) and the polyunsaturated linolenic acid (C18:3) promoted aflatoxin production, while C18:3, but not C18:0, inhibited aflatoxin biosynthesis after exposure to air for several hours. Further experiments showed that autoxidated C18:3 promoted mycelial growth, sporulation, and kojic acid production, but inhibited the expression of genes in the AF biosynthetic gene cluster. Mass spectrometry analyses of autoxidated C18:3 fractions that were able to inhibit aflatoxin biosynthesis led to the identification of multiple oxylipin species. These results may help to clarify the role of fatty acids in aflatoxin biosynthesis, and may explain why controversial results have been obtained for fatty acids in the past.

  20. Foreign gene recruitment to the fatty acid biosynthesis pathway in diatoms.

    PubMed

    Chan, Cheong Xin; Baglivi, Francesca L; Jenkins, Christina E; Bhattacharya, Debashish

    2013-09-01

    Diatoms are highly successful marine and freshwater algae that contribute up to 20% of global carbon fixation. These species are leading candidates for biofuel production owing to ease of culturing and high fatty acid content. To assist in strain improvement and downstream applications for potential use as a biofuel, it is important to understand the evolution of lipid biosynthesis in diatoms. The evolutionary history of diatoms is however complicated by likely multiple endosymbioses involving the capture of foreign cells and horizontal gene transfer into the host genome. Using a phylogenomic approach, we assessed the evolutionary history of 12 diatom genes putatively encoding functions related to lipid biosynthesis. We found evidence of gene transfer likely from a green algal source for seven of these genes, with the remaining showing either vertical inheritance or evolutionary histories too complicated to interpret given current genome data. The functions of horizontally transferred genes encompass all aspects of lipid biosynthesis (initiation, biosynthesis, and desaturation of fatty acids) as well as fatty acid elongation, and are not restricted to plastid-targeted proteins. Our findings demonstrate that the transfer, duplication, and subfunctionalization of genes were key steps in the evolution of lipid biosynthesis in diatoms and other photosynthetic eukaryotes. This target pathway for biofuel research is highly chimeric and surprisingly, our results suggest that research done on related genes in green algae may have application to diatom models.

  1. Indole-3-Acetic Acid Biosynthesis in Colletotrichum gloeosporioides f. sp. aeschynomene

    PubMed Central

    Robinson, M.; Riov, J.; Sharon, A.

    1998-01-01

    We characterized the biosynthesis of indole-3-acetic acid by the mycoherbicide Colletotrichum gloeosporioides f. sp. aeschynomene. Auxin production was tryptophan dependent. Compounds from the indole-3-acetamide and indole-3-pyruvic acid pathways were detected in culture filtrates. Feeding experiments and in vitro assay confirmed the presence of both pathways. Indole-3-acetamide was the major pathway utilized by the fungus to produce indole-3-acetic acid in culture. PMID:9835603

  2. Analysis of putative nonulosonic acid biosynthesis pathways in Archaea reveals a complex evolutionary history.

    PubMed

    Kandiba, Lina; Eichler, Jerry

    2013-08-01

    Sialic acids and the other nonulosonic acid sugars, legionaminic acid and pseudaminic acid, are nine carbon-containing sugars that can be detected as components of the glycans decorating proteins and other molecules in Eukarya and Bacteria. Yet, despite the prevalence of N-glycosylation in Archaea and the variety of sugars recruited for the archaeal version of this post-translational modification, only a single report of a nonulosonic acid sugar in an archaeal N-linked glycan has appeared. Hence, to obtain a clearer picture of nonulosonic acid sugar biosynthesis capability in Archaea, 122 sequenced genomes were scanned for the presence of genes involved in the biogenesis of these sugars. The results reveal that while Archaea and Bacteria share a common route of sialic acid biosynthesis, numerous archaeal nonulosonic acid sugar biosynthesis pathway components were acquired from elsewhere via various routes. Still, the limited number of Archaea encoding components involved in the synthesis of nonulosonic acid sugars implies that such saccharides are not major components of glycans in this domain.

  3. Dietary Polyunsaturated Fatty Acids and Inflammation: The Role of Phospholipid Biosynthesis

    PubMed Central

    Raphael, William; Sordillo, Lorraine M.

    2013-01-01

    The composition of fatty acids in the diets of both human and domestic animal species can regulate inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some ω-6 polyunsaturated fatty acids (PUFA), and subsequent incorporation into membrane phospholipids. Inflammation, however, can be resolved with ingestion of other fatty acids, such as ω-3 PUFA. The influence of dietary PUFA on phospholipid composition is influenced by factors that control phospholipid biosynthesis within cellular membranes, such as preferential incorporation of some fatty acids, competition between newly ingested PUFA and fatty acids released from stores such as adipose, and the impacts of carbohydrate metabolism and physiological state. The objective of this review is to explain these factors as potential obstacles to manipulating PUFA composition of tissue phospholipids by specific dietary fatty acids. A better understanding of the factors that influence how dietary fatty acids can be incorporated into phospholipids may lead to nutritional intervention strategies that optimize health. PMID:24152446

  4. Isolated etioplasts as test system for inhibitors of fatty acid biosynthesis

    SciTech Connect

    Lichtenthaler, H.K.; Kobek, K. )

    1989-04-01

    Isolated intact chloroplasts of mono- and dicotyledonous plants possess the capacity for de novo fatty acid biosynthesis, starting from {sup 14}C-acetate. These can be taken as test system for herbicides affecting fatty acid biosynthesis as shown earlier in our laboratory. The incorporation rates of acetate into the total fatty acids depend on the photosynthetic cofactors ATP and NADPH and amount in the light to 33 kBq (oat) and 39 kBq (pea) per mg chlorophyll x h, whereas in the dark only ca. 10% of these rates are obtained. In order to establish a test system, which is fully independent of light, we isolated and characterized etioplast fractions from oat and pea seedlings with a very high capacity of de novo fatty acid biosynthesis (500 and 400 kBq per mg carotenoids in a 20 min period). This activity was blocked by herbicides such as cycloxydim, sethoxydim and diclofop in a dose-dependent manner. This new test system has the great advantage that one can verify whether inhibitors of photosynthesis affect fatty acid biosynthesis.

  5. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    PubMed

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  6. Effect of precipitation, geographical location and biosynthesis on New Zealand milk powder bulk and fatty acids D/H ratios

    NASA Astrophysics Data System (ADS)

    Frew, R.; Emad Ehtesham, R.; Van Hale, R.; Hayman, A.; Baisden, T.

    2012-04-01

    D/H ratio measurements provide useful information for the investigation of biogeochemical influences on natural and agricultural produce, particularly with application to food traceability and authentication. Numerous studies have shown that variation of a product's D/H ratio is influenced by both environmental factors and biological processes. This study investigates the D/H ratio of New Zealand milk powder and individual fatty acids, and causal determinants of isotopic variation. One of the key environmental factors is precipitation, and the D/H ratio "isoscaping" of NZ has been undertaken. New Zealand provides a unique geography for these kinds of study in terms of proximity to the ocean and natural geographical variability from sea level to elevations as high as 3700 m. Milk powder samples were collected from different geographical regions from milk processing units, which were supplied by producers in the immediate region. H/D ratios of bulk milk powder and of individual fatty acids were determined. Initial comparison of the precipitation and milk powder bulk D/H data show a very good differentiation from north to southernmost parts of New Zealand and a relation between rain and milk bulk D/H abundance ratio. Almost 98% of milk FAs are in the form of triglycerides that have been extracted and hydrolysed to free FAs. Free FAs were esterified and analyzed with GC-IRMS. Individual FAs show variation in D/H ratio, and all values are depleted relative to the precipitation data. The difference in D/H ratio amongst individual FAs reflects the geographical environment and biological processes i.e. micro-organisms activity in the rumen of the cow. Short chain FAs (less than 8 carbons), particularly C4 (Butyric acid), appear to be key determinants. The variation in the data can be rationalized using statistical multivariate analysis.

  7. Direct biosynthesis of adipic acid from a synthetic pathway in recombinant Escherichia coli.

    PubMed

    Yu, Jia-Le; Xia, Xiao-Xia; Zhong, Jian-Jiang; Qian, Zhi-Gang

    2014-12-01

    The C6 dicarboxylic acid, adipic acid, is an important platform chemical in industry. Biobased production of adipic acid is a promising alternative to the current petrochemical route. Here, we report biosynthesis of adipic acid using an artificial pathway inspired by the reversal of beta-oxidation of dicarboxylic acids. The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction, dehydration, hydrogenation, and release of adipic acid from its thioester. The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipic acid into the culture medium. The production titer was increased by approximately 20-fold through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions and increasing availability of the condensation substrates. This work demonstrates direct biosynthesis of adipic acid via non-natural synthetic pathway, which may enable its renewable production.

  8. Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure

    PubMed Central

    Lewis, Amanda L.; Desa, Nolan; Hansen, Elizabeth E.; Knirel, Yuriy A.; Gordon, Jeffrey I.; Gagneux, Pascal; Nizet, Victor; Varki, Ajit

    2009-01-01

    Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phylogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of ≈1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the “unique” Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars. PMID:19666579

  9. Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae).

    PubMed

    Chen, Guanqun; Wang, Baobei; Han, Danxiang; Sommerfeld, Milton; Lu, Yinghua; Chen, Feng; Hu, Qiang

    2015-01-01

    Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H. pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H. pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H. pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H. pluvialis.

  10. Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering.

    PubMed

    Beld, Joris; Lee, D John; Burkart, Michael D

    2015-01-01

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

  11. Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

    SciTech Connect

    Beld, Joris; Lee, D. John; Burkart, Michael D.

    2014-10-20

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

  12. Fatty Acid Biosynthesis Revisited: Structure Elucidation and Metabolic Engineering

    PubMed Central

    Beld, Joris; Lee, D. John

    2014-01-01

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases’ many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field. PMID:25360565

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

    SciTech Connect

    Hayashi, H.; Miwa, A. )

    1989-11-01

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

  14. Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.

    PubMed

    Meyer, Astrid; Kirsch, Helene; Domergue, Frédéric; Abbadi, Amine; Sperling, Petra; Bauer, Jörg; Cirpus, Petra; Zank, Thorsten K; Moreau, Hervé; Roscoe, Thomas J; Zähringer, Ulrich; Heinz, Ernst

    2004-10-01

    In algae, the biosynthesis of docosahexaenoic acid (22:6omega3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5omega3; EPA) to 22:5omega3, which is required as a substrate for the final Delta4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Delta6-)C18-PUFAs and one specific for (Delta5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Delta6- and Delta5-elongases from T. pseudonana and O. tauri, respectively, with the Delta5- and Delta4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops.

  15. Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis

    PubMed Central

    Tyszka-Czochara, Malgorzata; Konieczny, Pawel; Majka, Marcin

    2017-01-01

    The efficacy of cancer treatments is often limited and associated with substantial toxicity. Appropriate combination of drug targeting specific mechanisms may regulate metabolism of tumor cells to reduce cancer cell growth and to improve survival. Therefore, we investigated the effects of anti-diabetic drug Metformin (Met) and a natural compound caffeic acid (trans-3,4-dihydroxycinnamic acid, CA) alone and in combination to treat an aggressive metastatic human cervical HTB-34 (ATCC CRL­1550) cancer cell line. CA at concentration of 100 µM, unlike Met at 10 mM, activated 5'-adenosine monophosphate-activated protein kinase (AMPK). What is more, CA contributed to the fueling of mitochondrial tricarboxylic acids (TCA) cycle with pyruvate by increasing Pyruvate Dehydrogenase Complex (PDH) activity, while Met promoted glucose catabolism to lactate. Met downregulated expression of enzymes of fatty acid de novo synthesis, such as ATP Citrate Lyase (ACLY), Fatty Acid Synthase (FAS), Fatty Acyl-CoA Elongase 6 (ELOVL6), and Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells. In conclusion, CA mediated reprogramming of glucose processing through TCA cycle via oxidative decarboxylation. The increased oxidative stress, as a result of CA treatment, sensitized cancer cells and, acting on cell biosynthesis and bioenergetics, made HTB-34 cells more susceptible to Met and successfully inhibited neoplastic cells. The combination of Metformin and caffeic acid to suppress cervical carcinoma cells by two independent mechanisms may provide a promising approach to cancer treatment. PMID:28230778

  16. Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis.

    PubMed Central

    Rajgarhia, V B; Strohl, W R

    1997-01-01

    The structure of the Streptomyces sp. strain C5 daunorubicin type II polyketide synthase (PKS) gene region is different from that of other known type II PKS gene clusters. Directly downstream of the genes encoding ketoacylsynthase alpha and beta (KS alpha, KS beta) are two genes (dpsC, dpsD) encoding proteins of unproven function, both absent from other type II PKS gene clusters. Also in contrast to other type II PKS clusters, the gene encoding the acyl carrier protein (ACP), dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS alpha and KS beta. In this work, we demonstrate that the minimal genes required to produce aklanonic acid in heterologous hosts are dpsG (ACP), dauI (regulatory activator), dpsA (KS alpha), dpsB (KS beta), dpsF (aromatase), dpsE (polyketide reductase), and dauG (putative deoxyaklanonic acid oxygenase). The two unusual open reading frames, dpsC (KASIII homolog lacking a known active site) and dpsD (acyltransferase homolog), are not required to synthesize aklanonic acid. Additionally, replacement of dpsD or dpsCD in Streptomyces sp. strain C5 with a neomycin resistance gene (aphI) results in mutant strains that still produced anthracyclines. PMID:9098068

  17. Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

    PubMed

    Goodrich-Tanrikulu, M; Stafford, A E; Lin, J T; Makapugay, M I; Fuller, G; McKeon, T A

    1994-10-01

    New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

  18. Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

    DOE PAGES

    Beld, Joris; Lee, D. John; Burkart, Michael D.

    2014-10-20

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understandingmore » of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.« less

  19. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Głuszuk, Katarzyna; Surażyński, Arkadiusz

    2014-01-01

    Aim The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Results Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts. PMID:25342885

  20. Metalloproteinase-mediated release of human Fas ligand

    PubMed Central

    1995-01-01

    Fas ligand (FasL) is a type II integral membrane protein homologous with tumor necrosis factor (TNF). Recent studies indicate that TNF is processed to yield the soluble cytokine by metalloproteinases at the cell surface of activated macrophages and T cells. In the present study, we investigated whether FasL is also released by metalloproteinases. Treatment with hydroxamic acid inhibitors of matrix metalloproteinases specifically led to accumulation of membrane-type FasL (p40) on the surface of human FasL cDNA transfectants and activated human T cells, as estimated by surface immunofluorescence and immunoprecipitation with newly established anti-human FasL monoclonal antibodies. This surface accumulation of mFasL was associated with the decrease of soluble FasL (p27) in the supernatant as estimated by quantitative ELISA and immunoprecipitation with anti-human FasL monoclonal antibodies. These results indicate that human FasL is efficiently released from the cell surface by metalloproteinases like TNF. PMID:7500022

  1. Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species.

    PubMed

    Onaka, Hiroyasu; Mori, Yukiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2011-01-01

    Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.

  2. Phorbic Acid Biosynthesis in the Latex Vessel System of Euphorbia

    PubMed Central

    Nordal, Arnold; Benson, A. A.

    1969-01-01

    Evidence is presented that phorbic acid is formed in the latex producing cell system, rather than in photosynthetic or chlorophyll-free tissues of Euphorbia resinifera Berg. When a branch of the plant was kept first in a 14CO2 atmosphere with 12 hr light-dark periods for 2 days and then left under natural conditions in the air outside for at least 2 to 3 days, radioactive phorbic acid was found in the latex. Phorbic acid synthesis appeared to be independent of the photosynthetic and respiratory activities of the plant. Besides phorbic acid 2 other major radioactive compounds were recognized in the latex, a glycoside or oligosaccharide, and a lipid belonging to the group of triterpenoid compounds characteristic of the latex in several species of Euphorbia. Images PMID:16657036

  3. Biosynthesis of pyruvic acid from glucose by Blastobotrys adeninivorans.

    PubMed

    Kamzolova, Svetlana V; Morgunov, Igor G

    2016-09-01

    The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid.

  4. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

    PubMed Central

    2013-01-01

    Background Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial

  5. The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

    PubMed Central

    Jamet, Stevie; Slama, Nawel; Domingues, Joana; Laval, Françoise; Texier, Pauline; Eynard, Nathalie; Quémard, Annaik; Peixoto, Antonio; Lemassu, Anne; Daffé, Mamadou; Cam, Kaymeuang

    2015-01-01

    Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs. PMID:26701652

  6. The Biosynthesis of Erucic Acid in Developing Embryos of Brassica rapa1

    PubMed Central

    Bao, Xiaoming; Pollard, Mike; Ohlrogge, John

    1998-01-01

    The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid. PMID:9733537

  7. Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

    PubMed

    Gupta, Vipul; Liu, Shujie; Ando, Hideki; Ishii, Ryohei; Tateno, Shumpei; Kaneko, Yuki; Yugami, Masato; Sakamoto, Satoshi; Yamaguchi, Yuki; Nureki, Osamu; Handa, Hiroshi

    2013-12-01

    Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

  8. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    PubMed

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  9. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68.

    PubMed

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  10. Effect of oxidoreduction potential on aroma biosynthesis by lactic acid bacteria in nonfat yogurt.

    PubMed

    Martin, F; Cachon, R; Pernin, K; De Coninck, J; Gervais, P; Guichard, E; Cayot, N

    2011-02-01

    The aim of this study was to investigate the effect of oxidoreduction potential (Eh) on the biosynthesis of aroma compounds by lactic acid bacteria in non-fat yogurt. The study was done with yogurts fermented by Lactobacillus bulgaricus and Streptococcus thermophilus. The Eh was modified by the application of different gaseous conditions (air, nitrogen, and nitrogen/hydrogen). Acetaldehyde, dimethyl sulfide, diacetyl, and pentane-2,3-dione, as the major endogenous odorant compounds of yogurt, were chosen as tracers for the biosynthesis of aroma compounds by lactic acid bacteria. Oxidative conditions favored the production of acetaldehyde, dimethyl sulfide, and diketones (diacetyl and pentane-2,3-dione). The Eh of the medium influences aroma production in yogurt by modifying the metabolic pathways of Lb. bulgaricus and Strep. thermophilus. The use of Eh as a control parameter during yogurt production could permit the control of aroma formation.

  11. The role of cis-carotenoids in abscisic acid biosynthesis.

    PubMed

    Parry, A D; Babiano, M J; Horgan, R

    1990-08-01

    Evidence has been obtained which is consistent with 9'-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography Rts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9'-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9'-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9'-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15-20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9'-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D2O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.

  12. Histidine Regulates Seed Oil Deposition through Abscisic Acid Biosynthesis and β-Oxidation.

    PubMed

    Ma, Huimin; Wang, Shui

    2016-10-01

    The storage compounds are deposited into plant seeds during maturation. As the model oilseed species, Arabidopsis (Arabidopsis thaliana) has long been studied for seed oil deposition. However, the regulation of this process remains unclear. Through genetic screen with a seed oil body-specific reporter, we isolated low oil1 (loo1) mutant. LOO1 was mapped to HISTIDINE BIOSYNTHESIS NUMBER 1A (HISN1A). HISN1A catalyzes the first step of His biosynthesis. Oil significantly decreased, and conversely proteins markedly increased in hisn1a mutants, indicating that HISN1A regulates both oil accumulation and the oil-protein balance. HISN1A was predominantly expressed in embryos and root tips. Accordingly, the hisn1a mutants exhibited developmental phenotype especially of seeds and roots. Transcriptional profiling displayed that β-oxidation was the major metabolic pathway downstream of HISN1A β-Oxidation was induced in hisn1a mutants, whereas it was reduced in 35S:HISN1A-transgenic plants. In plants, seed storage oil is broken-down by β-oxidation, which is controlled by abscisic acid (ABA). We found that His activated genes of ABA biosynthesis and correspondingly advanced ABA accumulation. Exogenous ABA rescued the defects of hisn1a mutants, whereas mutation of ABA DEFICIENT2, a key enzyme in ABA biosynthesis, blocked the effect of His on β-oxidation, indicating that ABA mediates His regulation in β-oxidation. Intriguingly, structural analysis showed that a potential His-binding domain was present in the general amino acid sensors GENERAL CONTROL NON-DEREPRESSIBLE2 and PII, suggesting that His may serve as a signal molecule. Taken together, our study reveals that His promotes plant seed oil deposition through ABA biosynthesis and β-oxidation.

  13. A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

    PubMed Central

    Cabruja, Matías; Mondino, Sonia; Tsai, Yi Ting; Lara, Julia; Gramajo, Hugo

    2017-01-01

    Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria. PMID:28228470

  14. 2-Keto acids based biosynthesis pathways for renewable fuels and chemicals.

    PubMed

    Tashiro, Yohei; Rodriguez, Gabriel M; Atsumi, Shota

    2015-03-01

    Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i.e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.

  15. Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis.

    PubMed

    Joo, Hyoe-Jin; Yim, Yong-Hyeon; Jeong, Pan-Young; Jin, You-Xun; Lee, Jeong-Eui; Kim, Heekyeong; Jeong, Seul-Ki; Chitwood, David J; Paik, Young-Ki

    2009-07-29

    Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.

  16. BnWRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed.

    PubMed

    Wu, Xue-Long; Liu, Zhi-Hong; Hu, Zhang-Hua; Huang, Rui-Zhi

    2014-06-01

    Photosynthesis in "green" seeds, such as rapeseed, soybean, and Arabidopsis, plays a substantial role in the improved efficiency of oil accumulation. However, the molecular mechanism underpinning the coordinated expression of fatty acid (FA) biosynthesis- and photosynthesis-related genes in such developing seeds remains to be elucidated. Here, we found that seed-specific overexpression of BnWRI1, a WRI1 homolog from rapeseed (Brassica napus cv. ZGY2), results in enhanced chlorophyll content in developing seeds and increased oil content and seed mass in matured seeds. BnWRI1 was co-expressed with BnBCCP and BnCAB, two marker genes of FA biosynthesis and photosynthesis during seed development, respectively. Overexpression of BnWRI1 increased expression of both marker genes. Further, the nuclear-localized BnWRI1 protein was found to act as a transcription activator. It could bind to the GT1-element and/or GCC-box, which are widespread in the upstream regions of genes involved in FA biosynthesis and photosynthesis pathways. Accordingly, BnWRI1 could interact with promoters of BCCP2 and LHB1B2 in vivo. These results suggested that BnWRI1 may coordinate FA biosynthesis and photosynthesis pathways in developing seeds via directly stimulating expression of GT1-element and/or GCC-box containing genes.

  17. Formula feeding potentiates docosahexaenoic and arachidonic acid biosynthesis in term and preterm baboon neonates.

    PubMed

    Sarkadi-Nagy, Eszter; Wijendran, Vasuki; Diau, Guan Yeu; Chao, Angela Chueh; Hsieh, Andrea T; Turpeinen, Anu; Lawrence, Peter; Nathanielsz, Peter W; Brenna, J Thomas

    2004-01-01

    Infant formulas supplemented with docosahexaenoic acid (DHA) and arachidonic acid (ARA) are now available in the United States; however, little is known about the factors that affect biosynthesis. Baboon neonates were assigned to one of four treatments: term, breast-fed; term, formula-fed; preterm (155 of 182 days gestation), formula-fed; and preterm, formula+DHA/ARA-fed. Standard formula had no DHA/ARA; supplemented formula had 0.61%wt DHA (0.3% of calories) and 1.21%wt ARA (0.6% of calories), and baboon breast milk contained 0.68 +/- 0.22%wt DHA and 0.62 +/- 0.12%wt ARA. At 14 days adjusted age, neonates received a combined oral dose of [U-13C]alpha-linolenic acid (LNA*) and [U-13C]linoleic acid (LA*), and tissues were analyzed 14 days after dose. Brain accretion of linolenic acid-derived DHA was approximately 3-fold greater for the formula groups than for the breast-fed group, and dietary DHA partially attenuated excess DHA synthesis among preterms. A similar, significant pattern was found in other organs. Brain linoleic acid-derived ARA accretion was significantly greater in the unsupplemented term group but not in the preterm groups compared with the breast-fed group. These data show that formula potentiates the biosynthesis/accretion of DHA/ARA in term and preterm neonates compared with breast-fed neonates and that the inclusion of DHA/ARA in preterm formula partially restores DHA/ARA biosynthesis to lower, breast-fed levels. Current formula DHA concentrations are inadequate to normalize long-chain polyunsaturated fatty acids synthesis to that of breast-fed levels.

  18. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis

    PubMed Central

    Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M.; Liu, Jie; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.

    2011-01-01

    The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (Vmax/km) for any of the complexes is 70–6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex–mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis. PMID:22160716

  19. Biosynthesis of car1ssol and carissic Acid.

    PubMed

    Nizami, S S; Khan, M A; Naim, Z; Islam, M N; Azeem, S W

    1993-01-01

    Carissa carandas belongs to family apocynaceae which consists of 300 genera and 1000 species. It is a large shrub with simple thorn and commonly cultivated throughout Pakistan for hedges and is called "Kakronda". The different parts of this plant have been used for various systems of medicine (Kirtikar et al., 1993). Cardiotonic activity was found in root of this plant (Rastogi et al., 1966; Rastogi et al., 1967; Vohra et al., 1963). This plant has been mentioned in the old chemical literature as purgative, stomachic, antihelmintics and antidote for snake-bite (Kirtikar., 1933). The physical characteristics of oil from the fruits of Carissa carandas were determined by using standard methods. In addition to this a study of sugars and amino acids from the fruits of this plant was also undertaken by the present authors (Naim et al., 1986). Our studies in the chemical investigation on this plant had led to the isolation of two new triterpene carissol Ia (Naim et al., 1985) and carissic acid lb (Naim et al., 1988).

  20. RGD-FasL Induces Apoptosis in Hepatocellular Carcinoma

    PubMed Central

    Liu, Zhongchen; Wang, Juan; Yin, Ping; Qiu, Jinhua; Liu, Ruizhen; Li, Wenzhu; Fan, Xin; Cheng, Xiaofeng; Chen, Caixia; Zhang, Jiakai; Zhuang, Guohong

    2009-01-01

    Despite impressive results obtained in animal models, the clinical use of Fas ligand (FasL) as an anticancer drug is limited by severe toxicity. Systemic toxicity of death ligands may be prevented by using genes encoding membrane-bound death ligands and by targeted transgene expression through either targeted transduction or targeted transcription. Selective induction of tumor cell death is a promising anticancer strategy. A fusion protein is created by fusing the extracellular domain of Fas ligand (FasL) to the peptide arginine-glycine-aspartic acid (RGD) that selectively targets avβ3-integrins on tumor endothelial cells. The purpose of this study is to evaluate the effects of RGD-FasL on tumor growth and survival in a murine hepatocellular carcinoma (HCC) tumor model. Treatment with RGD-FasL displaying an obvious suppressive effect on the HCC tumor model as compared to that with FasL (p < 0.05) and resulted in a more additive effect on tumor growth delay in this model. RGD-FasL treatment significantly enhanced mouse survival and caused no toxic effect, such as weight loss, organ failure, or other treatment-related toxicities. Apoptosis was detected by flow cytometric analysis and TUNEL assays; those results also showed that RGD-FasL is a more potent inducer of cell apoptosis for H22 and H9101 cell lines than FasL (p < 0.05). In conclusion, RGD-FasL appears to be a low-toxicity selective inducer of tumor cell death, which merits further investigation in preclinical and clinical studies. Furthermore, this approach offers a versatile technology for complexing target ligands with therapeutic recombinant proteins. To distinguish the anti-tumor effects of FasL in vivo, tumor and liver tissues were harvested to examine for evidence of necrotic cells, tumor cells, or apoptotic cells by Hematoxylin and eosin (H&E) staining. PMID:19728930

  1. 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

  2. Characterization of a Pipecolic Acid Biosynthesis Pathway Required for Systemic Acquired Resistance.

    PubMed

    Ding, Pingtao; Rekhter, Dmitrij; Ding, Yuli; Feussner, Kirstin; Busta, Lucas; Haroth, Sven; Xu, Shaohua; Li, Xin; Jetter, Reinhard; Feussner, Ivo; Zhang, Yuelin

    2016-10-01

    Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 (SARD4), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ(1)-piperideine-2-carboxylic acid (P2C). In Escherichia coli, expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use l-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment.

  3. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    PubMed Central

    Braga, Daniel; Hoffmeister, Dirk

    2016-01-01

    Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis. PMID:28144348

  4. PGC-1alpha activates CYP7A1 and bile acid biosynthesis.

    PubMed

    Shin, Dong-Ju; Campos, Jose A; Gil, Gregorio; Osborne, Timothy F

    2003-12-12

    Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

  5. [Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca].

    PubMed

    Yu, Shu-lin; Huang, Lu-qi; Yuan, Yuan; Qi, Lin-jie; Liu, Da-hui

    2015-03-01

    To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients.

  6. Biosynthesis of the 1,3,4,6-hexanetetracarboxylic acid subunit of methanofuran

    SciTech Connect

    White, R.H.

    1987-06-02

    /sup 2/H- and /sup 13/C-labeled precursors were used to establish the pathway for the biosynthesis of the 1,3,4,6-hexanetetracarboxylic acid (TCA) component of methanofuran, which is found in some methanogenic bacteria. The extent and position of incorporation of label into TCA were measured from the mass spectrum of the tetramethyl ester of TCA that was prepared from methanofuran present in cells grown in the presence of labeled acetate. (2,2,2-/sup 2/H/sub 3/)Acetate was found to incorporate deuterium into two separate sites of the TCA molecule, with one on each side of the symmetrical molecule. One site was found to be labeled 37% with deuterium, the same as for the glutamic acid present in the cells; the other site was labeled 77% with deuterium, the same as for the malonate-derived compounds in the cells. An analog of TCA, 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid, found in methanofuran isolated from Methanobrevibacter smithii, was found to incorporate /sup 13/C/sub 2/ units from (1,2-/sup 13/C/sub 2/)acetate into three positions of the molecule. One of the acetate /sup 13/C/sub 2/ units was incorporated into the non-hydroxyl-containing side of the molecule (carbons 4, 5, and 6 and the C-6 carboxylic acid group), and two acetate units were incorporated into the hydroxyl-containing side of the molecule (carbons 1, 2, and 3 and the C-1 carboxylic acid group). On the basis of this and additional information, it is concluded that TCA is biosynthesized by the condensation of ..cap alpha..-ketoglutaric acid with malonic acid to form 1,1,2,4-butanetetracarboxylic acid, which is further condensed with a second molecule of malonate, in a series of reactions analogous to those observed during fatty acid biosynthesis, to form TCA.

  7. Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles1[OPEN

    PubMed Central

    Vitulo, Nicola; Diretto, Gianfranco; Block, Maryse; Jouhet, Juliette; Meneghesso, Andrea; Valle, Giorgio; Giuliano, Giovanni; Maréchal, Eric

    2016-01-01

    The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level. PMID:27325666

  8. Biosynthesis of the respiratory toxin bongkrekic acid in the pathogenic bacterium Burkholderia gladioli.

    PubMed

    Moebius, Nadine; Ross, Claudia; Scherlach, Kirstin; Rohm, Barbara; Roth, Martin; Hertweck, Christian

    2012-09-21

    Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli λ Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like β-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant.

  9. Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

    PubMed

    Creelman, R A; Bell, E; Mullet, J E

    1992-07-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

  10. Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase.

    PubMed

    Sun, Jing; Lin, Yuheng; Shen, Xiaolin; Jain, Rachit; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2016-05-01

    3-Phenylpropionic acid (3PPA) and 3-(4-hydroxyphenyl) propionic acid (HPPA) are important commodity aromatic acids widely used in food, pharmaceutical and chemical industries. Currently, 3PPA and HPPA are mainly manufactured through chemical synthesis, which contains multiple steps involving toxic solvents and catalysts harmful to environment. Therefore, replacement of such existing petroleum-derived approaches with simple and environmentally friendly biological processes is highly desirable for manufacture of these chemicals. Here, for the first time we demonstrated the de novo biosynthesis of 3PPA and HPPA using simple carbon sources in E. coli by extending the cinnamic acids biosynthesis pathways through biological hydrogenation. We first screened 11 2-enoate reductases (ER) from nine microorganisms, leading to efficient conversion of cinnamic acid and p-coumaric acid to 3PPA and HPPA, respectively. Surprisingly, we found a strictly oxygen-sensitive Clostridia ER capable of functioning efficiently in E. coli even under aerobic conditions. On this basis, reconstitution of the full pathways led to the de novo production of 3PPA and HPPA and the accumulation of the intermediates (cinnamic acid and p-coumaric acid) with cell toxicity. To address this problem, different expression strategies were attempted to optimize individual enzyme׳s expression level and minimize intermediates accumulation. Finally, the titers of 3PPA and HPPA reached 366.77mg/L and 225.10mg/L in shake flasks, respectively. This study not only demonstrated the potential of microbial approach as an alternative to chemical process, but also proved the possibility of using oxygen-sensitive enzymes under aerobic conditions.

  11. Caffeic Acid Phenethyl Ester and Its Amide Analogue Are Potent Inhibitors of Leukotriene Biosynthesis in Human Polymorphonuclear Leukocytes

    PubMed Central

    Boudreau, Luc H.; Maillet, Jacques; LeBlanc, Luc M.; Jean-François, Jacques; Touaibia, Mohamed; Flamand, Nicolas; Surette, Marc E.

    2012-01-01

    Background 5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases. Methodology/Principal Findings Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC50 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC50 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC50 values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis. Conclusions CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis. PMID:22347509

  12. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications.

  13. A Novel Role for an ECF Sigma Factor in Fatty Acid Biosynthesis and Membrane Fluidity in Pseudomonas aeruginosa

    PubMed Central

    Boechat, Ana Laura; Kaihami, Gilberto Hideo; Politi, Mario José; Lépine, François; Baldini, Regina L.

    2013-01-01

    Extracytoplasmic function (ECF) sigma factors are members of cell-surface signaling systems, abundant in the opportunistic pathogen Pseudomonas aeruginosa. Twenty genes coding for ECF sigma factors are present in P. aeruginosa sequenced genomes, most of them being part of TonB systems related to iron uptake. In this work, poorly characterized sigma factors were overexpressed in strain PA14, in an attempt to understand their role in the bacterium´s physiology. Cultures overexpressing SigX displayed a biphasic growth curve, reaching stationary phase earlier than the control strain, followed by subsequent growth resumption. During the first stationary phase, most cells swell and die, but the remaining cells return to the wild type morphology and proceed to a second exponential growth. This is not due to compensatory mutations, since cells recovered from late time points and diluted into fresh medium repeated this behavior. Swollen cells have a more fluid membrane and contain higher amounts of shorter chain fatty acids. A proteomic analysis was performed to identify differentially expressed proteins due to overexpression of sigX, revealing the induction of several fatty acid synthesis (FAS) enzymes. Using qRT-PCR, we showed that at least one isoform from each of the FAS pathway enzymes were upregulated at the mRNA level in the SigX overexpressing strain thus pointing to a role for this ECF sigma factor in the FAS regulation in P. aeruginosa. PMID:24386415

  14. Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Miltyk, Wojciech; Galicka, Elżbieta; Przylipiak, Jerzy; Zaręba, Ilona; Surazynski, Arkadiusz

    2015-01-01

    Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take

  15. Biosynthesis of Polyunsaturated Fatty Acids in Marine Invertebrates: Recent Advances in Molecular Mechanisms

    PubMed Central

    Monroig, Óscar; Tocher, Douglas R.; Navarro, Juan C.

    2013-01-01

    Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs. PMID:24152561

  16. Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli

    PubMed Central

    Zhang, Junli; Kang, Zhen; Chen, Jian; Du, Guocheng

    2015-01-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. Here, we identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are also the major regulatory targets of the heme biosynthesis pathway. Interestingly, up-regulation of hemD and hemF benefited ALA accumulation whereas overexpression of hemB, hemG and hemH diminished ALA accumulation. Accordingly, by combinatorial overexpression of the hemA, hemL, hemD and hemF with different copy-number plasmids, the titer of ALA was improved to 3.25 g l−1. Furthermore, in combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX. This work has great potential to be scaled-up for microbial production of ALA and provides new important insights into the regulatory mechanism of the heme biosynthesis pathway. PMID:25716896

  17. Biosynthesis of polyunsaturated fatty acids in marine invertebrates: recent advances in molecular mechanisms.

    PubMed

    Monroig, Óscar; Tocher, Douglas R; Navarro, Juan C

    2013-10-21

    Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs.

  18. Modulation of endogenous indole-3-acetic acid biosynthesis in bacteroids within Medicago sativa nodules.

    PubMed

    Bianco, C; Senatore, B; Arbucci, S; Pieraccini, G; Defez, R

    2014-07-01

    To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on Medicago plant growth and dry weight production, we increased the synthesis of IAA in both free-living and symbiosis-stage rhizobial bacteroids during Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was applied to modify an 85-bp promoter sequence, driving the expression of iaaM and tms2 genes for IAA biosynthesis. A positive correlation was found between the higher expression of IAA biosynthetic genes in free-living bacteria and the increased production of IAA under both free-living and symbiotic conditions. Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA concentration, showed a significant (up to 73%) increase in the shoot fresh weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated by the wild-type strain. When these plants were analyzed by confocal microscopy, using an anti-IAA antibody, the strongest signal was observed in bacteroids of Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the senescent nodule zone. We show here a simple system to modulate endogenous IAA biosynthesis in bacteria nodulating legumes suitable to investigate which is the maximum level of IAA biosynthesis, resulting in the maximal increase of plant growth.

  19. Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids.

    PubMed

    Layton, Donovan S; Trinh, Cong T

    2016-08-01

    Volatile organic acids are byproducts of fermentative metabolism, for example, anaerobic digestion of lignocellulosic biomass or organic wastes, and are often times undesired inhibiting cell growth and reducing directed formation of the desired products. Here, we devised a general framework for upgrading these volatile organic acids to high-value esters that can be used as flavors, fragrances, solvents, and biofuels. This framework employs the acid-to-ester modules, consisting of an AAT (alcohol acyltransferase) plus ACT (acyl CoA transferase) submodule and an alcohol submodule, for co-fermentation of sugars and organic acids to acyl CoAs and alcohols to form a combinatorial library of esters. By assembling these modules with the engineered Escherichia coli modular chassis cell, we developed microbial manufacturing platforms to perform the following functions: (i) rapid in vivo screening of novel AATs for their catalytic activities; (ii) expanding combinatorial biosynthesis of unique fermentative esters; and (iii) upgrading volatile organic acids to esters using single or mixed cell cultures. To demonstrate this framework, we screened for a set of five unique and divergent AATs from multiple species, and were able to determine their novel activities as well as produce a library of 12 out of the 13 expected esters from co-fermentation of sugars and (C2-C6) volatile organic acids. We envision the developed framework to be valuable for in vivo characterization of a repertoire of not-well-characterized natural AATs, expanding the combinatorial biosynthesis of fermentative esters, and upgrading volatile organic acids to high-value esters. Biotechnol. Bioeng. 2016;113: 1764-1776. © 2016 Wiley Periodicals, Inc.

  20. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    SciTech Connect

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  1. Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1

    PubMed Central

    Xu, Zhaoxian; Sun, Zhuzhen; Li, Sha; Xu, Zheng; Cao, Changhong; Xu, Zongqi; Feng, Xiaohai; Xu, Hong

    2015-01-01

    Poly(L-diaminopropionic acid) (PDAP) is one of the four homopoly(amino acid)s that have been discovered in nature. However, the molecular mechanism of PDAP biosynthesis has yet to be described. In this work, the general layout of the PDAP biosynthetic pathway is characterised in Streptomyces albulus PD-1 by genome mining, gene disruption, heterologous expression and in vitro feeding experiments. As a result, L-diaminopropionic acid (L-DAP), which is the monomer of PDAP, is shown to be jointly synthesised by two protein homologues of cysteine synthetase and ornithine cyclodeaminase. Then, L-DAP is assembled into PDAP by a novel nonribosomal peptide synthetase (NRPS) with classical adenylation and peptidyl carrier protein domains. However, instead of the traditional condensation or thioesterase domain of NRPSs, this NRPS has seven transmembrane domains surrounding three tandem soluble domains at the C-terminus. As far as we know, this novel single-module NRPS structure has only been reported in poly(ε-L-lysine) synthetase. The similar NRPS structure of PDAP synthetase and poly(ε-L-lysine) synthetase may be a common characteristic of homopoly(amino acid)s synthetases. In this case, we may discover and/or design more homopoly(amino acid)s by mining this kind of novel NRPS structure in the future. PMID:26632244

  2. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco

    SciTech Connect

    Yalpani, N.; Leon, J.; Lawton, M.A.; Raskin, I. )

    1993-10-01

    Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied Sa biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of Sa accumulation is accompanied by a corresponding increase in the levels of benzoic acid. [sup 14]C-Tracer studies with cell suspensions and mock- or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [[sup 14]C] benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogenesis-related-1 proteins and increased resistance to TMV in benzoic acid but no in 0-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid. 27 refs., 7 figs., 1 tab.

  3. Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

    PubMed Central

    Geisler, Christoph; Jarvis, Donald L.

    2012-01-01

    The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems. PMID:23022569

  4. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  5. The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis.

    PubMed

    Wang, Zhen-Yu; Xiong, Liming; Li, Wenbo; Zhu, Jian-Kang; Zhu, Jianhua

    2011-05-01

    Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.

  6. A Novel Two-Gene Requirement for the Octanoyltransfer Reaction of Bacillus subtilis Lipoic Acid Biosynthesis

    PubMed Central

    Martin, Natalia; Christensen, Quin H.; Mansilla, María C.; Cronan, John E.; de Mendoza, Diego

    2011-01-01

    SUMMARY The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM, and ywfL which we have renamed lplJ, lipM and lipL, respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to E. coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulfur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilis ΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coli ΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion paper (Christensen et al., 2011) provide evidence that LipM and LipL catalyze sequential reactions in a novel pathway for lipoic acid biosynthesis. PMID:21338420

  7. Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis

    PubMed Central

    Wilson, Regina; Kumar, Pradeep; Parashar, Vijay; Vilchèze, Catherine; Veyron-Churlet, Romain; Freundlich, Joel S.; Barnes, S. Whitney; Walker, John R.; Szymonifka, Michael J.; Marchiano, Emily; Shenai, Shubhada; Colangeli, Roberto; Jacobs, William R.; Neiditch, Matthew B.; Kremer, Laurent

    2013-01-01

    We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis (Mtb) by the novel mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser-55 site in Pks13 conferred TP-resistance in Mtb. Over-expression of wild-type pks13 resulted in TP-resistance and over-expression of the F79S pks13 mutant conferred high-level resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type Mtb, but to a much lesser extent in TP-resistant Mtb. TP treatment was bactericidal and equivalent to the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment exhibited sterilizing activity. Computational-docking identified a possible TP-binding groove within the Pks13 ACP domain. This study confirms that Mtb Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway. PMID:23770708

  8. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  9. Identification of a Desaturase Involved in Mycolic Acid Biosynthesis in Mycobacterium smegmatis

    PubMed Central

    Singh, Albel; Varela, Cristian; Bhatt, Kiranmai; Veerapen, Natacha; Lee, Oona Y. C.; Wu, Houdini H. T.; Besra, Gurdyal S.; Minnikin, David E.; Fujiwara, Nagatoshi; Teramoto, Kanae; Bhatt, Apoorva

    2016-01-01

    Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria. PMID:27741286

  10. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    PubMed

    Blatti, Jillian L; Beld, Joris; Behnke, Craig A; Mendez, Michael; Mayfield, Stephen P; Burkart, Michael D

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

  11. Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae.

    PubMed

    Nakata, Paul A; He, Cixin

    2010-03-01

    Although the biosynthesis of oxalic acid is known to occur in a number of bacteria, the mechanism(s) regulating its production remains largely unknown. To date, there is no report on the identification of an oxalic acid biosynthetic pathway gene from bacteria. In an attempt to identify such a gene(s), a mutant screen was conducted using the simple oxalic acid-producing phytopathogenic bacterium, Burkholderia glumae. Four mutants that failed to produce oxalic acid were isolated from a transposon-mutagenized B. glumae library and named Burkholderia oxalate defective (Bod)1. DNA sequence analysis revealed that each mutant contained an insertion event at different sites in the same ORF, which we referred to as the oxalate biosynthetic component (obc)A locus. Complementation of the Bod1 mutant with the obcA gene, however, resulted only in a partial restoration of the oxalic acid-producing phenotype. Further complementation studies utilizing a larger DNA fragment encompassing the obcA locus coupled with deletion mutagenesis led to the identification of another ORF that we named the obcB locus, which was essential for higher levels of oxalic acid production. Transcript analysis indicated that both obcA and obcB are coexpressed and encoded on a single polycistron message.

  12. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    SciTech Connect

    Viola, Ronald E.; Faehnle, Christopher R.; Blanco, Julio; Moore, Roger A.; Liu, Xuying; Arachea, Buenafe T.; Pavlovsky, Alexander G.

    2013-02-28

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate {beta}-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

  13. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis

    PubMed Central

    Nair, Ramesh B.; Bastress, Kristen L.; Ruegger, Max O.; Denault, Jeff W.; Chapple, Clint

    2004-01-01

    Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP+-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall–esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes. PMID:14729911

  14. Sialylation of the Fas Death Receptor by ST6Gal-I Provides Protection against Fas-mediated Apoptosis in Colon Carcinoma Cells*

    PubMed Central

    Swindall, Amanda F.; Bellis, Susan L.

    2011-01-01

    The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2–6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2–6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2–6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2–6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2–6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis. PMID:21550977

  15. Biosynthesis of cyclopropyl long-chain fatty acids from cyclopropanecarboxylic acid by mammalian tissues in vitro

    PubMed Central

    Duncombe, W. G.; Rising, T. J.

    1968-01-01

    1. Radioactivity from cyclopropane[14C]carboxylic acid is incorporated into fatty acids in vitro by rat and guinea-pig adipose tissue, by rat liver slices and by the supernatant fraction of rat liver homogenate. 2. The labelled acids are different from endogenous straight-chain fatty acids, and evidence is produced that they consist of a cyclopropyl ring in the ω-position, the remainder of the chain being built up from C2 units (not derived from cyclopropanecarboxylic acid) in the normal way via the malonate pathway. 3. It is suggested that these unnatural acids have some metabolic effect related to the hypoglycaemic action of cyclopropanecarboxylic acid. PMID:5685874

  16. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

    PubMed Central

    Scheler, Ulschan; Brandt, Wolfgang; Porzel, Andrea; Rothe, Kathleen; Manzano, David; Božić, Dragana; Papaefthimiou, Dimitra; Balcke, Gerd Ulrich; Henning, Anja; Lohse, Swanhild; Marillonnet, Sylvestre; Kanellis, Angelos K.; Ferrer, Albert; Tissier, Alain

    2016-01-01

    Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities. PMID:27703160

  17. Amino acids biosynthesis and nitrogen assimilation pathways: a great genomic deletion during eukaryotes evolution

    PubMed Central

    2011-01-01

    Background Besides being building blocks for proteins, amino acids are also key metabolic intermediates in living cells. Surprisingly a variety of organisms are incapable of synthesizing some of them, thus named Essential Amino Acids (EAAs). How certain ancestral organisms successfully competed for survival after losing key genes involved in amino acids anabolism remains an open question. Comparative genomics searches on current protein databases including sequences from both complete and incomplete genomes among diverse taxonomic groups help us to understand amino acids auxotrophy distribution. Results Here, we applied a methodology based on clustering of homologous genes to seed sequences from autotrophic organisms Saccharomyces cerevisiae (yeast) and Arabidopsis thaliana (plant). Thus we depict evidences of presence/absence of EAA biosynthetic and nitrogen assimilation enzymes at phyla level. Results show broad loss of the phenotype of EAAs biosynthesis in several groups of eukaryotes, followed by multiple secondary gene losses. A subsequent inability for nitrogen assimilation is observed in derived metazoans. Conclusions A Great Deletion model is proposed here as a broad phenomenon generating the phenotype of amino acids essentiality followed, in metazoans, by organic nitrogen dependency. This phenomenon is probably associated to a relaxed selective pressure conferred by heterotrophy and, taking advantage of available homologous clustering tools, a complete and updated picture of it is provided. PMID:22369087

  18. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

    NASA Astrophysics Data System (ADS)

    Scheler, Ulschan; Brandt, Wolfgang; Porzel, Andrea; Rothe, Kathleen; Manzano, David; Božić, Dragana; Papaefthimiou, Dimitra; Balcke, Gerd Ulrich; Henning, Anja; Lohse, Swanhild; Marillonnet, Sylvestre; Kanellis, Angelos K.; Ferrer, Albert; Tissier, Alain

    2016-10-01

    Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

  19. Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

    PubMed

    Cao, Peng-Fei; Wu, Chen-Gao; Dang, Zhi-Hao; Shi, Liang; Jiang, Ai-Liang; Ren, Ang; Zhao, Ming-Wen

    2017-01-01

    We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

  20. Stimulatory Effects of Acibenzolar-S-Methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells.

    PubMed

    Ncube, Efficient N; Steenkamp, Paul A; Madala, Ntakadzeni E; Dubery, Ian A

    2016-01-01

    Centella asiatica is a perrenial herb that grows in tropical regions with numerous medicinal properties mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs) that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analyzed using a UHPLC-qTOF-MS platform. Data was processed and analyzed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid) and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the effects of precursor molecules on the levels of the CGAs were insignificant. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs.

  1. Stimulatory Effects of Acibenzolar-S-Methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells

    PubMed Central

    Ncube, Efficient N.; Steenkamp, Paul A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Centella asiatica is a perrenial herb that grows in tropical regions with numerous medicinal properties mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs) that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analyzed using a UHPLC-qTOF-MS platform. Data was processed and analyzed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid) and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the effects of precursor molecules on the levels of the CGAs were insignificant. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs. PMID:27733862

  2. Role of Acid Metabolism in Streptomyces coelicolor Morphological Differentiation and Antibiotic Biosynthesis

    PubMed Central

    Viollier, Patrick H.; Minas, Wolfgang; Dale, Glenn E.; Folcher, Marc; Thompson, Charles J.

    2001-01-01

    Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent Km values of CitA for acetyl coenzyme A (acetyl-CoA) (32 μM) and oxaloacetate (17 μM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD+, NADH, ATP, ADP, isocitrate, or α-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade. PMID:11325948

  3. Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis.

    PubMed

    Viollier, P H; Minas, W; Dale, G E; Folcher, M; Thompson, C J

    2001-05-01

    Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.

  4. Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-Deprived Response Dependent on Intact Creatine Biosynthesis

    PubMed Central

    Tang, Xiaohu; Keenan, Melissa M.; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J. Will; Freedland, Stephen J.; Murphy, Susan K.; Chi, Jen-Tsan

    2015-01-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  5. Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis.

    PubMed

    Tang, Xiaohu; Keenan, Melissa M; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J Will; Freedland, Stephen J; Murphy, Susan K; Chi, Jen-Tsan

    2015-04-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  6. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival

    PubMed Central

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A.; Pascon, Renata C.

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro. PMID:26162077

  7. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival.

    PubMed

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A; Pascon, Renata C

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.

  8. In Vitro Stepwise Reconstitution of Amino Acid Derived Vinyl Isocyanide Biosynthesis: Detection of an Elusive Intermediate.

    PubMed

    Chang, Wei-Chen; Sanyal, Dev; Huang, Jhih-Liang; Ittiamornkul, Kuljira; Zhu, Qin; Liu, Xinyu

    2017-02-17

    In vitro reconstitution of a newly discovered isonitrile synthase (AmbI1 and AmbI2) and the detection of an elusive intermediate (S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1 in indolyl vinyl isocyanide biogenesis are reported. The characterization of iron/2-oxoglutarate (Fe/2OG) dependent desaturases IsnB and AmbI3 sheds light on the possible mechanism underlying stereoselective alkene installation to complete the biosynthesis of (E)- and (Z)-3-(2-isocyanovinyl)-1H-indole 2 and 5. Establishment of a tractable isonitrile synthase system (AmbI1 and AmbI2) paves the way to elucidate the enigmatic enzyme mechanism for isocyanide formation.

  9. An in vitro system from maize seedlings for tryptophan-independent indole-3-acetic acid biosynthesis

    SciTech Connect

    Oestin, A.; Ilic, N.; Cohen, J.D.

    1999-01-01

    The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [{sup 14}C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [{sup 14}C]Trp nor [{sup 14}C]serine substituted for [{sup 14}C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.

  10. Resistance to herbicides inhibiting the biosynthesis of very-long-chain fatty acids.

    PubMed

    Busi, Roberto

    2014-09-01

    Herbicides that act by inhibiting the biosynthesis of very-long-chain fatty acids (VLCFAs) have been used to control grass weeds in major crops throughout the world for the past 60 years. VLCFA-inhibiting herbicides are generally highly selective in crops, induce similar symptoms in susceptible grasses and can be found within the herbicide groups classified by the HRAC as K3 and N. Even after many years of continuous use, only 12 grass weed species have evolved resistance to VLCFA-inhibiting herbicides. Here, the cases of resistance that have evolved in major grass weed species belonging to the Avena, Echinochloa and Lolium genera in three different agricultural systems are reviewed. In particular we explore the possible reasons why VLCFA herbicides have been slow to select resistant weeds, outline the herbicide mode of action and discuss the resistance mechanisms that are most likely to have been selected.

  11. Biosynthesis, structure and biological activity of hydroxyeicosatetraenoic acids in Hydra vulgaris.

    PubMed Central

    Di Marzo, V; De Petrocellis, L; Gianfrani, C; Cimino, G

    1993-01-01

    Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11% of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 1H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 11-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be: (1) associated with the cytosolic fraction of Hydra homogenates; (2) dependent on AA concentration, incubation time and protein amount in the homogenates; (3) unaffected by co-incubation with the 5- and 12-lipoxygenase inhibitors, 5,8,11-eicosatriynoic acid and nordihydroguaiaretic acid, the cyclo-oxygenase inhibitor, indomethacin, or the cytochrome P-450 inhibitors, proadifen and methoxalen. These results strongly suggest the presence of a very active (R)-11-lipoxygenase in H. vulgaris. The activity of both R and S enantiomers of synthetic 9-, 11- and 12-HETE and of 'endogenous' 11-HETE was studied on tentacle regeneration and bud formation in decapitated Hydra. Although almost all compounds tested inhibited budding, only endogenous 11-HETE and synthetic (R)-11-HETE significantly enhanced the average number of tentacles, thus suggesting that this eicosanoid might be one of the cellular regulators of regeneration in H. vulgaris. PMID:8216222

  12. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.

  13. An Examination of the Carbon Isotope Effects Associated with Amino Acid Biosynthesis

    NASA Astrophysics Data System (ADS)

    Scott, James H.; O'Brien, Diane M.; Emerson, David; Sun, Henry; McDonald, Gene D.; Salgado, Antonio; Fogel, Marilyn L.

    2006-12-01

    Stable carbon isotope ratios (δ13C) were determined for alanine, proline, phenylalanine, valine, leucine, isoleucine, aspartate (aspartic acid and asparagine), glutamate (glutamic acid and glutamine), lysine, serine, glycine, and threonine from metabolically diverse microorganisms. The microorganisms examined included fermenting bacteria, organotrophic, chemolithotrophic, phototrophic, methylotrophic, methanogenic, acetogenic, acetotrophic, and naturally occurring cryptoendolithic communities from the Dry Valleys of Antarctica. Here we demonstrated that reactions involved in amino acid biosynthesis can be used to distinguish amino acids formed by life from those formed by nonbiological processes. The unique patterns of δ13C imprinted by life on amino acids produced a biological bias. We also showed that, by applying discriminant function analysis to the δ13C value of a pool of amino acids formed by biological activity, it was possible to identify key aspects of intermediary carbon metabolism in the microbial world. In fact, microorganisms examined in this study could be placed within one of three metabolic groups: (1) heterotrophs that grow by oxidizing compounds containing three or more carbon-to-carbon bonds (fermenters and organotrophs), (2) autotrophs that grow by taking up carbon dioxide (chemolitotrophs and phototrophs), and (3) acetoclastic microbes that grow by assimilation of formaldehyde or acetate (methylotrophs, methanogens, acetogens, and acetotrophs). Furthermore, we demonstrated that cryptoendolithic communities from Antarctica grouped most closely with the autotrophs, which indicates that the dominant metabolic pathways in these communities are likely those utilized for CO2 fixation. We propose that this technique can be used to determine the dominant metabolic types in a community and reveal the overall flow of carbon in a complex ecosystem.

  14. A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum

    PubMed Central

    Taura, Futoshi; Iijima, Miu; Yamanaka, Eriko; Takahashi, Hironobu; Kenmoku, Hiromichi; Saeki, Haruna; Morimoto, Satoshi; Asakawa, Yoshinori; Kurosaki, Fumiya; Morita, Hiroyuki

    2016-01-01

    Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum. PMID:27729920

  15. Linoleic acid biosynthesis and characterization of the. Delta. sup 12 desaturase in insects

    SciTech Connect

    Cripps, C.

    1988-01-01

    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 of 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.

  16. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

    PubMed Central

    Brose, Stephen A.; Golovko, Svetlana A.; Golovko, Mikhail Y.

    2016-01-01

    Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke. PMID:27965531

  17. Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria.

    PubMed

    Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun

    2015-12-02

    Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 Å and 2.25 Å resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme.

  18. RNA-seq based transcriptomic analysis uncovers α-linolenic acid and jasmonic acid biosynthesis pathways respond to cold acclimation in Camellia japonica

    PubMed Central

    Li, Qingyuan; Lei, Sheng; Du, Kebing; Li, Lizhi; Pang, Xufeng; Wang, Zhanchang; Wei, Ming; Fu, Shao; Hu, Limin; Xu, Lin

    2016-01-01

    Camellia is a well-known ornamental flower native to Southeast of Asia, including regions such as Japan, Korea and South China. However, most species in the genus Camellia are cold sensitive. To elucidate the cold stress responses in camellia plants, we carried out deep transcriptome sequencing of ‘Jiangxue’, a cold-tolerant cultivar of Camellia japonica, and approximately 1,006 million clean reads were generated using Illumina sequencing technology. The assembly of the clean reads produced 367,620 transcripts, including 207,592 unigenes. Overall, 28,038 differentially expressed genes were identified during cold acclimation. Detailed elucidation of responses of transcription factors, protein kinases and plant hormone signalling-related genes described the interplay of signal that allowed the plant to fine-tune cold stress responses. On the basis of global gene regulation of unsaturated fatty acid biosynthesis- and jasmonic acid biosynthesis-related genes, unsaturated fatty acid biosynthesis and jasmonic acid biosynthesis pathways were deduced to be involved in the low temperature responses in C. japonica. These results were supported by the determination of the fatty acid composition and jasmonic acid content. Our results provide insights into the genetic and molecular basis of the responses to cold acclimation in camellia plants. PMID:27819341

  19. Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation.

    PubMed

    Ncube, E N; Steenkamp, P A; Madala, N E; Dubery, I A

    2016-07-01

    Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.

  20. myo-Inositol 1-Phosphate Synthase Inhibition and Control of Uridine Diphosphate-d-glucuronic Acid Biosynthesis in Plants 12

    PubMed Central

    Loewus, Mary W.; Loewus, Frank

    1974-01-01

    Of the eight intermediates associated with the two pathways of UDP-d-glucuronic acid biosynthesis found in plants, only d-glucuronic acid inhibited myo-inositol 1-phosphate synthase (EC 5.5.1.4), formerly referred to as d-glucose 6-phosphate cycloaldolase. Inhibition was competitive. An attempt to demonstrate over-all reversibility of the synthase indicated that it was less than 5% reversible, if at all. PMID:16658890

  1. Salicylic acid sans aspirin in animals and man: persistence in fasting and biosynthesis from benzoic acid.

    PubMed

    Paterson, John R; Baxter, Gwendoline; Dreyer, Jacob S; Halket, John M; Flynn, Robert; Lawrence, James R

    2008-12-24

    Salicylic acid (SA), which is central to defense mechanisms in plants and the principal metabolite of aspirin, occurs naturally in man with higher levels of SA and its urinary metabolite salicyluric acid (SU) in vegetarians overlapping with levels in patients on low-dose aspirin regimens. SA is widely distributed in animal blood. Fasting for major colorectal surgery did not cause disappearance of SA from plasma, even in patients following total proctocolectomy. A (13)C(6) benzoic acid load ingested by six volunteers led, between 8 and 16 h, to a median 33.9% labeling of urinary salicyluric acid. The overall contribution of benzoic acid (and its salts) to the turnover of circulating SA thus requires further assessment. However, that SA appears to be, at least partially, an endogenous compound should lead to reassessment of its role in human (and animal) pathophysiology.

  2. Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

    PubMed

    Horváth, Edit; Csiszár, Jolán; Gallé, Ágnes; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2015-07-01

    The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.

  3. Chlorogenic Acid Biosynthesis Appears Linked with Suberin Production in Potato Tuber (Solanum tuberosum).

    PubMed

    Valiñas, Matías Ariel; Lanteri, María Luciana; ten Have, Arjen; Andreu, Adriana Balbina

    2015-05-20

    Potato (Solanum tuberosum L.) is a good source of dietary antioxidants. Chlorogenic acid (CGA) and caffeic acid (CA) are the most abundant phenolic acid antioxidants in potato and are formed by the phenylpropanoid pathway. A number of CGA biosynthetic routes that involve hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and/or hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) have been proposed, but little is known about their path in potato. CA production requires a caffeoyl shikimate esterase (CSE), and CA serves as a substrate of lignin precursor ferulic acid via the action of caffeic/5-hydroxyferulic acid O-methyltransferase (COMT I). CGA is precursor of caffeoyl-CoA and, via caffeoyl-CoA O-methyltransferase (CCoAOMT), of feruloyl-CoA. Feruloyl-CoA is required for lignin and suberin biosynthesis, crucial for tuber development. Here, metabolite and transcript levels of the mentioned and related enzymes, such as cinnamate 4-hydroxylase (C4H), were determined in the flesh and skin of fresh and stored tubers. Metabolite and transcript levels were higher in skin than in flesh, irrespective of storage. CGA and CA production appear to occur via p-coumaroyl-CoA, using HQT and CSE, respectively. HCT is likely involved in CGA remobilization toward suberin. The strong correlation between CGA and CA, the correspondence with C4H, HQT, CCoAOMT2, and CSE, and the negative correlation of HCT and COMT I in potato tubers suggest a major flux toward suberin.

  4. Metabolomics Analysis and Biosynthesis of Rosmarinic Acid in Agastache rugosa Kuntze Treated with Methyl Jasmonate

    PubMed Central

    Uddin, Md. Romij; Xu, Hui; Park, Woo Tae; Tuan, Pham Anh; Li, Xiaohua; Chung, Eunsook; Lee, Jai-Heon; Park, Sang Un

    2013-01-01

    This study investigated the effect of methyl jasmonate (MeJA) on metabolic profiles and rosmarinic acid (RA) biosynthesis in cell cultures of Agastache rugosa Kuntze. Transcript levels of phenylpropanoid biosynthetic genes, i.e., ArPAL, Ar4CL, and ArC4H, maximally increased 4.5-fold, 3.4-fold, and 3.5-fold, respectively, compared with the untreated controls, and the culture contained relatively high amounts of RA after exposure of cells to 50 µM MeJA. RA levels were 2.1-, 4.7-, and 3.9-fold higher after exposure to 10, 50, and 100 µM MeJA, respectively, than those in untreated controls. In addition, the transcript levels of genes attained maximum levels at different time points after the initial exposure. The transcript levels of ArC4H and Ar4CL were transiently induced by MeJA, and reached a maximum of up to 8-fold at 3 hr and 6 hr, respectively. The relationships between primary metabolites and phenolic acids in cell cultures of A. rugosa treated with MeJA were analyzed by gas chromatography coupled with time-of-flight mass spectrometry. In total, 45 metabolites, including 41 primary metabolites and 4 phenolic acids, were identified from A. rugosa. Metabolite profiles were subjected to partial least square-discriminate analysis to evaluate the effects of MeJA. The results indicate that both phenolic acids and precursors for the phenylpropanoid biosynthetic pathway, such as aromatic amino acids and shikimate, were induced as a response to MeJA treatment. Therefore, MeJA appears to have an important impact on RA accumulation, and the increased RA accumulation in the treated cells might be due to activation of the phenylpropanoid genes ArPAL, ArC4H, and Ar4CL. PMID:23724034

  5. A Single Amino Acid Change Is Responsible for Evolution of Acyltransferase Specificity in Bacterial Methionine Biosynthesis

    SciTech Connect

    Zubieta, C.; Arkus, K.A.J.; Cahoon, R.E.; Jez, J.M.

    2009-05-28

    Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 {angstrom} resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.

  6. Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling

    PubMed Central

    Ruiz, F. Xavier; Porté, Sergio; Parés, Xavier; Farrés, Jaume

    2012-01-01

    Several aldo–keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance. PMID:22529810

  7. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  8. Auxin Biosynthesis

    PubMed Central

    Zhao, Yunde

    2014-01-01

    lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then undergoes oxidative decarboxylation catalyzed by the YUCCA (YUC) family of flavin monooxygenases to produce IAA. This two-step auxin biosynthesis pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. The successful elucidation of a complete auxin biosynthesis pathway provides the necessary tools for effectively modulating auxin concentrations in plants with temporal and spatial precision. The progress in auxin biosynthesis also lays a foundation for understanding polar auxin transport and for dissecting auxin signaling mechanisms during plant development. PMID:24955076

  9. Coordinated Regulation of Species-Specific Hydroxycinnamic Acid Degradation and Siderophore Biosynthesis Pathways in Agrobacterium fabrum

    PubMed Central

    Baude, Jessica; Vial, Ludovic; Villard, Camille; Campillo, Tony; Lavire, Céline; Nesme, Xavier

    2016-01-01

    ABSTRACT The rhizosphere-inhabiting species Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to degrade hydroxycinnamic acids (HCAs), especially ferulic acid and p-coumaric acid, via the novel A. fabrum HCA degradation pathway. Gene expression profiles of A. fabrum strain C58 were investigated in the presence of HCAs, using a C58 whole-genome oligoarray. Both ferulic acid and p-coumaric acid caused variations in the expression of more than 10% of the C58 genes. Genes of the A. fabrum HCA degradation pathway, together with the genes involved in iron acquisition, were among the most highly induced in the presence of HCAs. Two operons coding for the biosynthesis of a particular siderophore, as well as genes of the A. fabrum HCA degradation pathway, have been described as being specific to the species. We demonstrate here their coordinated expression, emphasizing the interdependence between the iron concentration in the growth medium and the rate at which ferulic acid is degraded by cells. The coordinated expression of these functions may be advantageous in HCA-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. The present results confirm that there is cooperation between the A. fabrum-specific genes, defining a particular ecological niche. IMPORTANCE We previously identified seven genomic regions in Agrobacterium fabrum that were specifically present in all of the members of this species only. Here we demonstrated that two of these regions, encoding the hydroxycinnamic acid degradation pathway and the iron acquisition pathway, were regulated in a coordinated manner. The coexpression of these functions may be advantageous in hydroxycinnamic acid-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. These data support the view that bacterial genomic species

  10. Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32

    PubMed Central

    Stanley, Sarah A.; Kawate, Tomohiko; Iwase, Noriaki; Shimizu, Motohisa; Clatworthy, Anne E.; Kazyanskaya, Edward; Sacchettini, James C.; Ioerger, Thomas R.; Siddiqi, Noman A.; Minami, Shoko; Aquadro, John A.; Schmidt Grant, Sarah; Rubin, Eric J.; Hung, Deborah T.

    2013-01-01

    Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets. PMID:23798446

  11. Effect of nitrogen deficiency on ascorbic acid biosynthesis and recycling pathway in cucumber seedlings.

    PubMed

    Zhang, Xue; Yu, Hong Jun; Zhang, Xiao Meng; Yang, Xue Yong; Zhao, Wen Chao; Li, Qiang; Jiang, Wei Jie

    2016-11-01

    L-Ascorbic acid (AsA, ascorbate) is one of the most abundant natural antioxidants, and it is an important factor in the nutritional quality of cucumber. In this work, key enzymes involved in the ascorbic acid biosynthesis and recycling pathway in cucumber seedlings under nitrogen deficiency were investigated at the levels of transcription and enzyme activity. The activities of myo-inositol oxygenase (MIOX) and transcript levels of MIOXs increased dramatically, while the activities of ascorbate oxidase (AO) and glutathione reductase (GR) and transcript levels of AOs and GR2 decreased significantly in N-limited leaves, as did the ascorbate concentration, in nitrogen-deficient cucumber seedlings. The activities of other enzymes and transcript levels of other genes involved in the ascorbate recycling pathway and ascorbate synthesis pathways decreased or remained unchanged under nitrogen deficiency. These results indicate that nitrogen deficiency induced genes involved in the ascorbate-glutathione recycling and myo-inositol pathway in cucumber leaves. Thus, the AO, GR and MIOX involved in the pathways might play roles in AsA accumulation.

  12. Articulation of three core metabolic processes in Arabidopsis: Fatty acid biosynthesis, leucine catabolism and starch metabolism

    PubMed Central

    Mentzen, Wieslawa I; Peng, Jianling; Ransom, Nick; Nikolau, Basil J; Wurtele, Eve Syrkin

    2008-01-01

    Background Elucidating metabolic network structures and functions in multicellular organisms is an emerging goal of functional genomics. We describe the co-expression network of three core metabolic processes in the genetic model plant Arabidopsis thaliana: fatty acid biosynthesis, starch metabolism and amino acid (leucine) catabolism. Results These co-expression networks form modules populated by genes coding for enzymes that represent the reactions generally considered to define each pathway. However, the modules also incorporate a wider set of genes that encode transporters, cofactor biosynthetic enzymes, precursor-producing enzymes, and regulatory molecules. We tested experimentally the hypothesis that one of the genes tightly co-expressed with starch metabolism module, a putative kinase AtPERK10, will have a role in this process. Indeed, knockout lines of AtPERK10 have an altered starch accumulation. In addition, the co-expression data define a novel hierarchical transcript-level structure associated with catabolism, in which genes performing smaller, more specific tasks appear to be recruited into higher-order modules with a broader catabolic function. Conclusion Each of these core metabolic pathways is structured as a module of co-expressed transcripts that co-accumulate over a wide range of environmental and genetic perturbations and developmental stages, and represent an expanded set of macromolecules associated with the common task of supporting the functionality of each metabolic pathway. As experimentally demonstrated, co-expression analysis can provide a rich approach towards understanding gene function. PMID:18616834

  13. How do background ozone concentrations affect the biosynthesis of rosmarinic acid in Melissa officinalis?

    PubMed

    Döring, Anne S; Pellegrini, Elisa; Della Batola, Michele; Nali, Cristina; Lorenzini, Giacomo; Petersen, Maike

    2014-03-01

    Lemon balm (Melissa officinalis; Lamiaceae) plants were exposed to background ozone (O3) dosages (80ppb for 5h), because high background levels of O3 are considered to be as harmful as episodic O3 peaks. Immediately at the end of fumigation the plants appeared visually symptomless, but necrotic lesions were observed later. The biosynthesis of rosmarinic acid (RA) comprises eight enzymes, among them phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT) and rosmarinic acid synthase (RAS). The transcript levels of these genes have been investigated by quantitative RT-PCR. There was a quick up-regulation of all genes at 3h of O3 exposure, but at 24h from beginning of exposure (FBE) only RAS and PAL were up-regulated. The specific activity of RAS was closely correlated with a decrease of RA concentration in lemon balm leaves. The specific activity of PAL increased at 12h FBE to 163% in comparison to control levels. This work provides insight into the effect of O3 stress on the formation of the main phenolic ingredient of the pharmaceutically important plant M. officinalis.

  14. Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition.

    PubMed

    Zhou, Yongjin J; Buijs, Nicolaas A; Zhu, Zhiwei; Gómez, Diego Orol; Boonsombuti, Akarin; Siewers, Verena; Nielsen, Jens

    2016-11-30

    Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and/or insufficient precursor supply. Compartmentalization in cellular organelles can isolate synthetic pathways from competing pathways, and provide a compact and suitable environment for biosynthesis. Peroxisomes are cellular organelles where fatty acids are degraded, a process that is inhibited under typical fermentation conditions making them an interesting workhouse for production of fatty-acid-derived molecules. Here, we show that targeting synthetic pathways to peroxisomes can increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins up to 700%. In addition, we demonstrate that biosynthesis of these chemicals in the peroxisomes results in significantly decreased accumulation of byproducts formed by competing enzymes. We further demonstrate that production can be enhanced up to 3-fold by increasing the peroxisome population. The strategies described here could be used for production of other chemicals, especially acyl-CoA-derived molecules.

  15. Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

    PubMed Central

    Sengupta, Sudeshna; Goonewardena, Lakshani; Juturu, Veeresh

    2015-01-01

    cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroFFBR, aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars. PMID:26362984

  16. Retroconversion of docosapentaenoic acid (n-6): an alternative pathway for biosynthesis of arachidonic acid in Daphnia magna.

    PubMed

    Strandberg, Ursula; Taipale, Sami J; Kainz, Martin J; Brett, Michael T

    2014-06-01

    The aim of this study was to assess metabolic pathways for arachidonic acid (20:4n-6) biosynthesis in Daphnia magna. Neonates of D. magna were maintained on [(13)C] enriched Scenedesmus obliquus and supplemented with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the control treatment, without any supplementary fatty acids (FA) containing only trace amounts of 20:4n-6 (~0.3% of all FA). As expected, the highest proportion of 20:4n-6 (~6.3%) was detected in Daphnia that received liposomes supplemented with this FA. Higher availability of 18:2n-6 in the diet increased the proportion of 18:2n-6 in Daphnia, but the proportion of 20:4n-6 was not affected. Daphnia supplemented with 22:5n-6 contained ~3.5% 20:4n-6 in the lipids and FA specific stable isotope analyses validated that the increase in the proportion of 20:4n-6 was due to retroconversion of unlabeled 22:5n-6. These results suggest that chain shortening of 22:5n-6 is a more efficient pathway to synthesize 20:4n-6 in D. magna than elongation and desaturation of 18:2n-6. These results may at least partially explain the discrepancies noticed between phytoplankton FA composition and the expected FA composition in freshwater cladocerans. Finally, retroconversion of dietary 22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long chain n-6 FA in lake food webs, which might be important for the nutritional ecology of fish.

  17. Characterization of the formation of branched short-chain fatty acid:CoAs for bitter acid biosynthesis in hop glandular trichomes.

    PubMed

    Xu, Haiyang; Zhang, Fengxia; Liu, Baoxiu; Huhman, David V; Sumner, Lloyd W; Dixon, Richard A; Wang, Guodong

    2013-07-01

    Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K cat /K m = 4100 s(-1) M(-1)), whereas recombinant HlCCL4 specifically utilized isobutyric acid (Kcat/K m = 1800 s(-1) M(-1)) and 2-methylbutyric acid (Kcat/K m = 6900 s(-1) M(-1)) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

  18. 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.

  19. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    PubMed

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  20. Biosynthesis of Indoleacetic Acid from Tryptophan-14C in Cell-free Extracts of Pea Shoot Tips 1

    PubMed Central

    Moore, Thomas C.; Shaner, Coralie A.

    1967-01-01

    A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-14C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a 14C-product as IAA was by (a) co-chromatography of authentic IAA and 14C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall ≅ etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA. PMID:16656720

  1. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    PubMed Central

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  2. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

    PubMed

    Mohedano, Maria L; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  3. Mutant characterization and in vivo conditional repression identify aromatic amino acid biosynthesis to be essential for Aspergillus fumigatus virulence

    PubMed Central

    Sasse, Anna; Hamer, Stefanie N; Amich, Jorge; Binder, Jasmin; Krappmann, Sven

    2016-01-01

    Pathogenicity of the saprobe Aspergillus fumigatus strictly depends on nutrient acquisition during infection, as fungal growth determines colonisation and invasion of a susceptible host. Primary metabolism has to be considered as a valid target for antimycotic therapy, based on the fact that several fungal anabolic pathways are not conserved in higher eukaryotes. To test whether fungal proliferation during invasive aspergillosis relies on endogenous biosynthesis of aromatic amino acids, defined auxotrophic mutants of A. fumigatus were generated and assessed for their infectious capacities in neutropenic mice and found to be strongly attenuated in virulence. Moreover, essentiality of the complete biosynthetic pathway could be demonstrated, corroborated by conditional gene expression in infected animals and inhibitor studies. This brief report not only validates the aromatic amino acid biosynthesis pathway of A. fumigatus to be a promising antifungal target but furthermore demonstrates feasibility of conditional gene expression in a murine infection model of aspergillosis. PMID:26605426

  4. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli.

    PubMed

    Zhang, Haoran; Stephanopoulos, Gregory

    2016-07-01

    3-amino-benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli-E. coli co-culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co-culture system was found to improve 3AB production by 15 fold, compared to the mono-culture approach. Further engineering of the co-culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co-culture engineering can be a powerful new approach in the broad field of metabolic engineering.

  5. [Antitoxic properties of pantothenic acid derivatives, precursors of coenzyme A biosynthesis, with regard to kanamycin].

    PubMed

    Moĭseenok, A G; Dorofeev, B F; Sheĭbak, V M; Khomich, T I

    1984-11-01

    The effect of calcium pantothenate (CPN)B 4'-phospho-CPN (PCP), pantetheine (PT) and calcium S-sulfopantetheine (SPN) on acute toxicity of kanamycin sulfate was studied on albino mice. The above derivatives of pantothenic acid except PT lowered the antibiotic toxicity. The coefficient of the antitoxic effect (LD50/ED50) of SPN and PCP was 1.3-1.4 times higher than that of CPN. The combined use of kanamycin (1/5 of the LD50) with CPN, PCP or PT (30 mg/kg bw was equivalent to CPN) for 15 days prevented the increase in the total content of CoA and in the content of the fraction of free CoA and the precursors of its biosynthesis participating in the reaction of N-acetylation in the liver and brain. The contents of these substances were within the normal during the whole experiment. A certain increase in the activity of pantothenate kinase in the liver cytosol due to the use of kanamycin was eliminated by the simultaneous use of PCP and PT. The vitamin-containing compounds PCP and SPN were recommended for the clinical trials as agents preventing complications of kanamycin therapy.

  6. Regulation of ascorbic acid biosynthesis and recycling during root development in carrot (Daucus carota L.).

    PubMed

    Wang, Guang-Long; Xu, Zhi-Sheng; Wang, Feng; Li, Meng-Yao; Tan, Guo-Fei; Xiong, Ai-Sheng

    2015-09-01

    Ascorbic acid (AsA), also known as vitamin C, is an essential nutrient in fruits and vegetables. The fleshy root of carrot (Daucus carota L.) is a good source of AsA for humans. However, the metabolic pathways and molecular mechanisms involved in the control of AsA content during root development in carrot have not been elucidated. To gain insights into the regulation of AsA accumulation and to identify the key genes involved in the AsA metabolism, we cloned and analyzed the expression of 21 related genes during carrot root development. The results indicate that AsA accumulation in the carrot root is regulated by intricate pathways, of which the l-galactose pathway may be the major pathway for AsA biosynthesis. Transcript levels of the genes encoding l-galactose-1-phosphate phosphatase and l-galactono-1,4-lactone dehydrogenase were strongly correlated with AsA levels during root development. Data from this research may be used to assist breeding for improved nutrition, quality, and stress tolerance in carrots.

  7. Differently Localized Lysophosphatidic Acid Acyltransferases Crucial for Triacylglycerol Biosynthesis in the Oleaginous Alga Nannochloropsis.

    PubMed

    Nobusawa, Takashi; Hori, Koichi; Mori, Hiroshi; Kurokawa, Ken; Ohta, Hiroyuki

    2017-02-20

    Production of renewable bioenergy will be necessary to meet rising global fossil fuel demands. Members of the marine microalgae genus Nannochloropsis produce large amounts of oils (triacylglycerols; TAGs), and this genus is regarded as one of the most promising for biodiesel production. Recent genome sequencing and transcriptomic studies on Nannochloropsis have provided a foundation for understanding its oleaginous trait, but the mechanism underlying oil accumulation remains to be clarified. Here we report Nannochloropsis knockout strains of four extraplastidic lysophosphatidic acid acyltransferases (LPAT1-4), which catalyze a major de novo biosynthetic step of TAGs and membrane lipids. We found that the four LPATs are differently involved in lipid metabolic flow in Nannochloropsis. Double knockouts among the LPATs revealed the pivotal LPATs for TAG biosynthesis, and localization analysis indicated that the stramenopile-specific LPATs (LPAT3 and LPAT4) associated with TAG synthesis reside at the perimeter of lipid droplets. However, no homologous region has been found with other lipid droplet-associated proteins. Lipid droplets are an organelle found in nearly all organisms, and recently they were shown to play important roles in cellular metabolism and signaling. Our results provide direct evidence for the importance of the perimeter of lipid droplet in TAG synthesis in addition to its known role in maintaining TAG stability, and these findings suggest that the oleaginous trait of Nannochloropsis is enabled by acquisition of LPATs at the perimeter of lipid droplets. This article is protected by copyright. All rights reserved.

  8. Pantothenic Acid Biosynthesis in the Parasite Toxoplasma gondii: a Target for Chemotherapy

    PubMed Central

    Mageed, Sarmad N.; Cunningham, Fraser; Hung, Alvin Wei; Silvestre, Hernani Leonardo; Wen, Shijun; Blundell, Tom L.; Abell, Chris

    2014-01-01

    Toxoplasma gondii is a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis in T. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed in T. gondii. A panel of inhibitors developed to target Mycobacterium tuberculosis pantothenate synthetase were tested and found to exhibit a range of values for inhibition of T. gondii growth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence, T. gondii encodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target. PMID:25049241

  9. Dissociation of cephamycin C and clavulanic acid biosynthesis by 1,3-diaminopropane in Streptomyces clavuligerus.

    PubMed

    Leite, Carla A; Cavallieri, André P; Baptista, Amanda S; Araujo, Maria L G C

    2016-01-01

    Streptomyces clavuligerus produces simultaneously cephamycin C (CephC) and clavulanic acid (CA). Adding 1,3-diaminopropane to culture medium stimulates production of beta-lactam antibiotics. However, there are no studies on the influence of this diamine on coordinated production of CephC and CA. This study indicates that 1,3-diaminopropane can dissociate CephC and CA productions. Results indicated that low diamine concentrations (below 1.25 g l(-1)) in culture medium increased CA production by 200%, but not that of CephC. Conversely, CephC production increased by 300% when 10 g l(-1) 1,3-diaminopropane was added to culture medium. Addition of just L-lysine (18.3 g l(-1)) to culture medium increased both biocompounds. On the other hand, while L-lysine plus 7.5 g l(-1) 1,3-diaminopropane increased volumetric production of CephC by 1100%, its impact on CA production was insignificant. The combined results suggest that extracellular concentration of 1,3-diaminopropane may trigger the dissociation of CephC and CA biosynthesis in S. clavuligerus.

  10. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  11. Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

    PubMed

    Kim, Yeon Bok; Shin, YouJin; Tuan, Pham Anh; Li, Xiaohua; Park, Yunji; Park, Nam-il; Park, Sang Un

    2014-01-01

    Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

  12. Deuterium NMR used to indicate a common mechanism for the biosynthesis of ricinoleic acid by Ricinus communis and Claviceps purpurea.

    PubMed

    Billault, Isabelle; Mantle, Peter G; Robins, Richard J

    2004-03-17

    Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct (2)H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative (2)H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.

  13. Metabolic engineering of Escherichia coli for 1-butanol biosynthesis through the inverted aerobic fatty acid β-oxidation pathway.

    PubMed

    Gulevich, Andrey Yu; Skorokhodova, Alexandra Yu; Sukhozhenko, Alexey V; Shakulov, Rustem S; Debabov, Vladimir G

    2012-03-01

    The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid β-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid β-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid β-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI(Q) attB-P(trc-ideal-4)-SD(φ10)-adhE(Glu568Lys) attB-P(trc-ideal-4)-SD(φ10)-atoB attB-P(trc-ideal-4)-SD(φ10)-fadB attB-P(trc-ideal-4)-SD(φ10)-fadE) synthesises 0.3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.

  14. Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha.

    PubMed

    Kageyama, Akito; Ishizaki, Kimitsune; Kohchi, Takayuki; Matsuura, Hideyuki; Takahashi, Kosaku

    2015-09-01

    Environmental stresses are effective triggers for the biosynthesis of various secondary metabolites in plants, and phytohormones such as jasmonic acid and abscisic acid are known to mediate such responses in flowering plants. However, the detailed mechanism underlying the regulation of secondary metabolism in bryophytes remains unclear. In this study, the induction mechanism of secondary metabolites in the model liverwort Marchantia polymorpha was investigated. Abscisic acid (ABA) and ultraviolet irradiation (UV-C) were found to induce the biosynthesis of isoriccardin C, marchantin C, and riccardin F, which are categorized as bisbibenzyls, characteristic metabolites of liverworts. UV-C led to the significant accumulation of ABA. Overexpression of MpABI1, which encodes protein phosphatase 2C (PP2C) as a negative regulator of ABA signaling, suppressed accumulation of bisbibenzyls in response to ABA and UV-C irradiation and conferred susceptibility to UV-C irradiation. These data show that ABA plays a significant role in the induction of bisbibenzyl biosynthesis, which might confer tolerance against UV-C irradiation in M. polymorpha.

  15. P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

    PubMed Central

    Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

    2017-01-01

    P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

  16. Molecular characterization of a mutation affecting abscisic acid biosynthesis and consequently stomatal responses to humidity in an agriculturally important species

    PubMed Central

    McAdam, Scott A. M.; Sussmilch, Frances C.; Brodribb, Timothy J.; Ross, John J.

    2015-01-01

    Mutants deficient in the phytohormone abscisic acid (ABA) have been instrumental in determining not only the biosynthetic pathway for this hormone, but also its physiological role in land plants. The wilty mutant of Pisum sativum is one of the classical, well-studied ABA-deficient mutants; however, this mutant remains uncharacterized at a molecular level. Using a candidate gene approach, we show that the wilty mutation affects the xanthoxin dehydrogenase step in ABA biosynthesis. To date, this step has only been represented by mutants in the ABA2 gene of Arabidopsis thaliana. Functional ABA biosynthesis appears to be critical for normal stomatal responses to changes in humidity in angiosperms, with wilty mutant plants having no increase in foliar ABA levels in response to a doubling in vapour pressure deficit, and no closure of stomata. Phylogenetic analysis of the ABA2 gene family from diverse land plants indicates that an ABA-biosynthesis-specific short-chain dehydrogenase (ABA2) evolved in the earliest angiosperms. The relatively recent origin of specificity in this step has important implications for both the evolution of ABA biosynthesis and action in land plants. PMID:26216469

  17. Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

    PubMed Central

    2009-01-01

    Background The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci). Results We report that the FabF homologue located within the fatty acid biosynthetic gene cluster of C. acetobutylicium functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in E. coli functionally replaced both the FabB and FabF proteins of the host in vivo and replaced E. coli FabB in a defined in vitro fatty acid synthesis system. In contrast the single C. acetobutylicium FabZ homologue, although able to functionally replace E. coli FabZ in vivo and in vitro, was unable to replace FabA, the key dehydratase-isomerase of E. coli unsaturated fatty acid biosynthesis in vivo and lacked isomerase activity in vitro. Conclusion Thus, C. acetobutylicium introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme. PMID:19493359

  18. FAS — EDRN Public Portal

    Cancer.gov

    FAS is a member of the TNF-receptor superfamily. The FAS protein is a receptor for TNFSF6/FASLG and has been shown to play a central role in the physiological regulation of programmed cell death, and has been implicated in the pathogenesis of various malignancies and diseases of the immune system. Several alternatively spliced transcript variants have been described, some of which are candidates for nonsense-mediated mRNA decay (NMD). The isoforms lacking the transmembrane domain may negatively regulate the apoptosis mediated by the full length isoform.

  19. Biosynthesis of 1-aminocyclopropane-1-carboxylic acid and ethylene from delta-aminolevulinic acid in ripening tomato fruits

    SciTech Connect

    El-Rayes, D.E.D.A.

    1987-01-01

    A new pathway for ethylene (C/sub 2/H/sub 4/) biosynthesis, which utilizes delta-aminolevulinic acid (ALA) as a precursor of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of C/sub 2/H/sub 4/, is presented. ALA enhanced ACC accumulation to 410% and C/sub 2/H/sub 4/ production to 232% of the control. The C/sub 2/H/sub 4/ production rate varied with the ALA concentration and the stage of tomato fruit development. As the ALA concentration increased from zero to 40 mM, the C/sub 2/H/sub 4/ production rate increased. Both treated and untreated pericarp discs from fruits at the pink stage of development yielded the largest C/sub 2/H/sub 4/ production rate. Radioactivity from (2,3-/sup 3/H)ALA was detected in both ACC and C/sub 2/H/sub 4/, and radioactivity from (4-/sup 14/C)ALA was detected in ACC and CO/sub 2/ but not in C/sub 2/H/sub 4/. However, radioactivity from (5-/sup 14/C)ALA was detected in CO/sub 2/, and its amount was greater than that obtained from (4-/sup 14/C)ALA. Neither ACC nor C/sub 2/H/sub 4/ showed any radioactivity when (5-/sup 14/C)ALA was supplied to the fruit discs. In addition, when (2,3-/sup 3/H)ALA or (4-/sup 14/C)ALA was supplied to the fruit discs, radioactivity was detected in other metabolites such as fumarate, succinate, malate, glutamate, glutamine, ..cap alpha..-ketoglutarate, and methionine, but the amount of radioactivity was insignificant as compared with the amount of radioactivity found in C/sub 2/H/sub 4/ and ACC.

  20. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  1. Inhibition of carnitine biosynthesis by valproic acid in rats--the biochemical mechanism of inhibition.

    PubMed

    Farkas, V; Bock, I; Cseko, J; Sandor, A

    1996-11-08

    The anticonvulsive drug, valproic acid (VPA), inhibits the biosynthesis of carnitine, and may contribute in this way to carnitine deficiency associated with VPA therapy. The conversion of [3H]-butyrobetaine into [3H]-carnitine was determined 60 min following a single intraperitoneal (i.p.) dose of 1.2 mmol/kg VPA in rats. The fraction of radioactivity found in [3H]-carnitine in the liver decreased from 63.2 +/- 1.50% to 39.2 +/- 1.11% (mean +/- SEM). Total carnitine in the liver also decreased, whereas the precursor butyrobetaine increased from 5.01 +/- 0.71 nmol/g to 8.22 +/- 0.82 nmol/g (mean +/- SEM). VPA also exhibited a dramatic effect on the conversion of an unlabeled loading amount of butyrobetaine. The increment in total carnitine caused by butyrobetaine in liver was reduced from 161 +/- 15.4 nmol/g to 53.2 +/- 5.11 nmol/g (mean +/- SEM). These data prove that VPA reduces the flux through butyrobetaine hydroxylase (EC 1.14.11.1.). The drug in vitro, however, did not inhibit the enzyme directly. Searching for the mechanism of action, we found that VPA decreased the level of alpha-ketoglutarate (alpha-KG; a cofactor of butyrobetaine hydroxylase) from 73.5 +/- 2.90 nmol/g to 52.9 +/- 2.2 nmol/g (mean +/- SEM) in the liver. The level of 1-glutamate showed a rather dramatic decrease in the liver. Moreover, alpha-KG proved to have a protective role against VPA in the [3H]-butyrobetaine conversion experiment.

  2. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro.

    PubMed

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-04-01

    Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell-cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase.

  3. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

    PubMed Central

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-01-01

    Abstract Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

  4. The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages

    PubMed Central

    Rossi, A; Pergola, C; Koeberle, A; Hoffmann, M; Dehm, F; Bramanti, P; Cuzzocrea, S; Werz, O; Sautebin, L

    2010-01-01

    BACKGROUND AND PURPOSE Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo. EXPERIMENTAL APPROACH Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model. KEY RESULTS Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes. CONCLUSIONS AND IMPLICATION Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed. PMID:20880396

  5. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis.

    PubMed

    Liu, Ying; Xu, Qiao-Xian; Xi, Pei-Yu; Chen, Hong-Hao; Liu, Chun-Sheng

    2013-05-01

    The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

  6. Immunohistochemical study on Fas and Fas ligand in skin wound healing.

    PubMed

    Guan, D W; Ohshima, T; Kondo, T

    2000-02-01

    An immunohistochemical study on the expression of Fas and Fas ligand (Fas L) was performed in order to examine the role of apoptosis through Fas-Fas L in mouse skin wound healing. After a 1-cm-long incision in the central dorsum skin, mice were sacrificed at intervals ranging from 0.5 to 240 h, followed by the sampling of wound margin. The expression of Fas and Fas L in the wound margins and in uninjured skin controls was studied using frozen sections. In uninjured skin controls, a very weak expression of Fas and Fas L was detected immunohistochemically in hair follicles, sebaceous glands and epidermal cells. In wounded specimens, polymorphonuclear cells and inflammatory mononuclear ones (round-shaped and spindle-shaped types) were evident. A single immunostaining showed that Fas or Fas L was detectable in inflammatory mononuclear cells involved in the skin wound healing process. Double immunostaining for Fas and Fas L revealed that inflammatory mononuclear cells co-expressed both antigens. In situ TUNEL combined with immunostaining showed that the inflammatory mononuclear cells expressing Fas or Fas L and the polymorphonuclear cells were TUNEL-stained, although neither Fas nor Fas L was detected in the polymorphonuclear cells. The number of TUNEL-positive, inflammatory mononuclear cells expressing Fas or Fas L per 0.01 x 0.01 cm2 was counted. The average number of 10 randomly selected microscope fields reached a peak at the fibro-proliferative phase of wound healing. These results indicate that apoptosis through Fas and Fas L may play an important role for reducing the cellularity during skin wound healing in mice.

  7. Salt-Related MYB1 Coordinates Abscisic Acid Biosynthesis and Signaling during Salt Stress in Arabidopsis1

    PubMed Central

    Wang, Ting; Tohge, Takayuki; Ivakov, Alexander; Mueller-Roeber, Bernd; Fernie, Alisdair R.; Mutwil, Marek; Schippers, Jos H.M.; Persson, Staffan

    2015-01-01

    Abiotic stresses, such as salinity, cause global yield loss of all major crop plants. Factors and mechanisms that can aid in plant breeding for salt stress tolerance are therefore of great importance for food and feed production. Here, we identified a MYB-like transcription factor, Salt-Related MYB1 (SRM1), that negatively affects Arabidopsis (Arabidopsis thaliana) seed germination under saline conditions by regulating the levels of the stress hormone abscisic acid (ABA). Accordingly, several ABA biosynthesis and signaling genes act directly downstream of SRM1, including SALT TOLERANT1/NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, RESPONSIVE TO DESICCATION26, and Arabidopsis NAC DOMAIN CONTAINING PROTEIN19. Furthermore, SRM1 impacts vegetative growth and leaf shape. We show that SRM1 is an important transcriptional regulator that directly targets ABA biosynthesis and signaling-related genes and therefore may be regarded as an important regulator of ABA-mediated salt stress tolerance. PMID:26243618

  8. The FAS Child: A Primer for Teachers.

    ERIC Educational Resources Information Center

    Wentz, Thomas L.; Larson, Julie

    1993-01-01

    This primer on fetal alcohol syndrome (FAS) distinguishes between the syndrome and fetal alcohol effects (FAE), offers a history of FAS, outlines medical criteria for diagnosis, rates of incidence, factors influencing incidence and severity, developmental stages of children with FAS, clinical features, and educational implications and approaches.…

  9. FasParser: a package for manipulating sequence data.

    PubMed

    Sun, Yan-Bo

    2017-03-18

    A computer software package called 'FasParser' was developed for manipulating sequence data. It can be used on personal computers to perform series of analyses, including counting and viewing differences between two sequences at both DNA and codon levels, identifying overlapping regions between two alignments, sorting of sequences according to their IDs or lengths, concatenating sequences of multiple loci for a particular set of samples, translating nucleotide sequences to amino acids, and constructing alignments in several different formats, as well as some extracting and filtrating of data for a particular FASTA file. Majority of these functions can be run in a batch mode, which is very useful for analyzing large data sets. This package can be used by a broad audience, and is designed for researchers that do not have programming experience in sequence analyses. The GUI version of FasParser can be downloaded from https://github.com/Sun-Yanbo/FasParser, free of charge.

  10. From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis[W][OA

    PubMed Central

    de Kraker, Jan-Willem; Gershenzon, Jonathan

    2011-01-01

    Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the kcat/Km for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism. PMID:21205930

  11. Methyl jasmonate stimulates biosynthesis of 2-phenylethylamine, phenylacetic acid and 2-phenylethanol in seedlings of common buckwheat.

    PubMed

    Horbowicz, Marcin; Wiczkowski, Wiesław; Sawicki, Tomasz; Szawara-Nowak, Dorota; Sytykiewicz, Hubert; Mitrus, Joanna

    2015-01-01

    Methyl jasmonate has a strong effect on secondary metabolizm in plants, by stimulating the biosynthesis a number of phenolic compounds and alkaloids. Common buckwheat (Fagopyrum esculentum Moench) is an important source of biologically active compounds. This research focuses on the detection and quantification of 2-phenylethylamine and its possible metabolites in the cotyledons, hypocotyl and roots of common buckwheat seedlings treated with methyl jasmonate. In cotyledons of buckwheat sprouts, only traces of 2-phenylethylamine were found, while in the hypocotyl and roots its concentration was about 150 and 1000-times higher, respectively. Treatment with methyl jasmonate resulted in a 4-fold increase of the 2-phenylethylamine level in the cotyledons of 7-day buckwheat seedlings, and an 11-fold and 5-fold increase in hypocotyl and roots, respectively. Methyl jasmonate treatment led also to about 4-fold increase of phenylacetic acid content in all examined seedling organs, but did not affect the 2-phenylethanol level in cotyledons, and slightly enhanced in hypocotyl and roots. It has been suggested that 2-phenylethylamine is a substrate for the biosynthesis of phenylacetic acid and 2-phenylethanol, as well as cinnamoyl 2-phenethylamide. In organs of buckwheat seedling treated with methyl jasmonate, higher amounts of aromatic amino acid transaminase mRNA were found. The enzyme can be involved in the synthesis of phenylpyruvic acid, but the presence of this compound could not be confirmed in any of the examined organs of common buckwheat seedling.

  12. Endurance exercise and conjugated linoleic acid (CLA) supplementation up-regulate CYP17A1 and stimulate testosterone biosynthesis.

    PubMed

    Barone, Rosario; Macaluso, Filippo; Catanese, Patrizia; Marino Gammazza, Antonella; Rizzuto, Luigi; Marozzi, Paola; Lo Giudice, Giuseppe; Stampone, Tomaso; Cappello, Francesco; Morici, Giuseppe; Zummo, Giovanni; Farina, Felicia; Di Felice, Valentina

    2013-01-01

    A new role for fat supplements, in particular conjugated linoleic acid (CLA), has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated in vivo by CLA supplementation associated with exercise. In vitro, Leydig tumour rat cells (R2C) supplemented with different concentrations of CLA exhibited increasing testosterone biosynthesis accompanied by increasing levels of CYP17A1 mRNA and protein. In vivo, trained mice showed an increase in free plasma testosterone and an up-regulation of CYP17A1 mRNA and protein. The effect of training on CYP17A1 expression and testosterone biosynthesis was significantly higher in the trained mice supplemented with CLA compared to the placebo. The results of the present study demonstrated that CLA stimulates testosterone biosynthesis via CYP17A1, and endurance training led to the synthesis of testosterone in vivo by inducing the overexpression of CYP17A1 mRNA and protein in the Leydig cells of the testis. This effect was enhanced by CLA supplementation. Therefore, CLA-associated physical activity may be used for its steroidogenic property in different fields, such as alimentary industry, human reproductive medicine, sport science, and anti-muscle wasting.

  13. The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato.

    PubMed

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong; Xiao, Han

    2015-03-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.

  14. Profiling and Quantifying Differential Gene Transcription Provide Insights into Ganoderic Acid Biosynthesis in Ganoderma lucidum in Response to Methyl Jasmonate

    PubMed Central

    Shi, Liang; Mu, Da-Shuai; Jiang, Ai-Liang; Han, Qin; Zhao, Ming-Wen

    2013-01-01

    Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum. PMID:23762280

  15. The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato1

    PubMed Central

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong

    2015-01-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. PMID:25637453

  16. PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng

    PubMed Central

    Rahimi, Shadi; Kim, Yu-Jin; Sukweenadhi, Johan; Zhang, Dabing; Yang, Deok-Chun

    2016-01-01

    Ginsenosides, the valuable pharmaceutical compounds in Panax ginseng, are triterpene saponins that occur mainly in ginseng plants. It was shown that in vitro treatment with the phytohormone jasmonic acid (JA) is able to increase ginsenoside production in ginseng plants. To understand the molecular link between JA biosynthesis and ginsenoside biosynthesis, we identified a JA biosynthetic 13-lipoxygenase gene (PgLOX6) in P. ginseng that promotes ginsenoside production. The expression of PgLOX6 was high in vascular bundles, which corresponds with expression of ginsenoside biosynthetic genes. Consistent with the role of PgLOX6 in synthesizing JA and promoting ginsenoside synthesis, transgenic plants overexpressing PgLOX6 in Arabidopsis had increased amounts of JA and methyl jasmonate (MJ), increased expression of triterpene biosynthetic genes such as squalene synthase (AtSS1) and squalene epoxidase (AtSE1), and increased squalene content. Moreover, transgenic ginseng roots overexpressing PgLOX6 had around 1.4-fold increased ginsenoside content and upregulation of ginsenoside biosynthesis-related genes including PgSS1, PgSE1, and dammarenediol synthase (PgDDS), which is similar to that of treatment with MJ. However, MJ treatment of transgenic ginseng significantly enhanced JA and MJ, associated with a 2.8-fold increase of ginsenoside content compared with the non-treated, non-transgenic control plant, which was 1.4 times higher than the MJ treatment effect on non-transgenic plants. These results demonstrate that PgLOX6 is responsible for the biosynthesis of JA and promotion of the production of triterpenoid saponin through up-regulating the expression of ginsenoside biosynthetic genes. This work provides insight into the role of JA in biosynthesizing secondary metabolites and provides a molecular tool for increasing ginsenoside production. PMID:27811076

  17. Biosynthesis of L-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalic acid occurs within individual calcium oxalate crystal idioblasts.

    PubMed

    Kostman, T A; Tarlyn, N M; Loewus, F A; Franceschi, V R

    2001-02-01

    L-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various (14)C-labeled compounds and examined by micro-autoradiography for incorporation of (14)C into calcium oxalate crystals. [(14)C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-(14)C]AsA also gave heavy labeling of crystals, whereas [6-(14)C]AsA gave no labeling. Labeled precursors of AsA (L-[1-(14)C]galactose; D-[1-(14)C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, D-[1-(14)C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > L-galactose > D-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via D-mannose and L-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments.

  18. Biosynthesis, biological effects, and receptors of hydroxyeicosatetraenoic acids (HETEs) and oxoeicosatetraenoic acids (oxo-ETEs) derived from arachidonic acid.

    PubMed

    Powell, William S; Rokach, Joshua

    2015-04-01

    Arachidonic acid can be oxygenated by a variety of different enzymes, including lipoxygenases, cyclooxygenases, and cytochrome P450s, and can be converted to a complex mixture of oxygenated products as a result of lipid peroxidation. The initial products in these reactions are hydroperoxyeicosatetraenoic acids (HpETEs) and hydroxyeicosatetraenoic acids (HETEs). Oxoeicosatetraenoic acids (oxo-ETEs) can be formed by the actions of various dehydrogenases on HETEs or by dehydration of HpETEs. Although a large number of different HETEs and oxo-ETEs have been identified, this review will focus principally on 5-oxo-ETE, 5S-HETE, 12S-HETE, and 15S-HETE. Other related arachidonic acid metabolites will also be discussed in less detail. 5-Oxo-ETE is synthesized by oxidation of the 5-lipoxygenase product 5S-HETE by the selective enzyme, 5-hydroxyeicosanoid dehydrogenase. It actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, suggesting that it may be important in eosinophilic diseases such as asthma. 5-Oxo-ETE also appears to stimulate tumor cell proliferation and may also be involved in cancer. Highly selective and potent OXE receptor antagonists have recently become available and could help to clarify its pathophysiological role. The 12-lipoxygenase product 12S-HETE acts by the GPR31 receptor and promotes tumor cell proliferation and metastasis and could therefore be a promising target in cancer therapy. It may also be involved as a proinflammatory mediator in diabetes. In contrast, 15S-HETE may have a protective effect in cancer. In addition to GPCRs, higher concentration of HETEs and oxo-ETEs can activate peroxisome proliferator-activated receptors (PPARs) and could potentially regulate a variety of processes by this mechanism. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

  19. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

    PubMed Central

    Rodrigues, Elisete P.; Soares, Cleiton de Paula; Galvão, Patrícia G.; Imada, Eddie L.; Simões-Araújo, Jean L.; Rouws, Luc F. M.; de Oliveira, André L. M.; Vidal, Márcia S.; Baldani, José I.

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and

  20. Structural Characterization of the Mycobacterium tuberculosis Biotin Biosynthesis Enzymes 7,8-Diaminopelargonic Acid Synthase and Dethiobiotin Synthetase†,‡

    PubMed Central

    Dey, Sanghamitra; Lane, James M.; Lee, Richard E.; Rubin, Eric J.; Sacchettini, James C.

    2010-01-01

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 Å and 1.85 Å. Superimposition of the DAPAS structures bound either to the SAM analog sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 Å, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb. PMID:20565114

  1. Activation of glycerol metabolic pathway by evolutionary engineering of Rhizopus oryzae to strengthen the fumaric acid biosynthesis from crude glycerol.

    PubMed

    Huang, Di; Wang, Ru; Du, Wenjie; Wang, Guanyi; Xia, Menglei

    2015-11-01

    Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L.

  2. Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm.

    PubMed

    Guerin, Chloé; Joët, Thierry; Serret, Julien; Lashermes, Philippe; Vaissayre, Virginie; Agbessi, Mawussé D T; Beulé, Thierry; Severac, Dany; Amblard, Philippe; Tregear, James; Durand-Gasselin, Tristan; Morcillo, Fabienne; Dussert, Stéphane

    2016-09-01

    Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for β-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

  3. DNA Methylation Perturbations in Genes Involved in Polyunsaturated Fatty Acid Biosynthesis Associated with Depression and Suicide Risk

    PubMed Central

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-yu; Cooper, Thomas B.; Burke, Ainsley K.; Oquendo, Maria A.; Mann, J. John; Sublette, M. Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  4. Alteration of Fas and Fas ligand expression during human visceral leishmaniasis

    PubMed Central

    Eidsmo, L; Wolday, D; Berhe, N; Sabri, F; Satti, I; El Hassan, A M; Sundar, S; Chiodi, F; Akuffo, H

    2002-01-01

    Several studies in murine systems have suggested a role of apoptosis in the pathogenesis of leishmaniasis. However, the role of apoptosis in visceral leishmaniasis in man has not been explored. In this study, we show that patients with visceral leishmaniasis demonstrate significant dysregulation of Fas and Fas ligand. Levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) were elevated in plasma of patients with active visceral leishmaniasis (VL) and individuals co-infected with VL-HIV-1 compared to healthy controls. The levels of sFas and sFasL were normalized 6 months after successful treatment. In VL patients, the expression of membrane bound Fas, and to a lower extent FasL, were up-regulated on Leishmania donovani-infected spleen cells, the site of parasite multiplication. Expression of Fas and FasL on peripheral blood mononuclear cells was within normal range, probably reflecting that the blood is not a normal site of L. donovani infection. Furthermore, this is suggested by the finding that in vitro infection of macrophages with L. donovani up-regulated Fas expression on the surface of infected cells and enhanced the levels of sFasL in supernatants from infected cultures. How this dysregulation may affect the pathogenesis of human visceral leishmaniasis is discussed. PMID:12390320

  5. Clinicopathological significance of Fas and Fas ligand expressions in esophageal cancer

    PubMed Central

    Wu, Guang-Zhou; Pan, Chun-Xia; Jiang, Dong; Zhang, Qiang; Li, Yin; Zheng, Shi-Ying

    2015-01-01

    Esophageal carcinomas have recently been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. However, the prognostic importance of Fas/FasL and their correlation with clinicopathological characteristics are yet to be delineated in this highly malignant carcinoma. Specimens from 106 esophageal squamous cell carcinoma patients were used for immuno-histochemical evaluation of Fas, FasL, and CD8 expressions. Fifty-two (49%) and 34 (32%) patients were positive for FasL and Fas, respectively. There were no associations between FasL expression and clinicopathological characteristics except lymph vessel invasion. Strong FasL expression correlated with significant (P < 0.001) decrease in tumor nest CD81 cells. However, neither FasL nor CD81 had any impact on patient survival. Strong Fas expression was correlated with depth of invasion (40.3% in pT1, T2 versus 20.5% in pT3, T4; P5 0.0308), histological differentiation (45.7% in well versus 25.4% in nonwell; P < 0.05), and lymph node metastasis (22.6% in positive versus 45.5% in negative; P < 0.01). Fas expression was one of the independent favorable prognosticators for patients’ survival (risk ratio, 3.26; P < 0.01) in esophageal SCC. Fas expression was an independent prognosticator for recurrencefree survival, whereas FasL expression did not influence the survival in esophageal squamous cell carcinoma. Down-regulation of tumor Fas may be the hallmark of immune privilege for the tumor, thus causing the patients’ poorer outcome. Tumor FasL may counterattack the host immune cells to such an extent that the prognosis is not affected. PMID:26609492

  6. Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

    PubMed

    Guerreiro, Joana F; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-12-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

  7. 6-Methyl-1,2,4-benzenetriol, a new intermediate in penicillic acid biosynthesis in Penicillium cyclopium

    SciTech Connect

    Sekiguchi, J.; Katayama, S.; Yamada, Y.

    1987-07-01

    Penicillic acid-negative mutants were obtained from a color mutant derived from Penicillium cyclopium NRRL 1888 through N-methyl-N'-nitro-N-nitrosoguanidine treatment. One mutant (SK2N6) accumulated 6-methyl-1,2,4-benzenetriol, which was not previously known to be a metabolite of P. cyclopium, in addition to orsellinic acid and orcinol. The radioactivity of (1-/sup 14/C)acetic acid was rapidly incorporated into 6-methyl-1,2,4-benzenetriol in a culture of P. cyclopium SK2N6. Moreover, the radioactivity of (/sup 14/C)6-methyl-1,2,4-benzenetriol was efficiently incorporated into penicillic acid in a culture of P. cyclopium NRRL 1888. These data indicate that 6-methyl-1,2,4-benzenetriol is a precursor for penicillic acid biosynthesis. The results on the addition of 1,4-dihydroxy-6-methoxy-2-methylbenzene, 6-methoxy-2-methylbenzoquinone (1,4), and 1-O-methylorcinol to a culture of P. cyclopium SK2N6 indicated that only the former two compounds are converted to penicillic acid. Thus, a new portion of the penicillic acid biosynthetic pathway is proposed.

  8. Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis.

    PubMed

    Gannoun-Zaki, Laila; Alibaud, Laeticia; Kremer, Laurent

    2013-01-01

    The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

  9. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis

    PubMed Central

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  10. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    PubMed

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  11. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89.

    PubMed

    Yuwen, Lei; Zhang, Feng-Li; Chen, Qi-Hua; Lin, Shuang-Jun; Zhao, Yi-Lei; Li, Zhi-Yong

    2013-01-01

    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid sequence of AADC have suggested that L-tryptophan and L-phenylalanine are the potential substrates of AADC. The enzymatic kinetic experiment of the recombinant AADC proved that L-tryptophan is a more reactive substrate of AADC than L-phenylalanine. Meanwhile, the AADC-catalyzed conversion of L-tryptophan into tryptamine was confirmed by means of HPLC and LC/MS. Thus during bacillamide C biosynthesis, the decarboxylation of L-tryptophan to tryptamine is likely conducted first under AADC catalysis, followed by the amidation of tryptamine with the carboxylic product of NRPS gene cluster.

  12. Silencing of BnTT1 family genes affects seed flavonoid biosynthesis and alters seed fatty acid composition in Brassica napus.

    PubMed

    Lian, Jianping; Lu, Xiaochun; Yin, Nengwen; Ma, Lijuan; Lu, Jing; Liu, Xue; Li, Jiana; Lu, Jun; Lei, Bo; Wang, Rui; Chai, Yourong

    2017-01-01

    TRANSPARENT TESTA1 (TT1) is a zinc finger protein that contains a WIP domain. It plays important roles in controlling differentiation and pigmentation of the seed coat endothelium, and can affect the expression of early biosynthetic genes and late biosynthetic genes of flavonoid biosynthesis in Arabidopsis thaliana. In Brassica napus (AACC, 2n=38), the functions of BnTT1 genes remain unknown and few studies have focused on their roles in fatty acid (FA) biosynthesis. In this study, BnTT1 family genes were silenced by RNA interference, which resulted in yellow rapeseed, abnormal testa development (a much thinner testa), decreased seed weight, and altered seed FA composition in B. napus. High-throughput sequencing of genes differentially expressed between developing transgenic B. napus and wild-type seeds revealed altered expression of numerous genes involved in flavonoid and FA biosynthesis. As a consequence of this altered expression, we detected a marked decrease of oleic acid (C18:1) and notable increases of linoleic acid (C18:2) and α-linolenic acid (C18:3) in mature transgenic B. napus seeds by gas chromatography and near-infrared reflectance spectroscopy. Meanwhile, liquid chromatography-mass spectrometry showed reduced accumulation of flavonoids in transgenic seeds. Therefore, we propose that BnTT1s are involved in the regulation of flavonoid biosynthesis, and may also play a role in FA biosynthesis in B. napus.

  13. Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability

    PubMed Central

    Rossin, A; Durivault, J; Chakhtoura-Feghali, T; Lounnas, N; Gagnoux-Palacios, L; Hueber, A-O

    2015-01-01

    The death receptor Fas undergoes a variety of post-translational modifications including S-palmitoylation. This protein acylation has been reported essential for an optimal cell death signaling by allowing both a proper Fas localization in cholesterol and sphingolipid-enriched membrane nanodomains, as well as Fas high-molecular weight complexes. In human, S-palmitoylation is controlled by 23 members of the DHHC family through their palmitoyl acyltransferase activity. In order to better understand the role of this post-translational modification in the regulation of the Fas-mediated apoptosis pathway, we performed a screen that allowed the identification of DHHC7 as a Fas-palmitoylating enzyme. Indeed, modifying DHHC7 expression by specific silencing or overexpression, respectively, reduces or enhances Fas palmitoylation and DHHC7 co-immunoprecipitates with Fas. At a functional level, DHHC7-mediated palmitoylation of Fas allows a proper Fas expression level by preventing its degradation through the lysosomes. Indeed, the decrease of Fas expression obtained upon loss of Fas palmitoylation can be restored by inhibiting the lysosomal degradation pathway. We describe the modification of Fas by palmitoylation as a novel mechanism for the regulation of Fas expression through its ability to circumvent its degradation by lysosomal proteolysis. PMID:25301068

  14. The status of Fas and Fas ligand expression can predict recurrence of hepatocellular carcinoma

    PubMed Central

    Ito, Y; Monden, M; Takeda, T; Eguchi, H; Umeshita, K; Nagano, H; Nakamori, S; Dono, K; Sakon, M; Nakamura, M; Tsujimoto, M; Nakahara, M; Nakao, K; Yokosaki, Y; Matsuura, N

    2000-01-01

    The status of Fas and Fas ligand (Fas L) expression was investigated in this study for 103 hepatocellular carcinomas (HCC). We studied the expression of the following three factors, Fas and Fas L expression in carcinoma cells and Fas L expression in stromal mononuclear cells (defined as stromal Fas L index). Fas expression in HCC cells was significantly decreased in cases with poor differentiation (P< 0.0001) and of larger size (P = 0.0058). Fas L expression in carcinoma cells was observed exclusively in moderately or poorly differentiated cases. Furthermore, each factor had prognostic significance for disease-free survival (DFS) (P< 0.0001, P = 0.0222 and 0.0027 respectively). We then scored the results of each factor and defined the total score as ‘Fas-Fas L risk score’. The P -value of the score for DFS was even lower than that of the clinical stage by multivariate analysis. These results suggest that the evaluation of Fas and Fas ligand expression potentially has a significant prognostic value for DFS of HCC patients, in addition to the clinical stage, and can be regarded as a new prognostic marker. © 2000 Cancer Research Campaign PMID:10735508

  15. Widespread Occurrence of Secondary Lipid Biosynthesis Potential in Microbial Lineages

    PubMed Central

    Shulse, Christine N.; Allen, Eric E.

    2011-01-01

    Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as “Pfa synthases”. In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of “secondary lipids” to describe these biosynthetic pathways and products, a proposition consistent with the “secondary metabolite” vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages. PMID:21629834

  16. Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.

    PubMed Central

    Braus, G H

    1991-01-01

    This review focuses on the gene-enzyme relationships and the regulation of different levels of the aromatic amino acid biosynthetic pathway in a simple eukaryotic system, the unicellular yeast Saccharomyces cerevisiae. Most reactions of this branched pathway are common to all organisms which are able to synthesize tryptophan, phenylalanine, and tyrosine. The current knowledge about the two main control mechanisms of the yeast aromatic amino acid biosynthesis is reviewed. (i) At the transcriptional level, most structural genes are regulated by the transcriptional activator GCN4, the regulator of the general amino acid control network, which couples transcriptional derepression to amino acid starvation of numerous structural genes in multiple amino acid biosynthetic pathways. (ii) At the enzyme level, the carbon flow is controlled mainly by modulating the enzyme activities at the first step of the pathway and at the branch points by feedback action of the three aromatic amino acid end products. Implications of these findings for the relationship of S. cerevisiae to prokaryotic as well as to higher eukaryotic organisms and for general regulatory mechanisms occurring in a living cell such as initiation of transcription, enzyme regulation, and the regulation of a metabolic branch point are discussed. PMID:1943992

  17. 4-Coumaroyl and caffeoyl shikimic acids inhibit 4-coumaric acid:coenzyme A ligases and modulate metabolic flux for 3-hydroxylation in monolignol biosynthesis of Populus trichocarpa.

    PubMed

    Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Chen, Hsi-Chuan; Liu, Jie; Loziuk, Philip; Song, Jina; Williams, Cranos; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2015-01-01

    Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography-tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.

  18. Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance.

    PubMed

    Jenkins, Benjamin J; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A; Summerhill, Keith; Griffin, Julian L; Vetter, Walter; Autio, Kaija J; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M; de Schryver, Evelyn; Van Veldhoven, Paul P; Ronis, Martin; Koulman, Albert

    2017-03-23

    Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1(-/-) mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1(-/-) only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

  19. Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

    PubMed Central

    Jenkins, Benjamin J.; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A.; Summerhill, Keith; Griffin, Julian L.; Vetter, Walter; Autio, Kaija J.; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M.; de Schryver, Evelyn; Van Veldhoven, Paul P.; Ronis, Martin; Koulman, Albert

    2017-01-01

    Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease. PMID:28332596

  20. Hyaluronic Acid--an "Old" Molecule with "New" Functions: Biosynthesis and Depolymerization of Hyaluronic Acid in Bacteria and Vertebrate Tissues Including during Carcinogenesis.

    PubMed

    Tsepilov, R N; Beloded, A V

    2015-09-01

    Hyaluronic acid is an evolutionarily ancient molecule commonly found in vertebrate tissues and capsules of some bacteria. Here we review modern data regarding structure, properties, and biological functions of hyaluronic acid in mammals and Streptococcus spp. bacteria. Various aspects of biogenesis and degradation of hyaluronic acid are discussed, biosynthesis and degradation metabolic pathways for glycosaminoglycan together with involved enzymes are described, and vertebrate and bacterial hyaluronan synthase genes are characterized. Special attention is given to the mechanisms underlying the biological action of hyaluronic acid as well as the interaction between polysaccharide and various proteins. In addition, all known signaling pathways involving hyaluronic acid are outlined. Impaired hyaluronic acid metabolism, changes in biopolymer molecular weight, hyaluronidase activity, and enzyme isoforms often accompany carcinogenesis. The interaction between cells and hyaluronic acid from extracellular matrix that may be important during malignant change is discussed. An expected role for high molecular weight hyaluronic acid in resistance of naked mole rat to oncologic diseases and the protective role of hyaluronic acid in bacteria are discussed.

  1. Structure and conformational variability of the mycobacterium tuberculosis fatty acid synthase multienzyme complex.

    PubMed

    Ciccarelli, Luciano; Connell, Sean R; Enderle, Mathias; Mills, Deryck J; Vonck, Janet; Grininger, Martin

    2013-07-02

    Antibiotic therapy in response to Mycobacterium tuberculosis infections targets de novo fatty acid biosynthesis, which is orchestrated by a 1.9 MDa type I fatty acid synthase (FAS). Here, we characterize M. tuberculosis FAS by single-particle cryo-electron microscopy and interpret the data by docking the molecular models of yeast and Mycobacterium smegmatis FAS. Our analysis reveals a porous barrel-like structure of considerable conformational variability that is illustrated by the identification of several conformational states with altered topology in the multienzymatic assembly. This demonstrates that the barrel-like structure of M. tuberculosis FAS is not just a static scaffold for the catalytic domains, but may play an active role in coordinating fatty acid synthesis. The conception of M. tuberculosis FAS as a highly dynamic assembly of domains revises the view on bacterial type I fatty acid synthesis and might inspire new strategies for inhibition of de novo fatty acid synthesis in M. tuberculosis.

  2. Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat.

    PubMed

    Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation.

  3. Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat

    PubMed Central

    Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation. PMID:27148345

  4. Fractionation of Carbon Isotopes in Biosynthesis of Fatty Acids by A Piezophilic Bacterium Moritella Japonica DSK1

    NASA Astrophysics Data System (ADS)

    Fang, J.; Uhle, M.; Bartlett, D.; Kato, C.

    2005-12-01

    We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica DSK1. DSK1 was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using natural seawater supplied with glucose with the sole carbon source. DSk1 synthesized typical bacterial fatty acids (C14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed ΔδFA-glucose (-1.0 to -11.9%) at 0.1 MPa was comparable to or slightly higher than fractionations reported on surface bacteria. However, Bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation ΔδFA-glucose) at high pressures was much higher than that for surface bacteria: -15.7, -15.3, and -18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions affected by hydrostatic pressure and to different biosynthetic pathways for short-chain and long-chain fatty acids. Our results have important implications for marine biogeochemistry. The 13C depleted fatty acids in marine sediments and water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i.e., methane). The interpretation of carbon isotope signatures of marine lipids must be based on principles derived from piezophilic bacteria.

  5. Biosynthesis of Chloro-β-Hydroxytyrosine, a Nonproteinogenic Amino Acid of the Peptidic Backbone of Glycopeptide Antibiotics

    PubMed Central

    Puk, Oliver; Bischoff, Daniel; Kittel, Claudia; Pelzer, Stefan; Weist, Stefan; Stegmann, Efthimia; Süssmuth, Roderich D.; Wohlleben, Wolfgang

    2004-01-01

    The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block β-hydroxytyrosine (β-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of β-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated β-HT. In contrast, supplementation with 3-chloro-β-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free β-HT, most likely during heptapeptide synthesis. PMID:15342578

  6. Regulation of the Bacterial Cell Wall: Analysis of a Mutant of Bacillus subtilis Defective in Biosynthesis of Teichoic Acid

    PubMed Central

    Boylan, R. J.; Mendelson, N. H.; Brooks, D.; Young, F. E.

    1972-01-01

    Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis. PMID:4622900

  7. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  8. Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans.

    PubMed

    Sun, Quanxi; Liu, Jiang; Zhang, Qin; Qing, Xiaohe; Dobson, Gary; Li, Xinzheng; Qi, Baoxiu

    2013-09-01

    Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5Δ(5, 8, 11, 14, 17)) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.

  9. Soluble Fas and the −670 Polymorphism of Fas in Lupus Nephritis

    PubMed Central

    Bollain-y-Goytia, Juan José; Arellano-Rodríguez, Mariela; Torres-Del-Muro, Felipe de Jesús; Daza-Benítez, Leonel; Francisco Muñoz-Valle, José; Avalos-Díaz, Esperanza; Herrera-Esparza, Rafael

    2014-01-01

    This study was performed to clarify the role of soluble Fas (sFas) in lupus nephritis (LN) and establish a potential relationship between LN and the −670 polymorphism of Fas in 67 patients with systemic lupus erythematosus (SLE), including a subset of 24 LN patients with proteinuria. Additionally, a group of 54 healthy subjects (HS) was included. The allelic frequency of the −670 polymorphism of Fas was determined using PCR-RFLP analysis, and sFas levels were assessed by ELISA. Additionally, the WT-1 protein level in urine was measured. The Fas receptor was determined in biopsies by immunohistochemistry (IHC) and in situ hybridization (FISH) and apoptotic features by TUNEL. Results. The −670 Fas polymorphism showed that the G allele was associated with increased SLE susceptibility, with an odds ratio (OR) of 1.86. The sFas was significantly higher in LN patients with the G/G genotype, and this subgroup exhibited correlations between the sFas level and proteinuria and increased urinary WT-1 levels. LN group shows increased expression of Fas and apoptotic features. In conclusion, our results indicate that the G allele of the −670 polymorphism of Fas is associated with genetic susceptibility in SLE patients with elevated levels of sFas in LN with proteinuria. PMID:25505993

  10. Fas-FasL expression and myocardial cell apoptosis in patients with viral myocarditis.

    PubMed

    Huang, T F; Wu, X H; Wang, X; Lu, I J

    2016-06-20

    The aim of the current study was to investigate Fas and FasL expression and myocardial cell apoptosis in viral myocarditis patients. Human heart specimens were selected from patients who were autopsied between February 2012 and February 2015; of these, 25 patients were diagnosed with viral myocarditis. Another 15 cases with no diagnosis of myocarditis were selected for the control group. All tissue specimens were divided into two parts, one for reverse transcription-polymerase chain reaction analysis and the other for immunohistochemical and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. In situ detection of apoptosis was performed by the TUNEL method, which revealed that myocardial cells from the viral myocarditis group exhibited significant apoptosis, whereas no apoptotic cells were observed in the control group. The number of cells staining positive for Fas and FasL protein in the viral myocarditis group was significantly higher than that in the control group (P < 0.05). There was also a correlation between Fas and FasL protein expression levels and scores (r = 0.92, P < 0.05). The mRNA expression of Fas and FasL was significantly higher in the viral myocarditis group than in the control group (P < 0.05). In conclusion, the Fas-FasL system may be involved in the pathogenesis of viral myocarditis. Furthermore, cytotoxic T lymphocytes may mediate cardiac muscle cells apoptosis via Fas-FasL signaling, and thus participate in the pathogenesis of viral myocarditis.

  11. New tuberculostatic agents targeting nucleic acid biosynthesis: drug design using QSAR approaches.

    PubMed

    Bueno, Renata V; Braga, Rodolpho C; Segretti, Natanael D; Ferreira, Elizabeth I; Trossini, Gustavo H G; Andrade, Carolina H

    2014-01-01

    Worldwide, tuberculosis (TB) is the leading cause of death among curable infectious diseases. The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB is a growing global health concern and there is an urgent need for new anti-TB drugs. Enzymes involved in DNA and ATP biosynthesis are potential targets for tuberculostatic drug design, since these enzymes are essential for Mycobacterium tuberculosis growth. This review presents the current progress and applications of structure-activity relationship analysis for the discovery of innovative tuberculostatic agents as inhibitors of ribonucleotide reductase, DNA gyrase, ATP synthase, and thymidylate kinase enzymes, highlighting present challenges and new opportunities in TB drug design.

  12. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

    PubMed Central

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

  13. The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast.

    PubMed

    Daicho, Katsue; Maruyama, Hironori; Suzuki, Asuka; Ueno, Masaru; Uritani, Masahiro; Ushimaru, Takashi

    2007-07-01

    Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.

  14. Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria.

    PubMed

    Ahmad, S; Weisburg, W G; Jensen, R A

    1990-02-01

    A comprehensive phylogenetic tree for virtually the entire assemblage of enteric bacteria is presented. Character states of aromatic amino acid biosynthesis are used as criteria, and the results are compared with partial trees based upon sequencing of 16S rRNA, 5S rRNA, and tryptophan leader peptide. Three major clusters are apparent. Enterocluster 1 possesses a gene fusion (trpG-trpD) encoding anthranilate synthase: anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase of tryptophan biosynthesis. This cluster includes the genera Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, and Enterobacter. The remaining two clusters lack the trpG-trpD gene fusion, but differ in the presence (enterocluster 2) or absence (enterocluster 3) of the three-step overflow pathway to L-phenylalanine. Enterocluster 2 consists of the genera Serratia and Erwinia. Enterocluster 3 includes the genera Cedecea, Kluyvera, Edwardsiella, Hafnia, Yersinia, Proteus, Providencia, and Morganella. Within these three major clusters, a tentative hierarchy of subcluster ordering is formulated on the basis of all data available. This hierarchical framework is proposed as a general working basis for continued refinement of the phylogenetic relationships of enteric bacteria.

  15. A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis.

    PubMed

    Makris, Thomas M; Chakrabarti, Mrinmoy; Münck, Eckard; Lipscomb, John D

    2010-08-31

    The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.

  16. Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis.

    PubMed

    Nugteren, D H; Christ-Hazelhof, E

    1987-03-01

    Fatty acids from natural sources (mostly seed oils) were isolated and assayed for their effect on the bioconversion of arachidonic acid into prostaglandin E2, using sheep vesicular gland microsomes. Homologues and isomers of the naturally occurring fatty acids, obtained by chemical modification and/or organic synthetic methods, were also tested. Two very active cyclooxygenase inhibitors were discovered, namely jacarandic acid (8Z, 10E, 12Z-octadecatrienoic acid), isolated from Jacaranda mimosifolia, the concentration which gives 50% inhibition ([I]50) being 2.4 microM and the synthetic 8Z, 10E, 12E-octadecatrienoic acid, having an [I]50 of 1.0 microM. Under the conditions of the assay (75 microM substrate), earlier described potent inhibitors showed the following [I]50's: indomethacin: 1.3 microM; 9,12-octadecadiynoic acid: 1.3 microM, 8Z, 12E, 14Z-eicosatrienoic acid: 2.7 microM; 5,8,11,14-eicosatetraynoic acid: 4.4 microM. At a concentration of about half that of the substrate, the following naturally occurring fatty acids revealed inhibition ([I]50): columbinic acid (29 microM), calendulic acid (31 microM), liagoric acid (31 microM), ximenynic acid (39 microM), crepenynic acid (40 microM) and timnodonic acid (43 microM). Other fatty acids, and some of the above acids, were converted themselves more or less rapidly, mostly into conjugated monohydroxy fatty acids.

  17. Unexpected Formation of Low Amounts of (R)-Configurated anteiso-Fatty Acids in Rumen Fluid Experiments

    PubMed Central

    Abdurahman, Halima; Ruoff, Tanja; Kaffarnik, Stefanie; Steingass, Herbert; Vetter, Walter

    2017-01-01

    Anteiso-fatty acids (aFA) with odd carbon number are a class of branched-chain fatty acids (BCFA) mainly produced by bacteria. Bacterial sources are also made responsible for their occurrence in the low percent-range in lipids of ruminants (meat and milk) and fish. aFAs are chiral molecules and typically occur predominantly in form of (S)-enantiomers, and their primary precursor has been noted to be isoleucine. Yet, low proportions of (R)-aFAs were also detected in fish and cheese samples. Here we investigated the potential formation of (R)-aFAs by means of incubation experiments with rumen fluid from fistulated cows. Supplementation of rumen fluid with both L- and DL-isoleucine, resulted in a significant (α <0.05) increase of the aFA concentrations but in both cases enantiopure (S)-aFAs were observed. By contrast, incubations without addition of any isoleucine lead to a significant (α <0.05) formation of small proportions of (R)-aFAs similarly to those previously observed in fish and cheese. These results were consistently reproduced in three different years with rumen fluid from different cows fed different diets. All findings point to the existence of a further biosynthesis pathway of aFAs with different stereospecificity than the classic one using isoleucine as primer. PMID:28129363

  18. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance.

    PubMed Central

    Kaneda, T

    1991-01-01

    Branched-chain fatty acids of the iso and anteiso series occur in many bacteria as the major acyl constituents of membrane lipids. In addition, omega-cyclohexyl and omega-cycloheptyl fatty acids are present in several bacterial species. These two types of fatty acids are synthesized by the repeated condensation of malonyl coenzyme A with one of the branched-chain and cyclic primers by the same enzyme system. The pathway of de novo branched-chain fatty acid synthesis differs only in initial steps of synthesis from that of the common straight-chain fatty acid (palmitic acid) present in most organisms. The cell membranes composed largely of iso-, anteiso-, and omega-alicyclic acids support growth of bacteria, which inhabit normal as well as extreme environments. The occurrence of these types of fatty acids as major cellular fatty acids is an important criterion used to aid identification and classification of bacteria. PMID:1886522

  19. crl mutants of Saccharomyces cerevisiae resemble both mutants affecting general control of amino acid biosynthesis and omnipotent translational suppressor mutants.

    PubMed

    McCusker, J H; Haber, J E

    1988-06-01

    Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

  20. Characterization of a Citrus R2R3-MYB Transcription Factor that Regulates the Flavonol and Hydroxycinnamic Acid Biosynthesis

    PubMed Central

    Liu, Chaoyang; Long, Jianmei; Zhu, Kaijie; Liu, Linlin; Yang, Wei; Zhang, Hongyan; Li, Li; Xu, Qiang; Deng, Xiuxin

    2016-01-01

    Flavonols and hydroxycinnamic acids are important phenylpropanoid metabolites in plants. In this study, we isolated and characterized a citrus R2R3-MYB transcription factor CsMYBF1, encoding a protein belonging to the flavonol-specific MYB subgroup. Ectopic expression of CsMYBF1 in tomato led to an up-regulation of a series of genes involved in primary metabolism and the phenylpropanoid pathway, and induced a strong accumulation of hydroxycinnamic acid compounds but not the flavonols. The RNAi suppression of CsMYBF1 in citrus callus caused a down-regulation of many phenylpropanoid pathway genes and reduced the contents of hydroxycinnamic acids and flavonols. Transactivation assays indicated that CsMYBF1 activated several promoters of phenylpropanoid pathway genes in tomato and citrus. Interestingly, CsMYBF1 could activate the CHS gene promoter in citrus, but not in tomato. Further examinations revealed that the MYBPLANT cis-elements were essential for CsMYBF1 in activating phenylpropanoid pathway genes. In summary, our data indicated that CsMYBF1 possessed the function in controlling the flavonol and hydroxycinnamic acid biosynthesis, and the regulatory differences in the target metabolite accumulation between two species may be due to the differential activation of CHS promoters by CsMYBF1. Therefore, CsMYBF1 constitutes an important gene source for the engineering of specific phenylpropanoid components. PMID:27162196

  1. Function Analysis of Caffeoyl-CoA O-Methyltransferase for Biosynthesis of Lignin and Phenolic Acid in Salvia miltiorrhiza.

    PubMed

    Wang, Zhengjun; Ge, Qian; Chen, Chen; Jin, Xinxin; Cao, Xiaoyan; Wang, Zhezhi

    2017-02-01

    In this study, we cloned a full-length cDNA and the genomic DNA sequence of SmCCoAOMT (GenBank ID JQ007585) from Salvia miltiorrhiza. The 744-bp open-reading frame encodes a protein of 247 amino acids that shares 95 % similarity with one in Vitis vinifera. Real-time quantitative PCR analysis revealed that SmCCoAOMT is most highly expressed in the stems and can be induced by methyl jasmonate (MeJA) and XC-1 treatment. To evaluate its function in vivo, we generated RNA interference transgenic plants through Agrobacterium tumefaciens-mediated gene transfer. Compared with untransformed control plants, the transgenics had significantly less lignin and the expression of lignin-biosynthetic genes SmCCR and SmCOMT was depressed. In 90-day-old roots from plants of transgenic line M5, accumulations of rosmarinic acid and salvianolic acid B (Sal B) were greatly reduced by 0.89- and 0.69-fold, respectively. This low-Sal B phenotype was stable in the roots, with the level of accumulation being approximately 43.58 mg g(-1) dry weight, which was 52 % of the amount measured in the untransformed control. Our results suggest that SmCCoAOMT is involved in lignin biosynthesis and affects the accumulation of phenolic acids. This study also provides potential guidance for using lignin-related genes to genetically engineer Salvia miltiorrhiza.

  2. DmSAS is required for sialic acid biosynthesis in cultured Drosophila third instar larvae CNS neurons.

    PubMed

    Granell, Annelise E von Bergen; Palter, Karen B; Akan, Ihan; Aich, Udayanath; Yarema, Kevin J; Betenbaugh, Michael J; Thornhill, William B; Recio-Pinto, Esperanza

    2011-11-18

    Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster (Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems. Here we investigated whether sialic acid incorporation in cultured Dm central nervous system (CNS) neurons required endogenously expressed Dm sialic acid synthase (DmSAS). We compared neurons derived from wild type Dm larvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac(4)ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents via Click-iT chemistry. We found that most of the wild type Dm CNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed α2,6-sialyltransferase. These results confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dm cells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in Dm CNS neurons, and the observed subcellular distribution of the newly synthesized sialic acids offers insights into their biological function.

  3. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid W

    SciTech Connect

    Messing, S.; Gabelli, S; Echeverria, I; Vogel, J; Guan, J; Tan, B; Klee, H; McCarty, D; Amzela, M

    2010-01-01

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  4. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

    SciTech Connect

    Messing, Simon A.J.; Gabelli, Sandra B.; Echeverria, Ignacia; Vogel, Jonathan T.; Guan, Jiahn Chou; Tan, Bao Cai; Klee, Harry J.; McCarty, Donald R.; Amzel, L. Mario

    2011-09-06

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  5. In Vivo Studies of the Biosynthesis of [alpha]-Eleostearic Acid in the Seed of Momordica charantia L.

    PubMed Central

    Liu, L.; Hammond, E. G.; Nikolau, B. J.

    1997-01-01

    In vivo radiotracer experiments using 14C-labeled acetate, oleate, linoleate, and linolenate were conducted to investigate the biosynthesis of [alpha]-eleostearic acid in the seeds of Momordica charantia. With the exception of [14C]linolenate, all of these precursors radioactively labeled [alpha]-eleostearate. Kinetics of the time course of metabolism of the radioactive precursors indicate that linoleate is the acyl precursor of [alpha]-eleostearate and that its conversion to [alpha]-eleostearate occurs while the acyl moiety is esterified to PC. Pulse-chase experiments with 14C-labeled acetate or linoleate provided additional corroborative evidence that linoleoyl PC is the precursor of [alpha]-eleostearoyl PC. PMID:12223677

  6. Identification of microRNAs Actively Involved in Fatty Acid Biosynthesis in Developing Brassica napus Seeds Using High-Throughput Sequencing

    PubMed Central

    Wang, Jia; Jian, Hongju; Wang, Tengyue; Wei, Lijuan; Li, Jiana; Li, Chao; Liu, Liezhao

    2016-01-01

    Seed development has a critical role during the spermatophyte life cycle. In Brassica napus, a major oil crop, fatty acids are synthesized and stored in specific tissues during embryogenesis, and understanding the molecular mechanism underlying fatty acid biosynthesis during seed development is an important research goal. In this study, we constructed three small RNA libraries from early seeds at 14, 21, and 28 days after flowering (DAF) and used high-throughput sequencing to examine microRNA (miRNA) expression. A total of 85 known miRNAs from 30 families and 1160 novel miRNAs were identified, of which 24, including 5 known and 19 novel miRNAs, were found to be involved in fatty acid biosynthesis.bna-miR156b, bna-miR156c, bna-miR156g, novel_mir_1706, novel_mir_1407, novel_mir_173, and novel_mir_104 were significantly down-regulated at 21 DAF and 28 DAF, whereas bna-miR159, novel_mir_1081, novel_mir_19 and novel_mir_555 were significantly up-regulated. In addition, we found that some miRNAs regulate functional genes that are directly involved in fatty acid biosynthesis and that other miRNAs regulate the process of fatty acid biosynthesis by acting on a large number of transcription factors. The miRNAs and their corresponding predicted targets were partially validated by quantitative RT-PCR. Our data suggest that diverse and complex miRNAs are involved in the seed development process and that miRNAs play important roles in fatty acid biosynthesis during seed development. PMID:27822220

  7. Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia.

    PubMed

    Semiz, Asli; Sen, Alaattin

    2015-03-01

    Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61% identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis.

  8. Autoimmune lymphoproliferative syndrome (ALPS) in a patient with a new germline Fas gene mutation.

    PubMed

    Del-Rey, Manuel J; Manzanares, Javier; Bosque, Alberto; Aguiló, Juan I; Gómez-Rial, José; Roldan, Ernesto; Serrano, Antonio; Anel, Alberto; Paz-Artal, Estela; Allende, Luis M

    2007-01-01

    Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphoproliferation, autoimmune manifestations and expansion of TCRalphabeta+CD4-CD8- lymphocytes. The main pathogenic factor is a defective Fas-mediated apoptosis generally caused by mutations in the Fas gene. This report describes a new heterozygous Fas gene mutation in a boy with clinical and immunological features of ALPS. In vitro, T-cell blasts from the patient are completely resistant to the effects on the anti-Fas cytotoxic mAb CH-11, they also have a higher proliferation rate than T cells from healthy donors, while PHA-induced AICD is normal. The location of the mutation (I246S) found in the intracytoplasmic death domain, and the conservation of that residue in four different species from human suggest that I246 is an essential amino acid for Fas function. The patient has inherited the mutation from his father who also shows defective Fas-mediated apoptosis but the clinical and immunological manifestations are much less severe. These results provide evidence that the penetrance of genetic defects in Fas is variable and that other factors may influence the phenotype of the disease.

  9. Using modern tools to probe the structure-function relationship of fatty acid synthases

    PubMed Central

    Burkart, Michael D.

    2015-01-01

    Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory. PMID:25676190

  10. Posttranslational regulation of Fas ligand function

    PubMed Central

    Voss, Matthias; Lettau, Marcus; Paulsen, Maren; Janssen, Ottmar

    2008-01-01

    The TNF superfamily member Fas ligand acts as a prototypic death factor. Due to its ability to induce apoptosis in Fas (APO-1, CD95) expressing cells, Fas ligand participates in essential effector functions of the immune system. It is involved in natural killer cell- and T cell-mediated cytotoxicity, the establishment of immune privilege, and in termination of immune responses by induction of activation-induced cell death. In addition, Fas ligand-positive tumours may evade immune surveillance by killing Fas-positive tumour-infiltrating cells. Given these strong cytotoxic capabilities of Fas ligand, it is obvious that its function has to be strictly regulated to avoid uncontrolled damage. In hematopoietic cells, the death factor is stored in secretory lysosomes and is mobilised to the immunological synapse only upon activation. The selective sorting to and the release from this specific lysosomal compartment requires interactions of the Fas ligand cytosolic moiety, which mediates binding to various adapter proteins involved in trafficking and cytoskeletal reorganisation. In addition, Fas ligand surface expression is further regulated by posttranslational ectodomain shedding and subsequent regulated intramembrane proteolysis, releasing a soluble ectodomain cytokine into the extracellular space and an N-terminal fragment with a potential role in intracellular signalling processes. Moreover, other posttranslational modifications of the cytosolic domain, including phosphorylation and ubiquitylation, have been described to affect various aspects of Fas ligand biology. Since FasL is regarded as a potential target for immunotherapy, the further characterisation of its biological regulation and function will be of great importance for the development and evaluation of future therapeutic strategies. PMID:19114018

  11. Biosynthesis of tiglic, ethacrylic, and 2-methylbutyric acids in a carabid beetle, Pterostichus (Hypherpes) californicus.

    PubMed

    Attygalle, Athula B; Wu, Xiaogang; Will, Kipling W

    2007-05-01

    Tiglic, 2-methylbutyric, and ethacrylic acids are found in the pygidial gland defensive fluid of many carabid beetles. By injecting a deuterium-labeled precursor into the carabid beetle Pterostichus (Hypherpes) californicus, and analyzing the defensive fluid by gas chromatography/mass spectrometry, we were able to demonstrate that tiglic and ethacrylic acids are biosynthesized from isoleucine via 2-methylbutyric acid. Moreover, we observed that the injection of L-isoleucine induces an increased production of tiglic acid in P. californicus. A strong primary kinetic isotope effect was found to operate in the dehydrogenation step of 2-methylbutyric acid to tiglic and ethacrylic acids. Consequently, ethacrylic acid was found to preferentially accumulate the deuterium labeling from [2,3,4,4-(2)H(4)]isoleucine during our biosynthetic experiments.

  12. Engagement of Fas on Macrophages Modulates Poly I:C induced cytokine production with specific enhancement of IP-10.

    PubMed

    Lyons, Caitriona; Fernandes, Philana; Fanning, Liam J; Houston, Aileen; Brint, Elizabeth

    2015-01-01

    Viral double-stranded RNA (dsRNA) is recognised by pathogen recognition receptors such as Toll-Like Receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I), and results in cytokine and interferon production. Fas, a well characterised death receptor, has recently been shown to play a role in the inflammatory response. In this study we investigated the role of Fas in the anti-viral immune response. Stimulation of Fas on macrophages did not induce significant cytokine production. However, activation of Fas modified the response of macrophages to the viral dsRNA analogue poly I:C. In particular, poly I:C-induced IP-10 production was significantly enhanced. A similar augmentation of IP-10 by Fas was observed following stimulation with both poly A:U and Sendai virus. Fas activation suppressed poly I:C-induced phosphorylation of the MAP kinases p38 and JNK, while overexpression of the Fas adaptor protein, Fas-associated protein with death domain (FADD), activated AP-1 and inhibited poly I:C-induced IP-10 production. Consistent with an inhibitory role for AP-1 in IP-10 production, mutation of the AP-1 binding site on the IP-10 promoter resulted in augmented poly I:C-induced IP-10. These results demonstrate that engagement of the Fas receptor plays a role in modifying the innate immune response to viral RNA.

  13. Biosynthesis of poly(4-hydroxybutyrate) in recombinant Escherichia coli grown on glycerol is stimulated by propionic acid.

    PubMed

    Kämpf, Michael M; Thöny-Meyer, Linda; Ren, Qun

    2014-11-01

    One of the most promising polyhydroxyalkanoates (PHAs) for medical applications is poly(4-hydroxybutyrate) (P4HB) due to its biodegradability, biocompatibility and mechanical properties. Currently, the major hurdle for expanding P4HB applications is the production and recovery cost. In this study, we investigated the stimulating factors for P4HB biosynthesis with the ultimate goal of reducing production cost. We found that addition of propionic acid to the culture medium stimulates the P4HB accumulation in recombinant Escherichia coli JM109 grown on glycerol. This stimulating effect was significantly weakened by addition of exogenous methionine, whereas it was not influenced by addition of cysteine. These results suggest that propionic acid enhances P4HB synthesis by reducing the intracellular methionine pool. Utilizing these findings for P4HB production in batch cultures on glycerol, the volumetric yield of P4HB could be improved 4 fold from 0.9g/L to 3.7g/L by adding 2g/L propionic acid into the medium.

  14. Solubilization and purification of the glucosyltransferase involved in the biosynthesis of teichuronic acid by fragments of Micrococcus luteus cell membranes

    SciTech Connect

    Hildebrandt, K.M.; Anderson, J.S.

    1987-05-01

    Enzymes involved in the biosynthesis of teichuronic acid have been demonstrated in cytoplasmic membrane fragments recovered from lysozyme treated Micrococcus luteus cells. Solubilization of the glucosyltransferase activity was effected with aqueous solutions of Triton X-100, Nonidet P-40, Tween 20, or Thesit. Thesit proved most amenable for recovery of glucosyltransferase activity as well as spectrophotometric protein determinations. Recovery of the glucosyltranferase activity was aided during purification by inclusion of 15% glycerol, 0.75% Thesit, 20 mM magnesium ion and 2 mM 2-mercaptoethanol in all buffers. Glucosyltransferase activity was monitored by the transfer of (/sup 14/C)glucose from UDP-(/sup 14/C)glucose to an artificial acceptor. Although the natural acceptor is presumed to be an undecaprenyl diphosphate-activated oligosaccharide, alternate acceptors such as isolated cell wall fractions containing teichuronic acid served equally well. Highly purified teichuronic acid devoid of peptidoglycan was the most effective alternate acceptor. The glucosyltransferase was purified by ammonium sulfate precipitation followed by ion exchange chromatography on DEAE-cellulose yielding an overall 200-fold increase in specific activity.

  15. Phosphatidic acid: biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

    PubMed

    Bond, Peter

    2017-01-01

    The mechanistic target of rapamycin complex 1 (mTORC1) has received much attention in the field of exercise physiology as a master regulator of skeletal muscle hypertrophy. The multiprotein complex is regulated by various signals such as growth factors, energy status, amino acids and mechanical stimuli. Importantly, the glycerophospholipid phosphatidic acid (PA) appears to play an important role in mTORC1 activation by mechanical stimulation. PA has been shown to modulate mTOR activity by direct binding to its FKBP12-rapamycin binding domain. Additionally, it has been suggested that exogenous PA activates mTORC1 via extracellular conversion to lysophosphatidic acid and subsequent binding to endothelial differentiation gene receptors on the cell surface. Recent trials have therefore evaluated the effects of PA supplementation in resistance-trained individuals on strength and body composition. As research in this field is rapidly evolving, this review attempts to provide a comprehensive overview of its biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

  16. Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

    PubMed Central

    Somerville, H. J.; Peel, J. L.

    1967-01-01

    Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-14C]- or [2-14C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of 14C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C3 unit derived directly from lactate with a C1 unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA→crotonyl-CoA→glutaconate→glutamate. PMID:6069834

  17. Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica strain DSK1

    NASA Astrophysics Data System (ADS)

    Fang, Jiasong; Uhle, Maria; Billmark, Kaycie; Bartlett, Douglas H.; Kato, Chaki

    2006-04-01

    We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica strain DSK1. The bacterium was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using sterile-filtered natural seawater supplied with glucose as the sole carbon source. Strain DSK1 synthesized typical bacterial fatty acids (C 14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6 ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed Δδ FA-glucose (-1.0‰ to -11.9‰) at 0.1 MPa was comparable to or slightly higher than fractionations reported in surface bacteria. However, bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation (Δδ FA-glucose) at high pressures was much higher than that for surface bacteria: -15.7‰, -15.3‰, and -18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions that are affected by hydrostatic pressure and to biosynthetic pathways that are different for short-chain and long-chain fatty acids. A simple quantitative calculation suggests that in situ piezophilic bacterial contribution of polyunsaturated fatty acids to marine sediments is nearly two orders of magnitude higher than that of marine phytoplankton and that the carbon isotope imprint of piezophilic bacteria can override that of surface phytoplankton. Our results have important implications for marine biogeochemistry. Depleted fatty acids reported in marine sediments and the water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i

  18. Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment

    PubMed Central

    Gondor, Orsolya K.; Janda, Tibor; Soós, Vilmos; Pál, Magda; Majláth, Imre; Adak, Malay K.; Balázs, Ervin; Szalai, Gabriella

    2016-01-01

    Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid. PMID:27733857

  19. Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment.

    PubMed

    Gondor, Orsolya K; Janda, Tibor; Soós, Vilmos; Pál, Magda; Majláth, Imre; Adak, Malay K; Balázs, Ervin; Szalai, Gabriella

    2016-01-01

    Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

  20. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    PubMed Central

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

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

    PubMed Central

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

    1997-01-01

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

  2. Dietary enhancement of selected fatty acid biosynthesis in the digestive gland of Mytilus galloprovincialis Lmk.

    PubMed

    Ventrella, Vittoria; Pagliarani, Alessandra; Nesci, Salvatore; Trombetti, Fabiana; Pirini, Maurizio

    2013-01-30

    The fatty acid composition of the digestive gland from the mussel Mytilus galloprovincialis subjected to three different dietary regimens for 30 days was analyzed. Samples were collected at the beginning and end of the trial to obtain a comprehensive picture of fatty acid dynamics. Group A was unfed; group B received a diet consisting of 100% Thalassiosira weissflogii and, thus, similar to natural food; and group C received a diet consisting of 100% wheat germ conferring a 18:2ω-6 abundance. Results indicate that fatty acid composition of lipid and phospholipid classes was affected by dietary treatments. However, adult mussel homeostatic skills minimized effects, and thus, only wheat germ diet deeply modified the fatty acid composition. Furthermore, in group C, the occurrence of the non-methylene-interrupted trienoic fatty acids was indicative of de novo fatty acid synthesis presumably because of active fatty acid elongation and Δ5 desaturation system, also supported by the general ω-3 polyunsaturated fatty acid decrease.

  3. Transcriptional regulation of chlorogenic acid biosynthesis in carrot root slices exposed to UV-B light

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orange carrots are well known for their nutritional value as producers of ß-carotene, a Vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, f...

  4. Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Wang, Yunpeng; Sun, Tao; Gao, Xingyan; Shi, Mengliang; Wu, Lina; Chen, Lei; Zhang, Weiwen

    2016-03-01

    3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO2 directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L(-1) (348.8 mg/g dry cell weight) 3-HP directly from CO2 in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO2 in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO2 fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.

  5. Transcription of the Streptococcus pyogenes hyaluronic acid capsule biosynthesis operon is regulated by previously unknown upstream elements.

    PubMed

    Falaleeva, Marina; Zurek, Oliwia W; Watkins, Robert L; Reed, Robert W; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M; Korotkova, Natalia

    2014-12-01

    The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

  6. Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

    PubMed Central

    Min, Bokkee; Pelaschier, Joanne T.; Graham, David E.; Tumbula-Hansen, Debra; Söll, Dieter

    2002-01-01

    Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [Becker, H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837; and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & Söll, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838–12843]. A genetic knockout disrupting this pathway deprives D. radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy. The organism's capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA. Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine. PMID:11880622

  7. New Approaches to Target the Mycolic Acid Biosynthesis Pathway for the Development of Tuberculosis Therapeutics

    PubMed Central

    North, E. Jeffrey; Jackson, Mary; Lee, Richard E.

    2015-01-01

    Mycolic acids are the major lipid component of the unique mycobacterial cell wall responsible for the protection of the tuberculosis bacilli from many outside threats. Mycolic acids are synthesized in the cytoplasm and transported to the outer membrane as trehalose-containing glycolipids before being esterified to the arabinogalactan portion of the cell wall and outer membrane glycolipids. The large size of these unique fatty acids is a result of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids to the mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, including fatty acid synthesis (KasA, KasB, MabA, InhA, HadABC), mycolic acid modifying enzymes (SAM-dependent methyltransferases, aNAT), fatty acid activating and condensing enzymes (FadD32, Acc, Pks13), transporters (MmpL3) and tranferases (Antigen 85A-C) all of which are excellent potential drug targets. Not surprisingly, in recent years many new compounds have been reported to inhibit specific portions of this pathway, discovered through both phenotypic screening and target enzyme screening. In this review, we analyze the new and emerging inhibitors of this pathway discovered in the post-genomic era of tuberculosis drug discovery, several of which show great promise as selective tuberculosis therapeutics. PMID:24245756

  8. Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium, Oscillatoria limnetica

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.; Lee, B.; Sweeney, M. J.; Klein, H. P.

    1989-01-01

    The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C16:1) of aerobically grown O. limnetica was shown to contain both the delta 7 (79%) and delta 9 (21%) isomers, while the octadecenoic (C18:1) acid was entirely the delta 9 acid. Incorporation of [2-14C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the delta 7 and delta 9 C16:1 and the delta 9 C18:1. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both delta 7 and delta 9 C16:1 and delta 9 and delta 11 C18:1. The synthesis of these is characteristic of a bacterial-type, anaerobic pathway.

  9. Biosynthesis of 5-aminopentanoic acid and 2-piperidone from cadaverine and 1-piperideine in mouse.

    PubMed

    Callery, P S; Geelhaar, L A

    1984-12-01

    1-Piperideine, 5-aminopentanoic acid, and its lactam, 2-piperidone, were identified as metabolites of cadaverine in 10,000 g mouse liver supernatants to which diamine oxidase had been added. Both metabolites were also found when the cadaverine metabolite 1-piperideine was incubated with the preparation which suggested that 1-piperideine is an intermediate in the formation of 5-aminopentanoic acid and 2-piperidone. Identification of the metabolites was based on gas chromatography-mass spectrometric analysis in comparison to authentic standards. Mouse brain homogenates converted 1-piperideine to 5-aminopentanoic acid. The results suggest that the metabolic fate of cadaverine may provide precursors of pharmacologically active analogues of GABA.

  10. Transcriptional profiling of genes involved in ascorbic acid biosynthesis, recycling, and degradation during three leaf developmental stages in celery.

    PubMed

    Huang, Wei; Wang, Guang-Long; Li, Hui; Wang, Feng; Xu, Zhi-Sheng; Xiong, Ai-Sheng

    2016-12-01

    Ascorbic acid (AsA) is an important nutrient in the human body and performs various healthy functions. With considerable medicinal properties, celery (Apium graveolens L.) could be a good source of AsA for human health. However, the biosynthetic, recycling, and degradation pathways of AsA in celery have yet to be characterized. To study the metabolic pathways involved in AsA, the genes involved in AsA biosynthesis, recycling, and degradation were isolated from celery, and their expression profiles and AsA levels were analyzed in the leaf blades and petioles of two celery varieties at three different growth stages. AsA levels were higher in 'Ventura' compared with 'Liuhehuangxinqin' in both tissues possibly because of different transcription levels of genes, such as L-galactose dehydrogenase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), and glutathione reductase (GR). Results revealed that the D-mannose/L-galactose pathway may be the predominant pathway in celery, and the D-galacturonic acid pathway appeared to contribute largely to AsA accumulation in petioles than in leaf blades in 'Liuhehuangxinqin.' AsA contents are regulated by complex regulatory mechanisms and vary at different growth stages, tissues, and varieties in celery. The results provide novel insights into AsA metabolic pathways in leaf during celery growth and development.

  11. Ascorbic Acid Biosynthesis and Brackish Water Acclimation in the Euryhaline Freshwater White-Rimmed Stingray, Himantura signifer

    PubMed Central

    Wong, Samuel Z. H.; Ching, Biyun; Chng, You R.; Wong, Wai P.; Chew, Shit F.; Ip, Yuen K.

    2013-01-01

    L-gulono-γ-lactone oxidase (Gulo) catalyzes the last step of ascorbic acid biosynthesis, which occurs in the kidney of elasmobranchs. This study aimed to clone and sequence gulonolactone oxidase (gulo) from the kidney of the euryhaline freshwater stingray, Himantura signifer, and to determine the effects of acclimation from freshwater to brackish water (salinity 20) on its renal gulo mRNA expression and Gulo activity. We also examined the effects of brackish water acclimation on concentrations of ascorbate, dehydroascorbate and ascorbate + dehydroascorbate in the kidney, brain and gill. The complete cDNA coding sequence of gulo from the kidney of H. signifer contained 1323 bp coding for 440 amino acids. The expression of gulo was kidney-specific, and renal gulo expression decreased significantly by 67% and 50% in fish acclimated to brackish water for 1 day and 6 days, respectively. There was also a significant decrease in renal Gulo activity after 6 days of acclimation to brackish water. Hence, brackish water acclimation led to a decrease in the ascorbic acid synthetic capacity in the kidney of H. signifer. However, there were significant increases in concentrations of ascorbate and ascorbate + dehydroascorbate in the gills (after 1 or 6 days), and a significant increase in the concentration of ascorbate and a significant decrease in the concentration of dehydroascorbate in the brain (after 1 day) of fish acclimated to brackish water. Taken together, our results indicate that H. signifer might experience greater salinity-induced oxidative stress in freshwater than in brackish water, possibly related to its short history of freshwater invasion. These results also suggest for the first time a possible relationship between the successful invasion of the freshwater environment by some euryhaline marine elasmobranchs and the ability of these elasmobranchs to increase the capacity of ascorbic acid synthesis in response to hyposalinity stress. PMID:23825042

  12. Ascorbic Acid Biosynthesis and Brackish Water Acclimation in the Euryhaline Freshwater White-Rimmed Stingray, Himantura signifer.

    PubMed

    Wong, Samuel Z H; Ching, Biyun; Chng, You R; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2013-01-01

    L-gulono-γ-lactone oxidase (Gulo) catalyzes the last step of ascorbic acid biosynthesis, which occurs in the kidney of elasmobranchs. This study aimed to clone and sequence gulonolactone oxidase (gulo) from the kidney of the euryhaline freshwater stingray, Himantura signifer, and to determine the effects of acclimation from freshwater to brackish water (salinity 20) on its renal gulo mRNA expression and Gulo activity. We also examined the effects of brackish water acclimation on concentrations of ascorbate, dehydroascorbate and ascorbate + dehydroascorbate in the kidney, brain and gill. The complete cDNA coding sequence of gulo from the kidney of H. signifer contained 1323 bp coding for 440 amino acids. The expression of gulo was kidney-specific, and renal gulo expression decreased significantly by 67% and 50% in fish acclimated to brackish water for 1 day and 6 days, respectively. There was also a significant decrease in renal Gulo activity after 6 days of acclimation to brackish water. Hence, brackish water acclimation led to a decrease in the ascorbic acid synthetic capacity in the kidney of H. signifer. However, there were significant increases in concentrations of ascorbate and ascorbate + dehydroascorbate in the gills (after 1 or 6 days), and a significant increase in the concentration of ascorbate and a significant decrease in the concentration of dehydroascorbate in the brain (after 1 day) of fish acclimated to brackish water. Taken together, our results indicate that H. signifer might experience greater salinity-induced oxidative stress in freshwater than in brackish water, possibly related to its short history of freshwater invasion. These results also suggest for the first time a possible relationship between the successful invasion of the freshwater environment by some euryhaline marine elasmobranchs and the ability of these elasmobranchs to increase the capacity of ascorbic acid synthesis in response to hyposalinity stress.

  13. Genetic Adaptation of Fatty-Acid Metabolism: A Human-Specific Haplotype Increasing the Biosynthesis of Long-Chain Omega-3 and Omega-6 Fatty Acids

    PubMed Central

    Ameur, Adam; Enroth, Stefan; Johansson, Åsa; Zaboli, Ghazal; Igl, Wilmar; Johansson, Anna C.V.; Rivas, Manuel A.; Daly, Mark J.; Schmitz, Gerd; Hicks, Andrew A.; Meitinger, Thomas; Feuk, Lars; van Duijn, Cornelia; Oostra, Ben; Pramstaller, Peter P.; Rudan, Igor; Wright, Alan F.; Wilson, James F.; Campbell, Harry; Gyllensten, Ulf

    2012-01-01

    Omega-3 and omega-6 long-chain polyunsaturated fatty acids (LC-PUFAs) are essential for the development and function of the human brain. They can be obtained directly from food, e.g., fish, or synthesized from precursor molecules found in vegetable oils. To determine the importance of genetic variability to fatty-acid biosynthesis, we studied FADS1 and FADS2, which encode rate-limiting enzymes for fatty-acid conversion. We performed genome-wide genotyping (n = 5,652 individuals) and targeted resequencing (n = 960 individuals) of the FADS region in five European population cohorts. We also analyzed available genomic data from human populations, archaic hominins, and more distant primates. Our results show that present-day humans have two common FADS haplotypes—defined by 28 closely linked SNPs across 38.9 kb—that differ dramatically in their ability to generate LC-PUFAs. No independent effects on FADS activity were seen for rare SNPs detected by targeted resequencing. The more efficient, evolutionarily derived haplotype appeared after the lineage split leading to modern humans and Neanderthals and shows evidence of positive selection. This human-specific haplotype increases the efficiency of synthesizing essential long-chain fatty acids from precursors and thereby might have provided an advantage in environments with limited access to dietary LC-PUFAs. In the modern world, this haplotype has been associated with lifestyle-related diseases, such as coronary artery disease. PMID:22503634

  14. Biosynthesis of gallic acid in Rhus typhina: discrimination between alternative pathways from natural oxygen isotope abundance.

    PubMed

    Werner, Roland A; Rossmann, Andreas; Schwarz, Christine; Bacher, Adelbert; Schmidt, Hanns-Ludwig; Eisenreich, Wolfgang

    2004-10-01

    The biosynthetic pathway of gallic acid in leaves of Rhus typhina is studied by oxygen isotope ratio mass spectrometry at natural oxygen isotope abundance. The observed delta18O-values of gallic acid indicate an 18O-enrichment of the phenolic oxygen atoms of more than 30 per thousand above that of the leaf water. This enrichment implies biogenetical equivalence with oxygen atoms of carbohydrates but not with oxygen atoms introduced by monooxygenase activation of molecular oxygen. It can be concluded that all phenolic oxygen atoms of gallic acid are retained from the carbohydrate-derived precursor 5-dehydroshikimate. This supports that gallic acid is synthesized entirely or predominantly by dehydrogenation of 5-dehydroshikimate.

  15. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.

    PubMed

    Rodriguez, Angelica; Kildegaard, Kanchana R; Li, Mingji; Borodina, Irina; Nielsen, Jens

    2015-09-01

    Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93 ± 0.26 g L(-1) was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4(K229L), chorismate mutase ARO7(G141S) and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides.

  16. Biosynthesis: Imaging cell-wall biosynthesis live

    NASA Astrophysics Data System (ADS)

    Bugg, Timothy D. H.

    2013-01-01

    The biosynthesis of peptidoglycan is an important step in bacterial cell division and cell-wall maturation. Now it has been shown that fluorescent D-amino acids can be used to label the peptidoglycan cell wall of living bacteria, providing a new tool to study this important process.

  17. Methyl-branched poly(hydroxyalkanoate) biosynthesis from 13- methyltetradecanoic acid and mixed isostearic acid isomer substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas resinovorans, a known medium-chain-length (mcl-) poly(hydroxyalkanoate) (PHA) producer, was grown on 13-methyltetradecanoic acid (13-MTDA) and a mixture of isostearic acid (IA) isomers to produce methyl-branched mcl-PHA polymers. Shake flask experiments revealed polymer productivities (...

  18. De Novo Amino Acid Biosynthesis Contributes to Salmonella enterica Growth in Alfalfa Seedling Exudates

    PubMed Central

    Kwan, Grace; Pisithkul, Tippapha; Amador-Noguez, Daniel

    2014-01-01

    Salmonella enterica is a member of the plant microbiome. Growth of S. enterica in sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305 S. enterica proteins extracted 24 h postinoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over one-quarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important for S. enterica growth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor during S. enterica growth in association with roots. Collectively, these data suggest that S. enterica metabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth. PMID:25416761

  19. Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification.

    PubMed Central

    Kranz, R G; Gabbert, K K; Madigan, M T

    1997-01-01

    The photosynthetic bacterium Rhodobacter capsulatus can grow with short- to long-chain fatty acids as the sole carbon source (R. G. Kranz, K. K. Gabbert, T. A. Locke, and M. T. Madigan, Appl. Environ. Microbiol. 63:3003-3009, 1997). Concomitant with growth on fatty acids is the production to high levels of the polyester storage compounds called polyhydroxyalkanoates (PHAs). Here, we describe colony screening and selection systems to analyze the production of PHAs in R. capsulatus. A screen with Nile red dissolved in acetone distinguishes between PHA producers and nonproducers. Unlike the wild type, an R. capsulatus PhaC- strain with the gene encoding PHA synthase deleted is unable to grow on solid media containing high concentrations of certain fatty acids. It is proposed that this deficiency is due to the inability of the PhaC- strain to detoxify the surrounding medium by consumption of fatty acids and their incorporation into PHAs. This fatty acid toxicity phenotype is used in selection for the cloning and characterization of heterologous phaC genes. PMID:9251190

  20. NaCS-PDMDAAC immobilized cultivation of recombinant Dictyostelium discoideum for soluble human Fas ligand production.

    PubMed

    Zheng, Chao; Zeng, Xianhai; Danquah, Michael K; Lu, Yinghua

    2015-01-01

    Dictyostelium discoideum is a promising eukaryotic host for the expression of heterologous proteins requiring post-translational modifications. However, the dilute nature of D. discoideum cell culture limits applications for high value proteins production. D. discoideum cells, entrapped in sodium cellulose sulfate/poly-dimethyl-diallyl-ammonium chloride (NaCS-PDMDAAC) capsules were used for biosynthesis of the heterologous protein, soluble human Fas ligand (hFasL). Semi-continuous cultivations with capsules recycling were carried out in shake flasks. Also, a scaled-up cultivation of immobilized D. discoideum for hFasL production in a customized vitreous airlift bioreactor was conducted. The results show that NaCS-PDMDAAC capsules have desirable biophysical properties including biocompatibility with the D. discoideum cells and good mechanical stability throughout the duration of cultivation. A maximum cell density of 2.02 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 2.22 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 130.40 μg L(-1) (equivalent to a hFasL concentration of 1434.40 μg L(-1) in capsules) were obtained in shake flask cultivation with capsules recycling. Also, a maximum cell density of 1.72 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 1.89 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 106.10 μg L(-1) (equivalent to a hFasL concentration of 1167.10 μg L(-1) in capsules) were obtained after ∼170 h cultivation in the airlift bioreactor (with a working volume of 200 mL in a 315 mL bioreactor). As the article presents a premier work in the application of NaCS-PDMDAAC immobilized D. discoideum cells for the production of hFasL, more work is required to further optimize the system to generate higher cell densities and hFasL titers for large-scale applications.

  1. Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.

    PubMed Central

    Weih, F; Ryseck, R P; Chen, L; Bravo, R

    1996-01-01

    The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas. Images Fig. 1 Fig. 2 Fig. 4 PMID:8643610

  2. dFasArt: dynamic neural processing in FasArt model.

    PubMed

    Cano-Izquierdo, Jose-Manuel; Almonacid, Miguel; Pinzolas, Miguel; Ibarrola, Julio

    2009-05-01

    The temporal character of the input is, generally, not taken into account in the neural models. This paper presents an extension of the FasArt model focused on the treatment of temporal signals. FasArt model is proposed as an integration of the characteristic elements of the Fuzzy System Theory in an ART architecture. A duality between the activation concept and membership function is established. FasArt maintains the structure of the Fuzzy ARTMAP architecture, implying a static character since the dynamic response of the input is not considered. The proposed novel model, dynamic FasArt (dFasArt), uses dynamic equations for the processing stages of FasArt: activation, matching and learning. The new formulation of dFasArt includes time as another characteristic of the input. This allows the activation of the units to have a history-dependent character instead of being only a function of the last input value. Therefore, dFasArt model is robust to spurious values and noisy inputs. As experimental work, some cases have been used to check the robustness of dFasArt. A possible application has been proposed for the detection of variations in the system dynamics.

  3. Very long chain fatty acids (policosanols) and phytosterols affect plasma lipid levels and cholesterol biosynthesis in hamsters.

    PubMed

    Wang, Yanwen; Ebine, Naoyuki; Jia, Xiaoming; Jones, Peter J H; Fairow, Clint; Jaeger, Ralf

    2005-04-01

    The aim of the current study was to examine the effects of very long chain fatty acids (VLCFA) alone at 2 dietary levels, or in combination of VLCFA at the lower level with lecithin (LT) or phytosterols (PS), on lipid profiles and cholesterol biosynthesis in hamsters. Seventy-five male Golden Syrian hamsters, weighing 100 to 120 g, were fed a regular rodent chow for 2 weeks before being randomly assigned into 5 groups of 15 animals each fed semisynthetic diets for 4 weeks. Group 1 was given a control diet that contained 0.25% cholesterol and 5% fat with a polyunsaturated to saturated fatty acids ratio of 0.4. Groups 2 to 5 were fed the control diet and given 25 mg/kg BW per day of VLCFA (Licowax) (VLCFA25), 50 mg/kg BW per day of VLCFA (VLCFA50), 25 mg/kg BW per day of VLCFA+1000 mg/kg BW per day of LT (VLCFA25/LT), and 25 mg/kg BW per day of VLCFA+1000 mg/kg BW per day of PS (Cholestatin, VLCFA25/PS), respectively. Results showed that HDL-cholesterol (HDL-C) levels were not changed by VLCFA25, although increased by VLCFA50 (P<.05) relative to control. Total cholesterol (T-C) and non-HDL-C levels were not affected by VLCFA25 and VLCFA50 as compared with control. VLCFA25/LT had higher (P<.02) T-C and HDL-C levels than any other treatments and increased (P<.05) liver weight relative to control. In contrast, VLCFA25/PS reduced T-C (P=.0004) and non-HDL-C (P=.007) without effect on HDL-C levels compared with control. Triglyceride levels were not affected by any treatment. Cholesterol biosynthesis rate was higher (P<.05) in animals fed VLCFA25 and VLCFA50 than those fed control or VLCFA25/LT or VLCFA25/PS. Results suggest that PSs can decrease total and non-HDL-C cholesterol, whereas VLCFA may increase HDL-C in hamsters.

  4. Biochemical and genetic engineering of diatoms for polyunsaturated fatty acid biosynthesis.

    PubMed

    Li, Hong-Ye; Lu, Yang; Zheng, Jian-Wei; Yang, Wei-Dong; Liu, Jie-Sheng

    2014-01-07

    The role of diatoms as a source of bioactive compounds has been recently explored. Diatom cells store a high amount of fatty acids, especially certain polyunsaturated fatty acids (PUFAs). However, many aspects of diatom metabolism and the production of PUFAs remain unclear. This review describes a number of technical strategies, such as modulation of environmental factors (temperature, light, chemical composition of culture medium) and culture methods, to influence the content of PUFAs in diatoms. Genetic engineering, a newly emerging field, also plays an important role in controlling the synthesis of fatty acids in marine microalgae. Several key points in the biosynthetic pathway of PUFAs in diatoms as well as recent progresses are also a critical part and are summarized here.

  5. Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.

    2001-01-01

    Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372

  6. Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold1[OPEN

    PubMed Central

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

    2015-01-01

    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. PMID:26224803

  7. De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

    PubMed Central

    Greseth, Matthew D.; Traktman, Paula

    2014-01-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  8. D27E mutation of VTC1 impairs the interaction with CSN5B and enhances ascorbic acid biosynthesis and seedling growth in Arabidopsis.

    PubMed

    Li, Shenghui; Wang, Juan; Yu, Yanwen; Wang, Fengru; Dong, Jingao; Huang, Rongfeng

    2016-11-01

    Our previous investigation revealed that GDP-Man pyrophosphorylase (VTC1), a vital ascorbic acid (AsA) biosynthesis enzyme, could be degraded through interaction with the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) in the darkness, demonstrating the posttranscriptional regulation of light signal in AsA production. Here, we further report that a point mutation in D27E of VTC1 disables the interaction with CSN5B, resulting in enhancement of AsA biosynthesis and seedling growth in Arabidopsis thaliana. To identify the interaction sites with CSN5B, we first predicted the key amino acids in VTC1 via bioinformatics analysis. And then we biochemically and genetically demonstrated that the 27th Asp was the amino acid that influenced the interaction of VTC1 with CSN5B in plants. Moreover, transgenic lines overexpressing the site-specific mutagenesis from D27 (Asp) into E27 (Glu) in VTC1 showed enhanced AsA accumulation and reduced H2O2 content in Arabidopsis seedlings, compared with the lines overexpressing the mutation from D27 into N27 (Asn) in VTC1. In addition, this regulation of VTC1 D27E mutation promoted seedling growth. Together, our data reveal that the 27th amino acid of VTC1 confers a key regulation in the interaction with CSN5B and AsA biosynthesis, as well as in Arabidopsis seedling growth.

  9. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-02

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  10. Regulation of indole-3-acetic acid biosynthesis by branched-chain amino acids in Enterobacter cloacae UW5.

    PubMed

    Parsons, Cassandra V; Harris, Danielle M M; Patten, Cheryl L

    2015-09-01

    The soil bacterium Enterobacter cloacae UW5 produces the rhizosphere signaling molecule indole-3-acetic acid (IAA) via the indolepyruvate pathway. Expression of indolepyruvate decarboxylase, a key pathway enzyme encoded by ipdC, is upregulated by the transcription factor TyrR in response to aromatic amino acids. Some members of the TyrR regulon may also be controlled by branched-chain amino acids and here we show that expression from the ipdC promoter and production of IAA are downregulated by valine, leucine and isoleucine. Regulation of the IAA synthesis pathway by both aromatic and branched-chain amino acids suggests a broader role for this pathway in bacterial physiology, beyond plant interactions.

  11. Biosynthesis of terephthalic acid, isophthalic acid and their derivatives from the corresponding dinitriles by tetrachloroterephthalonitrile-induced Rhodococcus sp.

    PubMed

    He, Yu-Cai; Wu, Ya-Dong; Pan, Xue-He; Ma, Cui-Luan

    2014-02-01

    The nitrilase from Rhodococcus sp. CCZU10-1 catalyses the hydrolysis of dinitriles to acids without the formation of amides and cyanocarboxylic acids. It was induced by benzonitrile and its analogues (tetrachloroterephthalonitrile > ε-caprolactam > benzonitrile > phenylacetonitrile), and had activity towards aromatic nitriles (terephthalonitrile > tetrachloroterephthalonitrile > isophthalonitrile > tetrachloroisophthalonitrile > tetrafluoroterephthalonitrile > benzonitrile). After the optimization, the highest nitrilase induction [311 U/(g DCW)] was achieved with tetrachloroterephthalonitrile (1 mM) in the medium after 24 h at 30 °C after optimum enzyme activity was at pH 6.8 and at 30 °C. Efficient biocatalyst recycling was achieved by cell immobilization in calcium alginate, with a product-to-biocatalyst ratios of 776 g terephthalic acid/g DCW and 630 g isophthalic acid/g DCW.

  12. Desaturase and elongase limiting endogenous long chain polyunsaturated fatty acid biosynthesis

    PubMed Central

    Zhang, Ji Yao; Kothapalli, Kumar S.D.; Brenna, J. Thomas

    2016-01-01

    Purpose of Review Endogenous synthesis of the long chain polyunsaturated fatty acids (LCPUFA) is mediated by the fatty acid desaturase (FADS) gene cluster (11q12-13.1) and elongation of very long chain fatty acids 2 (ELOVL2) (6p24.2) and ELOVL5 (6p12.1). Though older biochemical work identified the product of one gene, FADS2, rate limiting for LCPUFA synthesis, recent studies suggest that polymorphisms in any of these genes can limit accumulation of product LCPUFA. Recent findings Genome-wide association study (GWAS) of Greenland Inuit show strong adaptation signals within FADS gene cluster, attributed to high omega-3 fatty acid intake, while GWAS found ELOVL2 associated with sleep duration, age and DNA methylation. ELOVL5 coding mutations cause spinocerebellar ataxia 38, and epigenetic marks were associated with depression and suicide risk. Two sterol response element binding sites were found on ELOVL5, a SREBP-1c target gene. Minor allele carriers of a 3 single nucleotide polymorphism (SNP) haplotype in ELOVL2 have decreased 22:6n-3 levels. Unequivocal molecular evidence shows mammalian FADS2 catalyzes direct Δ4-desaturation to yield 22:6n-3 and 22:5n-6. A SNP near FADS1 influences the levels of 5-lipoxygenase products and epigenetic alteration. Summary Genetic polymorphisms within FADS and ELOVL can limit LCPUFA product accumulation at any step of the biosynthetic pathway. PMID:26828581

  13. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

    SciTech Connect

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent

    2011-08-24

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

  14. Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Caenorhabditis elegans secretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, perception of which enables worms to gauge depletion of food or a high worm population density. As a result, worms enter the dauer state, a specific developmental stage capable of surviv...

  15. Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia).

    PubMed

    Muir, Ryann M; Ibáñez, Ana M; Uratsu, Sandra L; Ingham, Elizabeth S; Leslie, Charles A; McGranahan, Gale H; Batra, Neelu; Goyal, Sham; Joseph, Jorly; Jemmis, Eluvathingal D; Dandekar, Abhaya M

    2011-04-01

    Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

  16. Indole-3-Acetic Acid Produced by Burkholderia heleia Acts as a Phenylacetic Acid Antagonist to Disrupt Tropolone Biosynthesis in Burkholderia plantarii

    PubMed Central

    Wang, Mengcen; Tachibana, Seiji; Murai, Yuta; Li, Li; Lau, Sharon Yu Ling; Cao, Mengchao; Zhu, Guonian; Hashimoto, Makoto; Hashidoko, Yasuyuki

    2016-01-01

    Burkholderia heleia PAK1-2 is a potent biocontrol agent isolated from rice rhizosphere, as it prevents bacterial rice seedling blight disease caused by Burkholderia plantarii. Here, we isolated a non-antibacterial metabolite from the culture fluid of B. heleia PAK1-2 that was able to suppress B. plantarii virulence and subsequently identified as indole-3-acetic acid (IAA). IAA suppressed the production of tropolone in B. plantarii in a dose-dependent manner without any antibacterial and quorum quenching activity, suggesting that IAA inhibited steps of tropolone biosynthesis. Consistent with this, supplementing cultures of B. plantarii with either L-[ring-2H5]phenylalanine or [ring-2H2~5]phenylacetic acid revealed that phenylacetic acid (PAA), which is the dominant metabolite during the early growth stage, is a direct precursor of tropolone. Exposure of B. plantarii to IAA suppressed production of both PAA and tropolone. These data particularly showed that IAA produced by B. heleia PAK1-2 disrupts tropolone production during bioconversion of PAA to tropolone via the ring-rearrangement on the phenyl group of the precursor to attenuate the virulence of B. plantarii. B. heleia PAK1-2 is thus likely a microbial community coordinating bacterium in rhizosphere ecosystems, which never eliminates phytopathogens but only represses production of phytotoxins or bacteriocidal substances. PMID:26935539

  17. Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine

    SciTech Connect

    Husain, Zaheed; Almeciga, Ingrid; Delgado, Julio C.; Clavijo, Olga P.; Castro, Januario E.; Belalcazar, Viviana; Pinto, Clara; Zuniga, Joaquin; Romero, Viviana; Yunis, Edmond J. . E-mail: edmond_yunis@dfci.harvard.edu

    2006-08-01

    Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 {mu}M) in the presence of 0.1 mM H{sub 2}O{sub 2} demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.

  18. Biosynthesis of the wall acidic polysaccharide in Bacillus cereus AHU 1356.

    PubMed

    Kojima, N; Araki, Y; Ito, E

    1986-03-17

    Biosynthetic studies on an acidic polysaccharide, comprising galactose, rhamnose, N-acetylglucosamine and sn-glycerol 1-phosphate, were carried out with a membrane system obtained from Bacillus cereus AHU 1356. Incubation of the membranes with UDP-[14C]Gal, TDP-[14C]Rha and UDP-[14C]GlcNAc resulted in the formation of four or more labeled-sugar-linked lipids and a labeled polysaccharide. Data on structural analysis of the sugar moieties released from the glycolipids, together with results of enzymatic conversion of [14C]galactose-linked lipid and [14C]Rha-Gal-linked lipid to higher-oligosaccharide-linked lipids and polysaccharide, led to the conclusion that the acidic polysaccharide is probably synthesized through the following pathway: (sequence in text) The glycerophosphate residues seem to be derived from phosphatidylglycerol.

  19. Determination of Gymnemic Acid I as a Protein Biosynthesis Inhibitor Using Chemical Proteomics.

    PubMed

    Capolupo, Angela; Esposito, Roberta; Zampella, Angela; Festa, Carmen; Riccio, Raffaele; Casapullo, Agostino; Tosco, Alessandra; Monti, Maria Chiara

    2017-03-03

    The plant Gymnema sylvestre has been used widely in traditional medicine as a remedy for several diseases, and its leaf extract is known to contain a group of bioactive triterpene saponins belonging to the gymnemic acid class. Gymnemic acid I (1) is one of the main components among this group of secondary metabolites and is endowed with an interesting bioactivity profile. Since there is a lack of information about its specific biological targets, the full interactome of 1 was investigated through a quantitative chemical proteomic approach, based on stable-isotope dimethyl labeling. The ribosome complex was found to be the main partner of compound 1, and a full validation of the proteomics results was achieved by orthogonal approaches. Further biochemical and biological investigations revealed an inhibitory effect of 1 on the ribosome machinery.

  20. Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes

    NASA Astrophysics Data System (ADS)

    Monson, K. David; Hayes, J. M.

    1982-02-01

    Methods for the determination of 13C abundances at individual olefinic carbon positions have been developed, tested, and shown to perform accurately. (1) The double bond is oxidized with ozone; (2) silver oxide is used to cleave the resulting ozonide quantitatively to carboxylic-acid fragments; (3) a modified Schmidt decarboxylation is used to produce CO 2 quantitatively from the carboxyl groups of the separated cleavage products; (4) the CO 2 is utilized for mass spectrometric analysis. The results of intramolecular isotopic analyses are combined with molecular-average isotopic compositions determined by total combustion in order to show that fatty acids biosynthesized by Escherichia coli grown aerobically with glucose as the sole carbon source and harvested at late log phase are depleted by approximately 3%. in 13C relative to the glucose. This fractionation arises in the formation of acetylcoenzyme A by pyruvate dehydrogenase and is localized at the carboxyl position in the acetyl-CoA product. The isotopic order in that two-carbon subunit is carried through the biosynthesis of fatty acids so that alternate positions in the fatty-acid chains are depleted in 13C by an amount equal to twice the molecular-average depletion. The kinetic isotope effect at C-2 for pyruvate dehydrogenase in vivo is shown to be approximately 2.3%. While it appears that no other fractionation mechanism has controlled the overall depletion of 13C in these fatty acids, a separate process responsible for control of isotopic abundances in the carboxyl groups has been identified and described elsewhere [Monson K.D. and Hayes J.M. (1980) J. Biol. Chem. 255, 11435-11441]. It is concluded that kinetic, rather than thermodynamic, factors have controlled isotopic distributions in these cells and that kinetic factors will be dominant in most biological reactions.

  1. A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis[OPEN

    PubMed Central

    Salie, Matthew J.; Zhang, Ning; Xu, Dong; Thelen, Jay J.

    2016-01-01

    Acetyl-CoA carboxylase (ACCase) catalyzes the committed step of de novo fatty acid biosynthesis. In prokaryotes, green algae, and most plants, this enzyme is a heteromeric complex requiring four different subunits for activity. The plant complex is recalcitrant to conventional purification schemes and hence the structure and composition of the full assembly have been unclear. In vivo coimmunoprecipitation using subunit-specific antibodies identified a novel family of proteins in Arabidopsis thaliana annotated as biotin/lipoyl attachment domain containing (BADC) proteins. Results from yeast two-hybrid and coexpression in Escherichia coli confirmed that all three BADC isoforms interact with the two biotin carboxyl carrier protein (BCCP) isoforms of Arabidopsis ACCase. These proteins resemble BCCP subunits but are not biotinylated due to a mutated biotinylation motif. We demonstrate that BADC proteins significantly inhibit ACCase activity in both E. coli and Arabidopsis. Targeted gene silencing of BADC isoform 1 in Arabidopsis significantly increased seed oil content when normalized to either mass or individual seed. We conclude the BADC proteins are ancestral BCCPs that gained a new function as negative regulators of ACCase after initial loss of the biotinylation motif. A functional model is proposed. PMID:27559025

  2. Biosynthesis of high molecular weight hyaluronic acid by Streptococcus zooepidemicus using oxygen vector and optimum impeller tip speed.

    PubMed

    Lai, Zee-Wei; Rahim, Raha Abdul; Ariff, Arbakariya B; Mohamad, Rosfarizan

    2012-09-01

    The potential use of n-dodecane and n-hexadecane as oxygen vectors for enhancing hyaluronic acid (HA) biosynthesis by Streptococcus zooepidemicus ATCC 39920 was investigated using a 2-L stirred-tank bioreactor equipped with helical ribbon or Rushton turbine impellers. The volumetric fraction of the oxygen vector influenced the gas-liquid volumetric oxygen transfer coefficient (K(L)a) positively. Batch HA fermentation with 1% (v/v) n-dodecane or 0.5% (v/v) n-hexadecane addition was carried out at different impeller tip speeds. Even though cell growth was lower in the fermentation with oxygen vector addition, the HA productivity and molecular weight were higher when compared to the fermentation without oxygen vector at low impeller tip speed. The highest HA concentration (4.25 gHA/l) and molecular weight (1.54 × 10(7) Da) were obtained when 0.5% (v/v) n-hexadecane and 0.785 m/s impeller tip speed of helical ribbon were used.

  3. Evidence for a universal pathway of abscisic acid biosynthesis in higher plants from sup 18 O incorporation patterns

    SciTech Connect

    Zeevaart, J.A.D.; Heath, T.G.; Gage, D.A. )

    1989-12-01

    Previous labeling studies of abscisic acid (ABA) with {sup 18}O{sub 2} have been mainly conducted with water-stressed leaves. In this study, {sup 18}O incorporation into ABA of stressed leaves of various species was compared with {sup 18}O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), {sup 18}O was most abundant in the carboxyl group, whereas incorporation of a second and third {sup 18}O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in {sup 18}O{sub 2}. ABA from turgid bean leaves showed significant {sup 18}O incorporation, again with highest {sup 18}O enrichment in the carboxyl group. On the basis of {sup 18}O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precusor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.

  4. Environmental DNA-encoded antibiotics fasamycins A and B inhibit FabF in type II fatty acid biosynthesis.

    PubMed

    Feng, Zhiyang; Chakraborty, Debjani; Dewell, Scott B; Reddy, Boojala Vijay B; Brady, Sean F

    2012-02-15

    In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of E. faecalis OG1RF. Next-generation sequencing of these mutants, in conjunction with in vitro biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by fabF overexpression. On the basis of comparisons with known FASII inhibitors and in silico docking studies, the chloro-gem-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.

  5. Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

    PubMed Central

    Krause, Katrin; Henke, Catarina; Asiimwe, Theodore; Ulbricht, Andrea; Klemmer, Sandra; Schachtschabel, Doreen

    2015-01-01

    Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza. PMID:26231639

  6. Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis

    PubMed Central

    Chen, Sheng-Chia; Huang, Chi-Hung; Lai, Shu-Jung; Yang, Chia Shin; Hsiao, Tzu-Hung; Lin, Ching-Heng; Fu, Pin-Kuei; Ko, Tzu-Ping; Chen, Yeh

    2016-01-01

    The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme. PMID:26980148

  7. Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids

    PubMed Central

    Reynolds, Kyle B.; Taylor, Matthew C.; Zhou, Xue-Rong; Vanhercke, Thomas; Wood, Craig C.; Blanchard, Christopher L.; Singh, Surinder P.; Petrie, James R.

    2015-01-01

    Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation. PMID:25852716

  8. The biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Identification and functional analysis of CMAS-2.

    PubMed

    George, K M; Yuan, Y; Sherman, D R; Barry, C E

    1995-11-10

    The major mycolic acid produced by Mycobacterium tuberculosis contains two cis-cyclopropanes in the meromycolate chain. The gene whose product cyclopropanates the proximal double bond was cloned by homology to a putative cyclopropane synthase identified from the Mycobacterium leprae genome sequencing project. This gene, named cma2, was sequenced and found to be 52% identical to cma1 (which cyclopropanates the distal double bond) and 73% identical to the gene from M. leprae. Both cma genes were found to be restricted in distribution to pathogenic species of mycobacteria. Expression of cma2 in Mycobacterium smegmatis resulted in the cyclopropanation of the proximal double bond in the alpha 1 series of mycolic acids. Coexpression of both cyclopropane synthases resulted in cyclopropanation of both centers, producing a molecule structurally similar to the M. tuberculosis alpha-dicyclopropyl mycolates. Differential scanning calorimetry of purified cell walls and mycolic acids demonstrated that cyclopropanation of the proximal position raised the observed transition temperature by 3 degrees C. These results suggest that cyclopropanation contributes to the structural integrity of the cell wall complex.

  9. De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

    PubMed

    Gottardi, Manuela; Knudsen, Jan Dines; Prado, Lydie; Oreb, Mislav; Branduardi, Paola; Boles, Eckhard

    2017-03-29

    The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

  10. 2-Oxoglutarate: linking TCA cycle function with amino acid, glucosinolate, flavonoid, alkaloid, and gibberellin biosynthesis

    PubMed Central

    Araújo, Wagner L.; Martins, Auxiliadora O.; Fernie, Alisdair R.; Tohge, Takayuki

    2014-01-01

    The tricarboxylic acid (TCA) cycle intermediate 2-oxoglutarate (2-OG) is used as an obligatory substrate in a range of oxidative reactions catalyzed by 2-OG-dependent dioxygenases. These enzymes are widespread in nature being involved in several important biochemical processes. We have recently demonstrated that tomato plants in which the TCA cycle enzyme 2-OG dehydrogenase (2-ODD) was antisense inhibited were characterized by early senescence and modified fruit ripening associated with differences in the levels of bioactive gibberellin (GA). Accordingly, there is now compelling evidence that the TCA cycle plays an important role in modulating the rate of flux from 2-OG to amino acid metabolism. Here we discuss recent advances in the biochemistry and molecular biology of 2-OG metabolism occurring in different biological systems indicating the importance of 2-OG and 2-OG dependent dioxygenases not only in glucosinolate, flavonoid and alkaloid metabolism but also in GA and amino acid metabolism. We additionally summarize recent findings regarding the impact of modification of 2-OG metabolism on biosynthetic pathways involving 2-ODDs. PMID:25360142

  11. Type III polyketide synthase is involved in the biosynthesis of protocatechuic acid in Aspergillus niger.

    PubMed

    Lv, Yangyong; Xiao, Jing; Pan, Li

    2014-11-01

    Genomic studies have shown that not only plants but also filamentous fungi contain type III polyketide synthases. To study the function of type III polyketide synthase (AnPKSIII) in Aspergillus niger, a deletion strain (delAnPKSIII) and an overexpression strain (oeAnPKSIII) were constructed in A. niger MA169.4, a derivative of the wild-type (WT) A. niger ATCC 9029 that produces large quantities of gluconic acid. Alterations in the metabolites were analyzed by HPLC when the extract of the overexpression strain was compared with extracts of the WT and deletion strains. Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid, 3.2 mg/l) was isolated and identified as the main product of AnPKSIII when inductively expressed in A. niger MA169.4. The molecular weight of PCA was 154.1 (m/z 153.1 [M-H](-)), was detected by ESI-MS in the negative ionization mode, and (1)H and (13)C NMR data confirmed its structure.

  12. A Type II Pathway for Fatty Acid Biosynthesis Presents Drug Targets in Plasmodium falciparum

    PubMed Central

    Waller, Ross F.; Ralph, Stuart A.; Reed, Michael B.; Su, Vanessa; Douglas, James D.; Minnikin, David E.; Cowman, Alan F.; Besra, Gurdyal S.; McFadden, Geoffrey I.

    2003-01-01

    It has long been held that the malaria parasite, Plasmodium sp., is incapable of de novo fatty acid synthesis. This view has recently been overturned with the emergence of data for the presence of a fatty acid biosynthetic pathway in the relict plastid of P. falciparum (known as the apicoplast). This pathway represents the type II pathway common to plant chloroplasts and bacteria but distinct from the type I pathway of animals including humans. Specific inhibitors of the type II pathway, thiolactomycin and triclosan, have been reported to target this Plasmodium pathway. Here we report further inhibitors of the plastid-based pathway that inhibit Plasmodium parasites. These include several analogues of thiolactomycin, two with sixfold-greater efficacy than thiolactomycin. We also report that parasites respond very rapidly to such inhibitors and that the greatest sensitivity is seen in ring-stage parasites. This study substantiates the importance of fatty acid synthesis for blood-stage parasite survival and shows that this pathway provides scope for the development of novel antimalarial drugs. PMID:12499205

  13. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

    PubMed Central

    Reichau, Sebastian; Blackmore, Nicola J.; Jiao, Wanting; Parker, Emily J.

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal. PMID:27128682

  14. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids.

    PubMed

    Reichau, Sebastian; Blackmore, Nicola J; Jiao, Wanting; Parker, Emily J

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal.

  15. Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects1[C][W][OA

    PubMed Central

    Hamberger, Björn; Ohnishi, Toshiyuki; Hamberger, Britta; Séguin, Armand; Bohlmann, Jörg

    2011-01-01

    Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism. PMID:21994349

  16. Brassinosteroids Improve Quality of Summer Tea (Camellia sinensis L.) by Balancing Biosynthesis of Polyphenols and Amino Acids

    PubMed Central

    Li, Xin; Ahammed, Golam J.; Li, Zhi-Xin; Zhang, Lan; Wei, Ji-Peng; Shen, Chen; Yan, Peng; Zhang, Li-Ping; Han, Wen-Yan

    2016-01-01

    Summer grown green tea is less popular due to bitterness and high astringency, which are attributed to high levels of tea polyphenols (TP) and low levels of amino acids (AA) in tea leaves (Camellia sinensis L.). Brassinosteroids (BRs), a group of steroidal plant hormones can regulate primary and secondary metabolism in a range of plant species under both normal and stress conditions. However, specific effects of BRs on the photosynthesis of tea plants and the quality of summer green tea are largely unknown. Here we show that 24-epibrassinolide (EBR), a bioactive BR, promoted photosynthesis in tea plants in a concentration-dependent manner. Stimulation in photosynthesis by EBR resulted in an increased summer tea yield. Although all tested concentrations (0.01, 0.05, 0.1, 0.5, and 1.0 ppm) of EBR increased concentrations of TP and AA, a moderate concentration (0.5 ppm) caused the highest decrease in TP to AA ratio, an important feature of quality tea. Time-course analysis using 0.5 ppm EBR as foliar spray revealed that TP or AA concentration increased as early as 3 h after EBR application, reaching the highest peak at 24 h and that remained more or less stable. Importantly, such changes in TP and AA concentration by EBR resulted in a remarkably decreased but stable TP to AA ratio at 24 h and onward. Furthermore, concentrations of catechins and theanine increased, while that of caffeine remained unaltered following treatment with EBR. EBR improved activity of phenylalanine ammonia-lyase (PAL) and glutamine: 2-oxoglutarate aminotransferase (GOGAT) enzymes involved in catechins and theanine biosynthesis, respectively. Transcript analysis revealed that transcript levels of CsPAL and CsGS peaked as early as 6 h, while that of CsGOGAT peaked at 12 h following application of EBR, implying that EBR increased the concentration of TP and AA by inducing their biosynthesis. These results suggest a positive role of BR in enhancing green tea quality, which might have potential

  17. BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS

    EPA Science Inventory

    Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis ...

  18. Nonylphenol induces thymocyte apoptosis through Fas/FasL pathway by mimicking estrogen in vivo.

    PubMed

    Yao, Genhong; Hou, Yayi

    2004-05-01

    Nonylphenol (NP) is the final biodegradation product of nonylphenol polyethoxylates, which are widely used surfactants in domestic and industrial products. Nonylphenol has been reported to have estrogenic activity and shown to have potential reproductive toxicity. However, its influence on immune system function remains unclear. In this study, we investigated the effects of nonylphenol on apoptosis and Fas/FasL gene expression in rat thymus. Nonylphenol were given orally by gavages at 125, 250, and 375mg/kg per day. Negative and positive controls were treated with the vehicle and E(2) 10ng/kg per day, respectively. Atrophy of thymus was determined by in situ morphological examination using hematoxylin and eosin staining. Apoptotic cells were identified by terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labeling (TUNEL) assay. A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze Fas and FasL mRNA levels. The results showed that both nonylphenol and E(2) increased the rates of apoptotic death; reduced the expression of Fas; enhanced the expression of FasL. These findings demonstrated that nonylphenol with estrogen-like activity might affect the regulation of the immune function through thymocyte apoptosis. This apoptosis was mediated by altering the expression of Fas and FasL mRNA.

  19. Fas and FasL expression in the spinal cord following cord hemisection in the monkey.

    PubMed

    Jia, Liu; Yu, Zou; Hui, Li; Yu-Guang, Guan; Xin-Fu, Zhou; Chao, You; Yanbin, Xiyang; Xi, Zhan; Jun, Wang; Xin-Hua, Heng; Xin-Hua, Hen; Ting-Hua, Wang

    2011-03-01

    The changes of endogenous Fas/FasL in injured spinal cord, mostly in primates, are not well known. In this study, we investigated the temporal changes in the expression of Fas and FasL and explored their possible roles in the ventral horn of the spinal cord and associated precentral gyrus following T(11) spinal cord hemisection in the adult rhesus monkey. A significant functional improvement was seen with the time going on in monkeys subjected to cord hemisection. Apoptotic cells were also seen in the ventral horn of injured spinal cord with TUNEL staining, and a marked increase presents at 7 days post operation (dpo). Simultaneously, the number of Fas and FasL immunoreactive neurons in the spinal cords caudal and rostral to injury site and their intracellular optical density (OD) in the ipsilateral side of injury site at 7 dpo increased significantly more than that of control group and contralateral sides. This was followed by a decrease and returned to normal level at 60 dpo. No positive neurons were observed in precentral gyrus. The present results may provide some insights to understand the role of Fas/FasL in the spinal cord but not motor cortex with neuronal apoptosis and neuroplasticity in monkeys subjected to hemisection spinal cord injury.

  20. Relationship of Acute Lung Inflammatory Injury to Fas/FasL System

    PubMed Central

    Neff, Thomas A.; Guo, Ren-Feng; Neff, Simona B.; Sarma, J. Vidya; Speyer, Cecilia L.; Gao, Hongwei; Bernacki, Kurt D.; Huber-Lang, Markus; McGuire, Stephanie; Hoesel, L. Marco; Riedemann, Niels C.; Beck-Schimmer, Beatrice; Zetoune, Firas S.; Ward, Peter A.

    2005-01-01

    There is mounting evidence that apoptosis plays a significant role in tissue damage during acute lung injury. To evaluate the role of the apoptosis mediators Fas and FasL in acute lung injury, Fas (lpr)- or FasL (gld)-deficient and wild-type mice were challenged with intrapulmonary deposition of IgG immune complexes. Lung injury parameters (125I-albumin leak, accumulation of myeloperoxidase, and wet lung weights) were measured and found to be consistently reduced in both lpr and gld mice. In wild-type mice, lung injury was associated with a marked increase in Fas protein in lung. Inflamed lungs of wild-type mice showed striking evidence of activated caspase-3, which was much diminished in inflamed lungs from lpr mice. Intratracheal administration of a monoclonal Fas-activating antibody (Jo2) in wild-type mice induced MIP-2 and KC production in bronchoalveolar lavage fluids, and a murine alveolar macrophage cell line (MH-S) showed significantly increased MIP-2 production after incubation with this antibody. Bronchoalveolar lavage fluid content of MIP-2 and KC was substantially reduced in lpr mice after lung injury when compared to levels in wild-type mice. These data suggest that the Fas/FasL system regulates the acute lung inflammatory response by positively affecting CXC-chemokine production, ultimately leading to enhanced neutrophil influx and tissue damage. PMID:15743781

  1. Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots.

    PubMed

    Armendáriz, O; Gil-Monreal, M; Zulet, A; Zabalza, A; Royuela, M

    2016-05-01

    The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.

  2. Pale-Green Phenotype of atl31 atl6 Double Mutant Leaves Is Caused by Disruption of 5-Aminolevulinic Acid Biosynthesis in Arabidopsis thaliana

    PubMed Central

    Maekawa, Shugo; Takabayashi, Atsushi; Huarancca Reyes, Thais; Yamamoto, Hiroko; Tanaka, Ayumi; Sato, Takeo; Yamaguchi, Junji

    2015-01-01

    Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the pale-green leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant. PMID:25706562

  3. The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid ω-hydroxylase involved in suberin monomer biosynthesis

    PubMed Central

    Höfer, Rene; Briesen, Isabel; Beck, Martina; Pinot, Franck; Schreiber, Lukas; Franke, Rochus

    2008-01-01

    The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of ω-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid ω-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in ω-hydroxyacids with a chain length biosynthesis takes place in this sub-cellular compartment before intermediates are exported in the apoplast. PMID:18544608

  4. Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105

    PubMed Central

    Youssef, Noha H.; Wofford, Neil; McInerney, Michael J.

    2011-01-01

    Bacillus species produce extracellular, surface-active lipopeptides such as surfactin that have wide applications in industry and medicine. The steps involved in the synthesis of 3-hydroxyacyl-coenzyme A (CoA) substrates needed for surfactin biosynthesis are not understood. Cell-free extracts of Bacillus subtilis strain OKB105 synthesized lipopeptide biosurfactants in presence of l-amino acids, myristic acid, coenzyme A, ATP, and H2O2, which suggested that 3-hydroxylation occurs prior to CoA ligation of the long chain fatty acids (LCFAs). We hypothesized that YbdT, a cytochrome P450 enzyme known to beta-hydroxylate LCFAs, functions to form 3-hydroxy fatty acids for lipopeptide biosynthesis. An in-frame mutation of ybdT was constructed and the resulting mutant strain (NHY1) produced predominantly non-hydroxylated lipopeptide with diminished biosurfactant and beta-hemolytic activities. Mass spectrometry showed that 95.6% of the fatty acids in the NHY1 biosurfactant were non-hydroxylated compared to only ∼61% in the OKB105 biosurfactant. Cell-free extracts of the NHY1 synthesized surfactin containing 3-hydroxymyristic acid from 3-hydroxymyristoyl-CoA at a specific activity similar to that of the wild type (17 ± 2 versus 17.4 ± 6 ng biosurfactant min−1·ng·protein−1, respectively). These results showed that the mutation did not affect any function needed to synthesize surfactin once the 3-hydroxyacyl-CoA substrate was formed and that YbdT functions to supply 3-hydroxy fatty acid for surfactin biosynthesis. The fact that YbdT is a peroxidase could explain why biosurfactant production is rarely observed in anaerobically grown Bacillus species. Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions. PMID:21673922

  5. Fermentation and alternative oxidase contribute to the action of amino acid biosynthesis-inhibiting herbicides.

    PubMed

    Zulet, Amaia; Gil-Monreal, Miriam; Zabalza, Ana; van Dongen, Joost T; Royuela, Mercedes

    2015-03-01

    Acetolactate synthase inhibitors (ALS-inhibitors) and glyphosate (GLP) are two classes of herbicide that act by the specific inhibition of an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. The physiological effects that are detected after application of these two classes of herbicides are not fully understood in relation to the primary biochemical target inhibition, although they have been well documented. Interestingly, the two herbicides' toxicity includes some common physiological effects suggesting that they kill the treated plants by a similar pattern despite targeting different enzymes. The induction of aerobic ethanol fermentation and alternative oxidase (AOX) are two examples of these common effects. The objective of this work was to gain further insight into the role of fermentation and AOX induction in the toxic consequences of ALS-inhibitors and GLP. For this, Arabidopsis T-DNA knockout mutants of alcohol dehydrogenase (ADH) 1 and AOX1a were used. The results found in wild-type indicate that both GLP and ALS-inhibitors reduce ATP production by inducing fermentation and alternative respiration. The main physiological effects in the process of herbicide activity upon treated plants were accumulation of carbohydrates and total free amino acids. The effects of the herbicides on these parameters were less pronounced in mutants compared to wild-type plants. The role of fermentation and AOX regarding pyruvate availability is also discussed.

  6. Biosynthesis of highly pure poly-γ-glutamic acid for biomedical applications.

    PubMed

    Pereira, Catarina Leite; Antunes, Joana Costa; Gonçalves, Raquel Madeira; Ferreira-da-Silva, Frederico; Barbosa, Mário Adolfo

    2012-07-01

    The remarkable properties of poly-aminoacids, mainly their biocompatibility and biodegradability, have prompted an increasing interest in these polymers for biomedical applications. Poly-γ-glutamic acid (γ-PGA) is one of the most interesting poly-aminoacids with potential applications as a biomaterial. Here we describe the production and characterization of γ-PGA by Bacillus subtilis natto. The γ-PGA was produced with low molecular weight (10-50 kDa), high purity grade (>99 %) and a D: -/L: -glutamate ratio of 50-60/50-40 %. To evaluate the feasibility of using this γ-PGA as a biomaterial, chitosan (Ch)/γ-PGA nanoparticles were prepared by the coacervation method at pH ranging from 3.0 to 5.0, with dimensions in the interval 214-221 nm with a poly-dispersion index of ca. 0.2. The high purity of γ-PGA produced by this method, which is firstly described here, renders this biopolymer suitable for biomedical applications. Moreover, the Ch/γ-PGA nanocomplexes developed in this investigation can be combined with biologically active substances for their delivery in the organism. The fact that the assembly between Ch and γ-PGA relies on electrostatic interactions enables addition of other molecules that can be released into the medium through changes from acidic to physiological pH, without loss in biological activity.

  7. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis

    PubMed Central

    ZHAO, PENG; MAO, JUN-MIN; ZHANG, SHU-YUN; ZHOU, ZE-QUAN; TAN, YANG; ZHANG, YU

    2014-01-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a ‘chemopreventer’. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer. PMID:25009654

  8. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis.

    PubMed

    Zhao, Peng; Mao, Jun-Min; Zhang, Shu-Yun; Zhou, Ze-Quan; Tan, Yang; Zhang, Yu

    2014-08-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a 'chemopreventer'. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer.

  9. Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

    PubMed Central

    Ely, B; Pittard, J

    1979-01-01

    Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme. PMID:222728

  10. Recycling and refurbishing old antitubercular drugs: the encouraging case of inhibitors of mycolic acid biosynthesis.

    PubMed

    Belardinelli, Juan M; Morbidoni, Héctor R

    2013-04-01

    One of the first approaches undertaken in the quest for antitubercular compounds was that of understanding the mechanism of action of old drugs and proposing chemical modifications or other strategies to improve their activity, generally lost to the mechanisms of resistance developed by Mycobacterium tuberculosis. A leading case was the work carried out on a set of compounds with proven activity on the essential pathway of the synthesis of mycolic acids. As a result, different solutions were presented, improving the activity of those inhibitors or producing novel compounds acting on the same molecular target(s), but avoiding the most common resistance strategies developed by the tubercle bacilli. This review focuses on the activity of those compounds, developed following the completion of the studies on several of the classic antitubercular drugs.

  11. Visualizing the chain-flipping mechanism in fatty-acid biosynthesis

    DOE PAGES

    Beld, Joris; Cang, Hu; Burkart, Michael D.

    2014-10-29

    The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. In this paper, we exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, thus enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity was confirmed by the use of a dual solvatochromic cross-linking probe and solution-phase NMR spectroscopy. Finally, the chain-flipping mechanism was visualized by single-molecule fluorescence techniques, thus demonstrating specificity between themore » Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII.« less

  12. Visualizing the chain-flipping mechanism in fatty-acid biosynthesis

    SciTech Connect

    Beld, Joris; Cang, Hu; Burkart, Michael D.

    2014-10-29

    The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. In this paper, we exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, thus enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity was confirmed by the use of a dual solvatochromic cross-linking probe and solution-phase NMR spectroscopy. Finally, the chain-flipping mechanism was visualized by single-molecule fluorescence techniques, thus demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII.

  13. THE BIOSYNTHESIS AND CONTENT OF GAMMA-AMINOBUTYRIC ACID IN THE GOLDFISH RETINA

    PubMed Central

    Lam, Dominic M. K.

    1972-01-01

    Goldfish retinas incubated with L-glutamate-14C (UL) were found to synthesize γ-aminobutyric acid-14C (GABA-14C) The accumulation of newly synthesized GABA was enhanced by physiological stimulation of the retina with flashing light; and this increase was directly proportional to the logarithm of the light intensity. The total GABA content was also higher in light-stimulated than in dark-adapted retinas, although the glutamate content remained unchanged No differences were found in the cell-free activities of glutamate decarboxylase (EC 4 1.1 15) and GABA-glutamate transaminase (EC 2.6.1.19) extracted from light-stimulated and dark-adapted retinas. These findings, together with other physiological and morphologcal evidence, suggest that GABA plays a functional role in synaptic transmission in the goldfish retina PMID:4339278

  14. Biosynthesis of Polyunsaturated Fatty Acids in Sea Urchins: Molecular and Functional Characterisation of Three Fatty Acyl Desaturases from Paracentrotus lividus (Lamark 1816).

    PubMed

    Kabeya, Naoki; Sanz-Jorquera, Alicia; Carboni, Stefano; Davie, Andrew; Oboh, Angela; Monroig, Oscar

    2017-01-01

    Sea urchins are broadly recognised as a delicacy and their quality as food for humans is highly influenced by their diet. Lipids in general and the long-chain polyunsaturated fatty acids (LC-PUFA) in particular, are essential nutrients that determine not only the nutritional value of sea urchins but also guarantee normal growth and reproduction in captivity. The contribution of endogenous production (biosynthesis) of LC-PUFA in sea urchins remained unknown. Using Paracentrotus lividus as our model species, we aimed to characterise both molecularly and functionally the repertoire of fatty acyl desaturases (Fads), key enzymes in the biosynthesis of LC-PUFA, in sea urchins. Three Fads, namely FadsA, FadsC1 and FadsC2, were characterised. The phylogenetic analyses suggested that the repertoire of Fads within the Echinodermata phylum varies among classes. On one hand, orthologues of the P. lividus FadsA were found in other echinoderm classes including starfishes, brittle stars and sea cucumbers, thus suggesting that this desaturase is virtually present in all echinoderms. Contrarily, the FadsC appears to be sea urchin-specific desaturase. Finally, a further desaturase termed as FadsB exists in starfishes, brittle stars and sea cucumbers, but appears to be missing in sea urchins. The functional characterisation of the P. lividus Fads confirmed that the FadsA was a Δ5 desaturase with activity towards saturated and polyunsaturated fatty acids (FA). Moreover, our experiments confirmed that FadsA plays a role in the biosynthesis of non-methylene interrupted FA, a group of compounds typically found in marine invertebrates. On the other hand, both FadsC desaturases from P. lividus showed Δ8 activity. The present results demonstrate that P. lividus possesses desaturases that account for all the desaturation reactions required to biosynthesis the physiological essential eicosapentaenoic and arachidonic acids through the so-called "Δ8 pathway".

  15. Biosynthesis of Polyunsaturated Fatty Acids in Sea Urchins: Molecular and Functional Characterisation of Three Fatty Acyl Desaturases from Paracentrotus lividus (Lamark 1816)

    PubMed Central

    Carboni, Stefano; Davie, Andrew; Oboh, Angela

    2017-01-01

    Sea urchins are broadly recognised as a delicacy and their quality as food for humans is highly influenced by their diet. Lipids in general and the long-chain polyunsaturated fatty acids (LC-PUFA) in particular, are essential nutrients that determine not only the nutritional value of sea urchins but also guarantee normal growth and reproduction in captivity. The contribution of endogenous production (biosynthesis) of LC-PUFA in sea urchins remained unknown. Using Paracentrotus lividus as our model species, we aimed to characterise both molecularly and functionally the repertoire of fatty acyl desaturases (Fads), key enzymes in the biosynthesis of LC-PUFA, in sea urchins. Three Fads, namely FadsA, FadsC1 and FadsC2, were characterised. The phylogenetic analyses suggested that the repertoire of Fads within the Echinodermata phylum varies among classes. On one hand, orthologues of the P. lividus FadsA were found in other echinoderm classes including starfishes, brittle stars and sea cucumbers, thus suggesting that this desaturase is virtually present in all echinoderms. Contrarily, the FadsC appears to be sea urchin-specific desaturase. Finally, a further desaturase termed as FadsB exists in starfishes, brittle stars and sea cucumbers, but appears to be missing in sea urchins. The functional characterisation of the P. lividus Fads confirmed that the FadsA was a Δ5 desaturase with activity towards saturated and polyunsaturated fatty acids (FA). Moreover, our experiments confirmed that FadsA plays a role in the biosynthesis of non-methylene interrupted FA, a group of compounds typically found in marine invertebrates. On the other hand, both FadsC desaturases from P. lividus showed Δ8 activity. The present results demonstrate that P. lividus possesses desaturases that account for all the desaturation reactions required to biosynthesis the physiological essential eicosapentaenoic and arachidonic acids through the so-called “Δ8 pathway”. PMID:28052125

  16. Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids.

    PubMed

    Chang, Perng-Kuang; Hua, Sui Sheng T; Sarreal, Siov Bouy L; Li, Robert W

    2015-09-24

    The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 μL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids OPEN ACCESS Toxins 2015, 7 3888 were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in

  17. Structural characterisation of the fatty acid biosynthesis enzyme FabF from the pathogen Listeria monocytogenes

    PubMed Central

    Soares da Costa, Tatiana P.; Nanson, Jeffrey D.; Forwood, Jade K.

    2017-01-01

    Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes, providing an excellent platform for the rational design of novel inhibitors. By comparing the structure of L. monocytogenes FabF with other published bacterial FabF structures in complex with known inhibitors and substrates, we highlight conformational changes within the active site, which will need to be accounted for during drug design and virtual screening studies. This high-resolution structure of FabF represents an important step in the development of new classes of antimicrobial agents targeting FabF for the treatment of listeriosis. PMID:28045020

  18. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    PubMed Central

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-01-01

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition. PMID:27775558

  19. High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum.

    PubMed

    Cheng, Fangyu; Gong, Qianying; Yu, Huimin; Stephanopoulos, Gregory

    2016-03-01

    Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied Mw . The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA-hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal Mw was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects.

  20. Nitrile-hydrolyzing enzyme from Meyerozyma guilliermondii and its potential in biosynthesis of 3-hydroxypropionic acid.

    PubMed

    Zhang, Qiang; Gong, Jin-Song; Dong, Ting-Ting; Liu, Ting-Ting; Li, Heng; Dou, Wen-Fang; Lu, Zhen-Ming; Shi, Jin-Song; Xu, Zheng-Hong

    2017-03-11

    3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag(+), Pb(2+) and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

  1. Biosynthesis of gamma-linolenic acid in developing seeds of borage (Borago officinalis L.).

    PubMed

    Galle, A M; Joseph, M; Demandre, C; Guerche, P; Dubacq, J P; Oursel, A; Mazliak, P; Pelletier, G; Kader, J C

    1993-08-20

    delta 6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of gamma-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago officinalis. Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro delta 6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl-CoA was the precursor, but not when [14C]linoleoyl-CoA was the precursor although it stimulated the reaction. The in vitro delta 6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, delta 6-desaturase reached its highest activity 14 to 16 days after pollination.

  2. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis.

    PubMed

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-10-19

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism-congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  3. Energy metabolism and alginate biosynthesis in Pseudomonas aeruginosa: role of the tricarboxylic acid cycle.

    PubMed Central

    Schlictman, D; Kavanaugh-Black, A; Shankar, S; Chakrabarty, A M

    1994-01-01

    Infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is the leading cause of mortality among patients with cystic fibrosis. Alginate production by P. aeruginosa is not constitutive but is triggered by stresses such as starvation. The algR2 (also termed algQ) gene has been previously identified as being necessary for mucoidy; an algR2 mutant strain is unable to produce alginate when grown at 37 degrees C. We show here that the levels of phosphorylated succinyl coenzyme A synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa, are reduced in the algR2 mutant. We were able to correlate the lower level of phosphorylated Scs with a decrease in Scs activity. Western blots (immunoblots) also showed a decreased level of Ndk in the algR2 mutant, but the presence of another kinase activity sensitive to Tween 20 provides the missing Ndk function. The effect of AlgR2 on tricarboxylic acid (TCA) cycle enzymes appears to be specific for Scs, since none of the other TCA cycle enzymes measured showed a significant decrease in activity. Furthermore, the ability of the algR2 mutant to grow on TCA cycle intermediates, but not glucose, is impaired. These data indicate that AlgR2 is responsible for maintaining proper operation of the TCA cycle and energy metabolism. Images PMID:7928963

  4. Stem Cell Therapies for Intervertebral Disc Degeneration: Immune Privilege Reinforcement by Fas/FasL Regulating Machinery.

    PubMed

    Ma, Chi-Jiao; Liu, Xu; Che, Lu; Liu, Zhi-Heng; Samartzis, Dino; Wang, Hai-Qiang

    2015-01-01

    As a main contributing factor to low back pain, intervertebral disc degeneration (IDD) is the fundamental basis for various debilitating spinal diseases. The pros and cons of current treatment modalities necessitate biological treatment strategies targeting for reversing or altering the degeneration process in terms of molecules or genes. The advances in stem cell research facilitate the studies aiming for possible clinical application of stem cell therapies for IDD. Human NP cells are versatile with cell morphology full of variety, capable of synthesizing extracellular matrix components, engulfing substances by autophagy and phagocytosis, mitochondrial vacuolization indicating dysfunction, expressing Fas and FasL as significant omens of immune privileged sites. Human discs belong to immune privilege organs with functional FasL expression, which can interact with invasive immune cells by Fas-FasL regulatory machinery. IDD is characterized by decreased expression level of FasL with dysfunctional FasL, which in turn unbalances the interaction between NP cells and immune cells. Certain modulation factors might play a role in the process, such as miR-155. Accumulating evidence indicates that Fas-FasL network expresses in a variety of stem cells. Given the expression of functional FasL and insensitive Fas in stem cells (we term as FasL privilege), transplantation of stem cells into the disc may regenerate the degenerative disc by not only differentiating into NP-like cells, increasing extracellular matrix, but also reinforce immune privilege via interaction with immune cells by Fas-FasL network.

  5. Role of Fas/FasL in regulation of inflammation in vaginal tissue during HSV-2 infection.

    PubMed

    Krzyzowska, M; Shestakov, A; Eriksson, K; Chiodi, F

    2011-03-17

    To assess the role of Fas in lesion development during genital HSV-2 infection, we used a well-established HSV-2 murine model applied to MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) C57BL6 mice. In vitro infection of murine keratinocytes and epithelial cells was used to clarify molecular details of HSV-2 infection. Despite upregulation of Fas and FasL, HSV-2-infected keratinocytes and epithelial cells showed a moderate level of apoptosis due to upregulated expression of the anti-apoptotic factors Bcl-2, Akt kinase and NF-κB. Inflammatory lesions within the HSV-2-infected epithelium of C57BL6 mice consisted of infected cells upregulating Fas, FasL and Bcl-2, uninfected cells upregulating Fas and neutrophils expressing both Fas and FasL. Apoptosis was detected in HSV-2-infected cells and to even higher extent in non-infected cells surrounding HSV-2 infection sites. HSV-2 infection of Fas- and FasL-deficient mice led to increased apoptosis and stronger recruitment of neutrophils within the infection sites. We conclude that the Fas pathway participates in regulation of inflammatory response in the vaginal epithelium at the initial stage of HSV-2 infection.

  6. Expression of Tropodithietic Acid Biosynthesis Is Controlled by a Novel Autoinducer▿ †

    PubMed Central

    Geng, Haifeng; Belas, Robert

    2010-01-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda− mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda− mutants—tdaA and tdaH failed to respond—by placing wild-type (Tda+) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal. PMID:20601479

  7. Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

    PubMed

    Geng, Haifeng; Belas, Robert

    2010-09-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

  8. Biosynthesis and thermal properties of PHBV produced from levulinic acid by Ralstonia eutropha.

    PubMed

    Wang, Yuanpeng; Chen, Ronghui; Cai, JiYuan; Liu, Zhenggui; Zheng, Yanmei; Wang, Haitao; Li, Qingbiao; He, Ning

    2013-01-01

    Levulinic acid (LA) can be cost-effectively produced from a vast array of renewable carbohydrate-containing biomaterials. LA could facilitate the commercialization of the polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and PHBV-based products as carbon substrates. Therefore, this paper focused on the production of PHBV by Ralstonia eutropha with LA for hydroxyvalerate (HV) production, which plays an important role in enhancing the thermal properties of PHBV. Accordingly, the HV content of PHBV varied from 0-40.9% at different concentrations of LA. Stimulation of cell growth and PHBV accumulation were observed when 2-6 g L(-1) LA was supplied to the culture. The optimal nitrogen sources were determined to be 0.5 g L(-1) ammonium chloride and 2 g L(-1) casein peptone. It was determined that the optimal pH for cell growth and PHBV accumulation was 7.0. When the cultivation was performed in large scale (2 L fermenter) with a low DO concentration of 30% and a pH of 7.0, a high maximum dry cell weight of 15.53 g L(-1) with a PHBV concentration of 12.61 g L(-1) (53.9% HV), up to 81.2% of the dry cell weight, was obtained. The melting point of PHBV found to be decreased as the fraction of HV present in the polymer increased, which resulted in an improvement in the ductility and flexibility of the polymer. The results of this study will improve the understanding of the PHBV accumulation and production by R. eutropha and will be valuable for the industrial production of biosynthesized polymers.

  9. Biosynthesis and actions of 5-oxoeicosatetraenoic acid (5-oxo-ETE) on feline granulocytes.

    PubMed

    Cossette, Chantal; Gravel, Sylvie; Reddy, Chintam Nagendra; Gore, Vivek; Chourey, Shishir; Ye, Qiuji; Snyder, Nathaniel W; Mesaros, Clementina A; Blair, Ian A; Lavoie, Jean-Pierre; Reinero, Carol R; Rokach, Joshua; Powell, William S

    2015-08-01

    The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially hampered progress in this area. As an alternative, we have explored the possibility that the cat could serve as an appropriate animal model to investigate the role of 5-oxo-ETE. We found that feline peripheral blood leukocytes synthesize 5-oxo-ETE and that physiologically relevant levels of 5-oxo-ETE are present in bronchoalveolar lavage fluid from cats with experimentally induced asthma. 5-Oxo-ETE (EC50, 0.7nM) is a much more potent activator of actin polymerization in feline eosinophils than various other eicosanoids, including leukotriene (LT) B4 and prostaglandin D2. 5-Oxo-ETE and LTB4 induce feline leukocyte migration to similar extents at low concentrations (1nM), but at higher concentrations the response to 5-oxo-ETE is much greater. Although high concentrations of selective human OXE receptor antagonists blocked 5-oxo-ETE-induced actin polymerization in feline granulocytes, their potencies were about 200 times lower than for human granulocytes. We conclude that feline leukocytes synthesize and respond to 5-oxo-ETE, which could potentially play an important role in feline asthma, a common condition in this species. The cat could serve as a useful animal model to investigate the pathophysiological role of 5-oxo-ETE.

  10. Post-translational enzyme modification by the phosphopantetheinyl transferase is required for lysine and penicillin biosynthesis but not for roquefortine or fatty acid formation in Penicillium chrysogenum.

    PubMed

    García-Estrada, Carlos; Ullán, Ricardo V; Velasco-Conde, Tania; Godio, Ramiro P; Teijeira, Fernando; Vaca, Inmaculada; Feltrer, Raúl; Kosalková, Katarina; Mauriz, Elba; Martín, Juan F

    2008-10-15

    NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4'-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT(-)) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT(-) mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT(-) with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.

  11. Systems Biology of Lignin Biosynthesis in Populus trichocarpa: Heteromeric 4-Coumaric Acid:Coenzyme A Ligase Protein Complex Formation, Regulation, and Numerical Modeling[W

    PubMed Central

    Chen, Hsi-Chuan; Song, Jina; Wang, Jack P.; Lin, Ying-Chung; Ducoste, Joel; Shuford, Christopher M.; Liu, Jie; Li, Quanzi; Shi, Rui; Nepomuceno, Angelito; Isik, Fikret; Muddiman, David C.; Williams, Cranos; Sederoff, Ronald R.; Chiang, Vincent L.

    2014-01-01

    As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein–protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation. PMID:24619612

  12. Nicotiana attenuata SIPK, WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway1[W][OA

    PubMed Central

    Kallenbach, Mario; Alagna, Fiammetta; Baldwin, Ian Thomas; Bonaventure, Gustavo

    2010-01-01

    Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants. PMID:19897603

  13. Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

    PubMed Central

    Mosher, Roy H.; Paradkar, Ashish S.; Anders, Cecilia; Barton, Barry; Jensen, Susan E.

    1999-01-01

    Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis. PMID:10223939

  14. Region-specific vulnerability to lipid peroxidation and evidence of neuronal mechanisms for polyunsaturated fatty acid biosynthesis in the healthy adult human central nervous system.

    PubMed

    Naudí, Alba; Cabré, Rosanna; Dominguez-Gonzalez, Mayelin; Ayala, Victoria; Jové, Mariona; Mota-Martorell, Natalia; Piñol-Ripoll, Gerard; Gil-Villar, Maria Pilar; Rué, Montserrat; Portero-Otín, Manuel; Ferrer, Isidre; Pamplona, Reinald

    2017-02-07

    Lipids played a determinant role in the evolution of the brain. It is postulated that the morphological and functional diversity among neural cells of the human central nervous system (CNS) is projected and achieved through the expression of particular lipid profiles. The present study was designed to evaluate the differential vulnerability to oxidative stress mediated by lipids through a cross-regional comparative approach. To this end, we compared 12 different regions of CNS of healthy adult subjects, and the fatty acid profile and vulnerability to lipid peroxidation, were determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS), respectively. In addition, different components involved in PUFA biosynthesis, as well as adaptive defense mechanisms against lipid peroxidation, were also measured by western blot and immunohistochemistry, respectively. We found that: i) four fatty acids (18.1n-9, 22:6n-3, 20:1n-9, and 18:0) are significant discriminators among CNS regions; ii) these differential fatty acid profiles generate a differential selective neural vulnerability (expressed by the peroxidizability index); iii) the cross-regional differences for the fatty acid profiles follow a caudal-cranial gradient which is directly related to changes in the biosynthesis pathways which can be ascribed to neuronal cells; and iv) the higher the peroxidizability index for a given human brain region, the lower concentration of the protein damage markers, likely supported by the presence of adaptive antioxidant mechanisms. In conclusion, our results suggest that there is a region-specific vulnerability to lipid peroxidation and offer evidence of neuronal mechanisms for polyunsaturated fatty acid biosynthesis in the human central nervous system.

  15. The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

    PubMed

    Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

    2016-02-01

    In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

  16. Targeting the Fas/FasL system in Rheumatoid Arthritis therapy: Promising or risky?

    PubMed

    Calmon-Hamaty, Flavia; Audo, Rachel; Combe, Bernard; Morel, Jacques; Hahne, Michael

    2015-10-01

    Rheumatoid Arthritis (RA) is a chronic inflammatory disease affecting synovial joints. Tumor necrosis factor (TNF) α is a key component of RA pathogenesis and blocking this cytokine is the most common strategy to treat the disease. Though TNFα blockers are very efficient, one third of the RA patients are unresponsive or present side effects. Therefore, the development of novel therapeutic approaches is required. RA pathogenesis is characterized by the hyperplasia of the synovium, closely associated to the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLS), which invade and destroy the joint structure. Hence, depletion of RA FLS has been proposed as an alternative therapeutic strategy. The TNF family member Fas ligand (FasL) was reported to trigger apoptosis in FLS of arthritic joints by binding to its receptor Fas and therefore suggested as a promising candidate for targeting the hyperplastic synovial tissue. However, this cytokine is pleiotropic and recent data from the literature indicate that Fas activation might have a disease-promoting role in RA by promoting cell proliferation. Therefore, a FasL-based therapy for RA requires careful evaluation before being applied. In this review we aim to overview what is known about the apoptotic and non-apoptotic effects of Fas/FasL system and discuss its relevance in RA.

  17. In vivo analysis of Fas/FasL interactions in HIV-infected patients.

    PubMed Central

    Badley, A D; Dockrell, D H; Algeciras, A; Ziesmer, S; Landay, A; Lederman, M M; Connick, E; Kessler, H; Kuritzkes, D; Lynch, D H; Roche, P; Yagita, H; Paya, C V

    1998-01-01

    Recent insights into the pharmacological control of HIV replication and the molecular mechanisms of peripheral T cells homeostasis allowed us to investigate in vivo the mechanisms mediating T cell depletion in HIV-infected patients. Before the initiation of highly active antiretroviral therapy (HAART), a high degree of lymphoid tissue apoptosis is present, which is reduced upon HAART initiation (P < 0.001) and directly correlates with reduction of viral load and increases of peripheral T lymphocytes (P < 0.01). Because Fas/FasL interactions play a key role in peripheral T lymphocyte homeostasis, we investigated the susceptibility to Fas-mediated apoptosis in peripheral T lymphocytes and of FasL expression in lymphoid tissue before and during HAART. High levels of Fas-susceptibility found in peripheral CD4 T lymphocytes before HAART were significantly reduced after HAART, coinciding with decreases in viral load (P = 0.018) and increases in peripheral CD4 T lymphocyte counts (P < 0.01). However, the increased FasL expression in the lymphoid tissue of HIV-infected individuals was not reduced after HAART. These results demonstrate that lymphoid tissue apoptosis directly correlates with viral load and peripheral T lymphocyte numbers, and suggest that HIV-induced susceptibility to Fas-dependent apoptosis may play a key role in the regulation of T cell homeostasis in HIV-infected individuals. PMID:9649560

  18. Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes.

    PubMed

    Leite-de-Moraes, M C; Herbelin, A; Gouarin, C; Koezuka, Y; Schneider, E; Dy, M

    2000-10-15

    NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.

  19. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products1[OPEN

    PubMed Central

    Yuen, Macaire M.S.; Bohlmann, Jörg

    2016-01-01

    Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I–IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes. PMID:26936895

  20. An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

    PubMed Central

    Zelenaya-Troitskaya, O; Perlman, P S; Butow, R A

    1995-01-01

    The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA. Images PMID:7621838

  1. A lack of Fas/FasL signalling leads to disturbances in the antiviral response during ectromelia virus infection.

    PubMed

    Bień, K; Sobańska, Z; Sokołowska, J; Bąska, P; Nowak, Z; Winnicka, A; Krzyzowska, M

    2016-04-01

    Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the murine equivalent of human smallpox. Fas receptor-Fas ligand (FasL) signaling is involved in apoptosis of immune cells and virus-specific cytotoxicity. The Fas/FasL pathway also plays an important role in controlling the local inflammatory response during ECTV infection. Here, the immune response to the ECTV Moscow strain was examined in Fas (-) (lpr), FasL (-) (gld) and C57BL6 wild-type mice. During ECTV-MOS infection, Fas- and FasL mice showed increased viral titers, decreased total numbers of NK cells, CD4(+) and CD8(+) T cells followed by decreased percentages of IFN-γ expressing NK cells, CD4(+) and CD8(+) T cells in spleens and lymph nodes. At day 7 of ECTV-MOS infection, Fas- and FasL-deficient mice had the highest regulatory T cell (Treg) counts in spleen and lymph nodes in contrast to wild-type mice. Furthermore, at days 7 and 10 of the infection, we observed significantly higher numbers of PD-L1-expressing dendritic cells in Fas (-) and FasL (-) mice in comparison to wild-type mice. Experiments in co-cultures of CD4(+) T cells and bone-marrow-derived dendritic cells showed that the lack of bilateral Fas-FasL signalling led to expansion of Tregs. In conclusion, our results demonstrate that during ECTV infection, Fas/FasL can regulate development of tolerogenic DCs and Tregs, leading to an ineffective immune response.

  2. Immune privilege and FasL: two ways to inactivate effector cytotoxic T lymphocytes by FasL-expressing cells

    PubMed Central

    Li, Jie-Hui; Rosen, Dalia; Sondel, Paul; Berke, Gideon

    2002-01-01

    The theory that Fas ligand (FasL)-expressing tumours are immune-privileged and can directly counterattack Fas-expressing effector T lymphocytes has recently been questioned and several alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL-expressing tumours on in vivo-primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells. We have found that PEL populations undergo Fas-based apoptotic cell death when co-cultured with FasL-expressing tumour cells and that PEL destruction of cognate targets in a 51Cr-release assay was markedly inhibited by the pre-exposure to either cognate or non-cognate tumour cells expressing FasL. Furthermore, cytocidal function of PEL was markedly inhibited by preincubation with FasL-negative tumour cells, if and only if they were the cognate targets of the CTL; this CTL inhibition involved FasL–Fas interactions. The killing function of ‘bystander’ PELs, reactive to a third-party target cell, was inhibited by co-cultivation with PELs mixed with their cognate target. This activation-induced CTL fratricide was not influenced by the expression of FasL on the cognate target cells. These studies demonstrate the existence of two distinct pathways whereby FasL-expressing cells inhibit in vivo-primed FasL- and Fas-expressing CTLs: first, by FasL-based direct tumour counterattack, and second, by FasL-mediated activation-induced cell death of the CTLs, which is consistent with the concept that FasL expression in vivo could play a role in inducing immune privilege. PMID:11918688

  3. Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

    PubMed Central

    Holler, Nils; Tardivel, Aubry; Kovacsovics-Bankowski, Magdalena; Hertig, Sylvie; Gaide, Olivier; Martinon, Fabio; Tinel, Antoine; Deperthes, David; Calderara, Silvio; Schulthess, Therese; Engel, Jürgen; Schneider, Pascal; Tschopp, Jürg

    2003-01-01

    The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways. PMID:12556501

  4. The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis

    PubMed Central

    Eidsmo, Liv; Nylen, Susanne; Khamesipour, Ali; Hedblad, Mari-Anne; Chiodi, Francesca; Akuffo, Hannah

    2005-01-01

    Cutaneous leishmaniasis (CL), caused by the intracellular protozoan Leishmania major, is characterized by lesion formation and ulceration at the site of infection. The mechanism of ulcer formation during CL is not fully understood. The expression of Fas and FasL and the levels of apoptosis in skin biopsies and in restimulated blood mononuclear cells from patients with 1 to 7 months of L. major-induced CL were analyzed using immunohistochemistry and fluorescence-activated cell sorting analysis. The levels of soluble Fas and FasL were also analyzed by enzyme-linked immunosorbent assay. A substantial number of apoptotic keratinocytes were observed mainly in the superficial epidermis of morphologically active and healing CL skin samples. Fas expression was increased on epidermis in active CL, whereas Fas expression was similar in healing and healthy epidermis. FasL-expressing macrophages and T cells were found in subepidermal infiltrate, mainly in active disease. When CL peripheral blood mononuclear cells were restimulated with L. major, Fas was up-regulated on effector T cells, and high levels of sFasL were secreted. Supernatants from restimulated cultures induced apoptosis in human keratinocytes (HaCaT), possibly through Fas/FasL interactions. Our results indicate that FasL-expressing effector T cells and macrophages may act to induce apoptosis and ulcer formation in Fas-expressing keratinocytes during L. major infection. PMID:15793290

  5. Intracellular Fas ligand in normal and malignant breast epithelium does not induce apoptosis in Fas-sensitive cells

    PubMed Central

    Ragnarsson, G B; Mikaelsdottir, E K; Vidarsson, H; Jónasson, J G; Ólafsdóttir, K; Kristjánsdóttir, K; Kjartansson, J; Ögmundsdóttir, H M; Rafnar, T

    2000-01-01

    Fas ligand (FasL) is expressed on some cancers and may play a role in the immune evasion of the tumour. We used immuno-histochemistry to study the expression of Fas and FasL in tissue samples from breast cancer patients, as well as normal breast tissue. Our results show that Fas and FasL are co-expressed both in normal tissue and in breast tumours. Fas and FasL mRNA were expressed in fresh normal and malignant breast tissue, as well as cultured breast epithelium and breast cancer cell lines. Flow cytometry analysis of live cells failed to detect FasL on the surface of normal or malignant breast cells; however, both stained positive for FasL after permeabilization. Fas was detected on the surface of normal breast cells and T47D and MCF-10A cell lines but only intracellularly in other breast cell lines tested. Neither normal breast epithelium nor breast cell lines induced Fas-dependent apoptosis in Jurkat cells. Finally, 20 tumour samples were stained for apoptosis. Few apoptotic cells were detected and there was no increase in apoptotic cells on the borders between tumour cells and lymphocytes. We conclude that FasL is expressed intracellularly in both normal and malignant breast epithelium and unlikely to be important for the immune evasion of breast tumours. © 2000 Cancer Research Campaign http://www.bjcancer.com PMID:11104571

  6. Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

    PubMed

    Chen, Jia-Wen; Liu, Wan-Jun; Hu, Dong-Xiong; Wang, Xiang; Balamurugan, Srinivasan; Alimujiang, Adili; Yang, Wei-Dong; Liu, Jie-Sheng; Li, Hong-Ye

    2016-08-30

    Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to lack of suitable industrial strain with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT-overexpressing N. oceanica showed higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. GC-MS analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of EPA (C20:5) increased by 8%, which is a polyunsaturated fatty acid necessary for animal nutrition. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential. This article is protected by copyright. All rights reserved.

  7. A new version of the HBSC Family Affluence Scale - FAS III: Scottish Qualitative Findings from the International FAS Development Study.

    PubMed

    Hartley, Jane E K; Levin, Kate; Currie, Candace

    A critical review of the Family Affluence Scale (FAS) concluded that FAS II was no longer discriminatory within very rich or very poor countries, where a very high or a very low proportion of children were categorised as high FAS or low FAS respectively (Currie et al. 2008). The review concluded that a new version of FAS - FAS III - should be developed to take into account current trends in family consumption patterns across the European region, the US and Canada. In 2012, the FAS Development and Validation Study was conducted in eight countries - Denmark, Greenland, Italy, Norway, Poland, Romania, Slovakia and Scotland. This paper describes the Scottish qualitative findings from this study. The Scottish qualitative fieldwork comprising cognitive interviews and focus groups sampled from 11, 13 and 15 year-old participants from 18 of the most- and least- economically deprived schools. These qualitative results were used to inform the final FAS III recommendations.

  8. Characterization of the multiple molecular mechanisms underlying RsaL control of phenazine-1-carboxylic acid biosynthesis in the rhizosphere bacterium Pseudomonas aeruginosa PA1201.

    PubMed

    Sun, Shuang; Chen, Bo; Jin, Zi-Jing; Zhou, Lian; Fang, Yun-Ling; Thawai, Chitti; Rampioni, Giordano; He, Ya-Wen

    2017-03-18

    Phenazines are important secondary metabolites that have been found to affect a broad spectrum of organisms. Two almost identical gene clusters phz1 and phz2 are responsible for phenazines biosynthesis in the rhizobacterium Pseudomonas aeruginosa PA1201. Here, we show that the transcriptional regulator RsaL is a potent repressor of phenazine-1-carboxylic acid (PCA) biosynthesis. RsaL negatively regulates phz1 expression and positively regulates phz2 expression via multiple mechanisms. First, RsaL binds to a 25-bp DNA region within the phz1 promoter to directly repress phz1 expression. Second, RsaL indirectly regulates the expression of both phz clusters by decreasing the activity of the las and pqs quorum sensing (QS) systems, and by promoting the rhl QS system. Finally, RsaL represses phz1 expression through the downstream transcriptional regulator CdpR. RsaL directly binds to the promoter region of cdpR to positively regulate its expression, and subsequently CdpR regulates phz1 expression in a negative manner. We also show that RsaL represents a new mechanism for the turnover of the QS signal molecule N-3-oxododecanoyl-homoserine lactone (3-oxo-C12-HSL). Overall, this study elucidates RsaL control of phenazines biosynthesis and indicates that a PA1201 strain harboring deletions in both the rsaL and cdpR genes could be used to improve the industrial production of PCA.

  9. Transcriptomic analysis of the biosynthesis, recycling, and distribution of ascorbic acid during leaf development in tea plant (Camellia sinensis (L.) O. Kuntze)

    PubMed Central

    Li, Hui; Huang, Wei; Wang, Guang-Long; Wang, Wen-Li; Cui, Xin; Zhuang, Jing

    2017-01-01

    Ascorbic acid (AsA), known as vitamin C, is an essential nutrient for humans and mainly absorbed from food. Tea plant (Camellia sinensis (L.) O. Kuntze) leaves can be a dietary source of AsA for humans. However, experimental evidence on the biosynthesis, recycling pathway and distribution of AsA during leaf development in tea plants is unclear. To gain insight into the mechanism and distribution of AsA in the tea plant leaf, we identified 18 related genes involved in AsA biosynthesis and recycling pathway based on the transcriptome database of tea plants. Tea plant leaves were used as samples at different developmental stages. AsA contens in tea plant leaves at three developmental stages were measured by reversed-phase high-performance liquid chromatography (RP-HPLC). The correlations between expression levels of these genes and AsA contents during the development of tea plant leaves were discussed. Results indicated that the l-galactose pathway might be the primary pathway of AsA biosynthesis in tea plant leaves. CsMDHAR and CsGGP might play a regulatory role in AsA accumulation in the leaves of three cultivars of tea plants. These findings may provide a further glimpse to improve the AsA accumulation in tea plants and the commercial quality of tea. PMID:28393854

  10. Fatty acid profile in the seeds and seed tissues of Paeonia L. species as new oil plant resources

    PubMed Central

    Yu, Shuiyan; Du, Shaobo; Yuan, Junhui; Hu, Yonghong

    2016-01-01

    Most common plant oils have little α-linolenic acid (C18:3Δ9,12,15, ALA) and an unhealthy ω6/ω3 ratio. Here, fatty acids (FAs) in the seeds of 11 species of Paeonia L., including 10 tree peony and one herbaceous species, were explored using gas chromatograph–mass spectrometer. Results indicated that all Paeonia had a ω6/ω3 ratio less than 1.0, and high amounts of ALA (26.7–50%), oleic acid (C18:1Δ9, OA) (20.8–46%) and linoleic acid (C18:2Δ9,12, LA) (10–38%). ALA was a dominant component in oils of seven subsection Vaginatae species, whereas OA was predominant in two subsection Delavayanae species. LA was a subdominant oil component in P. ostii and P. obovata. Moreover, the FA composition and distribution of embryo (22 FAs), endosperm (14 FAs) and seed coat (6 FAs) in P. ostii, P. rockii and P. ludlowii were first reported. Peony species, particularly P. decomposita and P. rockii, can be excellent plant resources for edible oil because they provide abundant ALA to balance the ω6/ω3 ratio. The differences in the ALA, LA and OA content proportion also make the peony species a good system for detailed investigation of FA biosynthesis pathway and ALA accumulation. PMID:27240678

  11. Repression of gamma-aminobutyric acid type A receptor alpha1 polypeptide biosynthesis requires chronic agonist exposure.

    PubMed

    Miranda, J D; Barnes, E M

    1997-06-27

    Although it is well established that the number of gamma-aminobutyric acid type A (GABAA) receptors declines in cortical neurons exposed to GABAA receptor agonists, the mechanisms responsible for this use-dependent down-regulation remain unclear. Two hypotheses have been proposed: (i) agonist-evoked sequestration and degradation of surface GABAA receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [35S]Met/Cys pulse-chase labeling of chick cortical neurons in culture and immunoprecipitation and immunoblotting with an antibody (RP4) directed against a GABAA receptor alpha1-(331-381) fusion protein. Exposure of the cells to GABA or isoguvacine for 2 h to 4 days had no effect on the initial rate of 35S incorporation into the GABAA receptor 51-kDa alpha1 polypeptide, but this rate declined by 33% after a 7-day treatment. This is consistent with a previous report (Baumgartner, B. J., Harvey, R. J., Darlison, M. G., and Barnes, E. M. (1994) Mol. Brain Res. 26, 9-17) that a 7-day GABA treatment of this preparation produced a 45% reduction in the alpha1 subunit mRNA level, while a 4-day exposure had no detectable effect. On the other hand, after a 4-day exposure to these agonists, a 30% reduction in the level of the alpha1 polypeptide was observed on immunoblots, similar to that found previously for down-regulation of GABAA receptor ligand-binding sites. Thus, the de novo synthesis of GABAA receptor alpha1 subunits is subject to a delayed use-dependent repression that was observed after, rather than before, the decline in neuronal levels of the polypeptide. Pulse-chase experiments showed a monophasic degradation of the GABAA receptor 35S-alpha1 subunit with a t1/2 = 7.7 h, a process that was unaffected by the addition of GABA to neurons during the chase period. These nascent 35S-labeled polypeptides are presumably diluted into the neuronal pool of unlabeled unassembled alpha1 subunits, which was found to exceed by a 4:1 molar

  12. Differential expression of structural genes for the late phase of phytic acid biosynthesis in developing seeds of wheat (Triticum aestivum L.).

    PubMed

    Bhati, Kaushal Kumar; Aggarwal, Sipla; Sharma, Shivani; Mantri, Shrikant; Singh, Sudhir P; Bhalla, Sherry; Kaur, Jagdeep; Tiwari, Siddharth; Roy, Joy K; Tuli, Rakesh; Pandey, Ajay K

    2014-07-01

    In cereals, phytic acid (PA) or inositol hexakisphosphate (IP6) is a well-known phosphate storage compound as well as major chelator of important micronutrients (iron, zinc, calcium, etc.). Genes involved in the late phases of PA biosynthesis pathway are known in crops like maize, soybeans and barley but none have been reported from wheat. Our in silico analysis identified six wheat genes that might be involved in the biosynthesis of inositol phosphates. Four of the genes were inositol tetraphosphate kinases (TaITPK1, TaITPK2, TaITPK3, and TaITPK4), and the other two genes encode for inositol triphosphate kinase (TaIPK2) and inositol pentakisphosphate kinase (TaIPK1). Additionally, we identified a homolog of Zmlpa-1, an ABCC subclass multidrug resistance-associated transporter protein (TaMRP3) that is putatively involved in PA transport. Analyses of the mRNA expression levels of these seven genes showed that they are differentially expressed during seed development, and that some are preferentially expressed in aleurone tissue. These results suggest selective roles during PA biosynthesis, and that both lipid-independent and -dependent pathways are active in developing wheat grains. TaIPK1 and TaMRP3 were able to complement the yeast ScΔipk1 and ScΔycf1 mutants, respectively, providing evidence that the wheat genes have the expected biochemical functions. This is the first comprehensive study of the wheat genes involved in the late phase of PA biosynthesis. Knowledge generated from these studies could be utilized to develop strategies for generating low phyate wheat.

  13. Seasonal abscisic acid signal and a basic leucine zipper transcription factor, DkbZIP5, regulate proanthocyanidin biosynthesis in persimmon fruit.

    PubMed

    Akagi, Takashi; Katayama-Ikegami, Ayako; Kobayashi, Shozo; Sato, Akihiko; Kono, Atsushi; Yonemori, Keizo

    2012-02-01

    Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5.

  14. Inhibiting activities of the secondary metabolites of Phlomis brunneogaleata against parasitic protozoa and plasmodial enoyl-ACP Reductase, a crucial enzyme in fatty acid biosynthesis.

    PubMed

    Kirmizibekmez, Hasan; Calis, Ihsan; Perozzo, Remo; Brun, Reto; Dönmez, Ali A; Linden, Anthony; Rüedi, Peter; Tasdemir, Deniz

    2004-08-01

    Anti-plasmodial activity-guided fractionation of Phlomis brunneogaleata (Lamiaceae) led to the isolation of two new metabolites, the iridoid glycoside, brunneogaleatoside and a new pyrrolidinium derivative (2 S,4 R)-2-carboxy-4-( E)- p-coumaroyloxy-1,1-dimethylpyrrolidinium inner salt [(2 S,4 R)-1,1-dimethyl-4-( E)- p-coumaroyloxyproline inner salt]. Moreover, a known iridoid glycoside, ipolamiide, six known phenylethanoid glycosides, verbascoside, isoverbascoside, forsythoside B, echinacoside, glucopyranosyl-(1-->G (i)-6)-martynoside and integrifolioside B, two flavone glycosides, luteolin 7- O-beta- D-glucopyranoside ( 10) and chrysoeriol 7- O-beta- D-glucopyranoside ( 11), a lignan glycoside liriodendrin, an acetophenone glycoside 4-hydroxyacetophenone 4- O-(6'- O-beta- D-apiofuranosyl)-beta- D-glucopyranoside and three caffeic acid esters, chlorogenic acid, 3-O-caffeoylquinic acid methyl ester and 5- O-caffeoylshikimic acid were isolated. The structures of the pure compounds were elucidated by means of spectroscopic methods (UV, IR, MS, 1D and 2D NMR, [alpha] (D)) and X-ray crystallography. Compounds 10 and 11 were determined to be the major anti-malarial principles of the crude extract (IC (50) values of 2.4 and 5.9 micrograms/mL, respectively). They also exhibited significant leishmanicidal activity (IC (50) = 1.1 and 4.1 micrograms/mL, respectively). The inhibitory potential of the pure metabolites against plasmodial enoyl-ACP reductase (FabI), which is the key regulator of type II fatty acid synthases (FAS-II) in P. falciparum, was also assessed. Compound 10 showed promising FabI inhibiting effect (IC (50) = 10 micrograms/mL) and appears to be the first anti-malarial natural product targeting FabI of P. falciparum.

  15. Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition†

    PubMed Central

    Mohedano, M. Luz; Overweg, Karin; de la Fuente, Alicia; Reuter, Mark; Altabe, Silvia; Mulholland, Francis; de Mendoza, Diego; López, Paloma; Wells, Jerry M.

    2005-01-01

    The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism. PMID:15774879

  16. Transcription factors FabR and FadR regulate both aerobic and anaerobic pathways for unsaturated fatty acid biosynthesis in Shewanella oneidensis.

    PubMed

    Luo, Qixia; Shi, Miaomiao; Ren, Yedan; Gao, Haichun

    2014-01-01

    As genes for type II fatty acid synthesis are essential to the growth of Escherichia coli, its sole (anaerobic) pathway has significant potential as a target for novel antibacterial drug, and has been extensively studied. Despite this, we still know surprisingly little about fatty acid synthesis in bacteria because this anaerobic pathway in fact is not widely distributed. In this study, we show a novel model of unsaturated fatty acid (UFA) synthesis in Shewanella, emerging human pathogens in addition to well-known metal reducers. We identify both anaerobic and aerobic UFA biosynthesis pathways in the representative species, S. oneidensis. Uniquely, the bacterium also contains two regulators FabR and FadR, whose counterparts in other bacteria control the anaerobic pathway. However, we show that in S. oneidensis these two regulators are involved in regulation of both pathways, in either direct or indirect manner. Overall, our results indicate that the UFA biosynthesis and its regulation are far more complex than previously expected, and S. oneidensis serves as a good research model for further work.

  17. Role of Fas/FasL in regulation of inflammation in vaginal tissue during HSV-2 infection

    PubMed Central

    Krzyzowska, M; Shestakov, A; Eriksson, K; Chiodi, F

    2011-01-01

    To assess the role of Fas in lesion development during genital HSV-2 infection, we used a well-established HSV-2 murine model applied to MRL-Faslpr/J (Fas−/−) and C3-Faslgld/J (FasL−/−) C57BL6 mice. In vitro infection of murine keratinocytes and epithelial cells was used to clarify molecular details of HSV-2 infection. Despite upregulation of Fas and FasL, HSV-2-infected keratinocytes and epithelial cells showed a moderate level of apoptosis due to upregulated expression of the anti-apoptotic factors Bcl-2, Akt kinase and NF-κB. Inflammatory lesions within the HSV-2-infected epithelium of C57BL6 mice consisted of infected cells upregulating Fas, FasL and Bcl-2, uninfected cells upregulating Fas and neutrophils expressing both Fas and FasL. Apoptosis was detected in HSV-2-infected cells and to even higher extent in non-infected cells surrounding HSV-2 infection sites. HSV-2 infection of Fas- and FasL-deficient mice led to increased apoptosis and stronger recruitment of neutrophils within the infection sites. We conclude that the Fas pathway participates in regulation of inflammatory response in the vaginal epithelium at the initial stage of HSV-2 infection. PMID:21412278

  18. Contributions of Fas-Fas Ligand Interactions to the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System

    PubMed Central

    Parra, Beatriz; Lin, Mark T.; Stohlman, Stephen A.; Bergmann, Cornelia C.; Atkinson, Roscoe; Hinton, David R.

    2000-01-01

    The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication. PMID:10666278

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

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

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

  20. GABA tea prevents cardiac fibrosis by attenuating TNF-alpha and Fas/FasL-mediated apoptosis in streptozotocin-induced diabetic rats.

    PubMed

    Cherng, Shur-Hueih; Huang, Chih-Yang; Kuo, Wei-Wen; Lai, Shue-Er; Tseng, Chien-Yu; Lin, Yueh-Min; Tsai, Fuu-Jen; Wang, Hsueh-Fang

    2014-03-01

    GABA tea is a tea product that contains a high level of gamma-aminobutyric acid (GABA). This study investigated the effects of GABA tea on the heart in a diabetic rat model. Male Wistar rats were injected with 55mg/kg streptozotocin (STZ) to induce diabetes for 2weeks and then orally given dosages of 4.55 and 45.5mg/kg/day GABA tea extract for 6weeks. The results revealed that fasting blood glucose levels returned to normal levels in GABA tea-treated diabetic rats, but not in the untreated diabetic rats. Additionally, GABA tea effectively inhibited cardiac fibrosis induced by STZ. Further experiments showed that the STZ-induced protein levels of tumor necrosis factor-alpha (TNF-alpha), Fas, activated caspase-8 and caspase-3 were significantly inhibited by the GABA tea treatment. Therefore, our data suggest that the inhibiting effect of GABA tea on STZ-induced cardiac fibrosis in diabetic rats may be mediated by reducing blood glucose and further attenuating TNF-alpha expression and/or Fas/Fas ligand (FasL)-mediated apoptosis. These findings will provide implications for the potential anti-diabetic properties of GABA tea.

  1. Roles of proinflammatory cytokines and the Fas/Fas ligand interaction in the pathogenesis of inflammatory myopathies.

    PubMed

    Kondo, Masahiro; Murakawa, Yohko; Harashima, Nanae; Kobayashi, Shotai; Yamaguchi, Shuhei; Harada, Mamoru

    2009-09-01

    Within the lesions of inflammatory myopathies, muscle fibres and invading mononuclear cells express Fas and Fas ligand (FasL), respectively. However, the roles of the Fas/FasL interaction in the pathogenesis of inflammatory myopathies are not fully understood. In the present study, we investigated the roles of proinflammatory cytokines and the Fas/FasL system in the pathogenesis of inflammatory myopathies. In vitro culturing of muscle cells with the proinflammatory cytokines interferon-gamma, tumour necrosis factor-alpha, and interleukin (IL)-1beta synergistically increased Fas expression, susceptibility to Fas-mediated apoptosis, and the expression of cytoplasmic caspases 8 and 3. In addition, culturing of muscle cells with activated CD4(+) T cells induced muscle cell apoptosis, which was partially inhibited by anti-FasL antibody. We also tested the possibility that T helper (Th) 17, which is an IL-17-producing helper T-cell subset that plays crucial roles in autoimmune and inflammatory responses, participates in the pathogenesis of inflammatory myopathies. Interestingly, in vitro culturing of dendritic cells with anti-Fas immunoglobulin M (IgM) or activated CD4(+) T cells induced the expression of mRNA for IL-23p19, but not for IL-12p35, in addition to proinflammatory cytokines. Furthermore, IL-23p19 and IL-17 mRNAs were detected in the majority of biopsy samples from patients with inflammatory myopathies. Taken together, these results suggest that proinflammatory cytokines enhance Fas-mediated apoptosis of muscle cells, and that the Fas/FasL interaction between invading dendritic cells and CD4(+) T cells induces local production of IL-23 and proinflammatory cytokines, which can promote the proliferation of Th17 cells and enhance Fas-mediated apoptosis of muscle cells, respectively.

  2. A Fas(hi) Lymphoproliferative Phenotype Reveals Non-Apoptotic Fas Signaling in HTLV-1-Associated Neuroinflammation.

    PubMed

    Menezes, Soraya Maria; Leal, Fabio E; Dierckx, Tim; Khouri, Ricardo; Decanine, Daniele; Silva-Santos, Gilvaneia; Schnitman, Saul V; Kruschewsky, Ramon; López, Giovanni; Alvarez, Carolina; Talledo, Michael; Gotuzzo, Eduardo; Nixon, Douglas F; Vercauteren, Jurgen; Brassat, David; Liblau, Roland; Vandamme, Anne Mieke; Galvão-Castro, Bernardo; Van Weyenbergh, Johan

    2017-01-01

    Human T-cell lymphotropic virus (HTLV)-1 was the first human retrovirus to be associated to cancer, namely adult T-cell leukemia (ATL), but its pathogenesis remains enigmatic, since only a minority of infected individuals develops either ATL or the neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A functional FAS -670 polymorphism in an interferon (IFN)-regulated STAT1-binding site has been associated to both ATL and HAM/TSP susceptibility. Fas(hi) T stem cell memory (Tscm) cells have been identified as the hierarchical apex of ATL, but have not been investigated in HAM/TSP. In addition, both FAS and STAT1 have been identified in an IFN-inducible HAM/TSP gene signature, but its pathobiological significance remains unclear. We comprehensively explored Fas expression (protein/mRNA) and function in lymphocyte activation, apoptosis, proliferation, and transcriptome, in PBMC from a total of 47 HAM/TSP patients, 40 asymptomatic HTLV-1-infected individuals (AC), and 58 HTLV-1 -uninfected healthy controls. Fas surface expression followed a two-step increase from HC to AC and from AC to HAM/TSP. In HAM/TSP, Fas levels correlated positively to lymphocyte activation markers, but negatively to age of onset, linking Fas(hi) cells to earlier, more aggressive disease. Surprisingly, increased lymphocyte Fas expression in HAM/TSP was linked to decreased apoptosis and increased lymphoproliferation upon in vitro culture, but not to proviral load. This Fas(hi) phenotype is HAM/TSP-specific, since both ex vivo and in vitro Fas expression was increased as compared to multiple sclerosis (MS), another neuroinflammatory disorder. To elucidate the molecular mechanism underlying non-apoptotic Fas signaling in HAM/TSP, we combined transcriptome analysis with functional assays, i.e., blocking vs. triggering Fas receptor in vitro with antagonist and agonist-, anti-Fas mAb, respectively. Treatment with agonist anti-Fas mAb restored apoptosis

  3. Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme.

    PubMed Central

    Heaton, M P; Neuhaus, F C

    1992-01-01

    The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid. PMID:1385594

  4. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts.

  5. Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

    PubMed

    Holland, William L; Bikman, Benjamin T; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M; Bulchand, Sarada; Knotts, Trina A; Shui, Guanghou; Clegg, Deborah J; Wenk, Markus R; Pagliassotti, Michael J; Scherer, Philipp E; Summers, Scott A

    2011-05-01

    Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

  6. Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis.

    PubMed

    Chollet, Aurélien; Stigliani, Jean-Luc; Pasca, Maria Rosalia; Mori, Giorgia; Lherbet, Christian; Constant, Patricia; Quémard, Annaïk; Bernadou, Jean; Pratviel, Geneviève; Bernardes-Génisson, Vania

    2016-11-01

    Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

  7. Involvement of Fas and FasL in Ectromelia virus-induced apoptosis in mouse brain.

    PubMed

    Krzyzowska, Małgorzata; Cymerys, Joanna; Winnicka, Anna; Niemiałtowski, Marek

    2006-02-01

    In this study we showed that the virulent Moscow strain of Ectromelia virus (ECTV-MOS) infection leads to induction of apoptosis in the BALB/c mouse central nervous system. ECTV-MOS-infected cells and inflammation sites were found in brain parenchyma between 5 and 15 days after footpad infection with ECTV-MOS. Infected cells consisted of microglia and monocytes, astrocytes and oligodendrocytes and these type of cells underwent apoptosis within 5-15 days post infection (d.p.i.). The highest number of apoptotic cells was found at 5 and 10 d.p.i. and represented mainly microglia (61.4% and 38.6% of apoptotic cells, respectively) and astrocytes (21% and 8.9%, respectively). The number of apoptotic oligodendrocytes was 5.4% and 4.5%, respectively. Fluorometric assays demonstrated involvement of caspase-1, -3 and -8 but not caspase-9 in apoptosis in ECTV-MOS-infected mouse brains. Expression of Fas/FasL was significantly increased on ECTV-MOS-infected cells between 5 and 15 d.p.i., whereas Fas was up-regulated also on the surrounding, non-infected cells. Taking together we may conclude that ECTV-MOS infection of microglia and astrocytes leads to local inflammation resulting in Fas/FasL up-regulation and apoptosis, which limits mouse central nervous system infection with ECTV-MOS.

  8. Molecular characterization and expression analysis of GlHMGS, a gene encoding hydroxymethylglutaryl-CoA synthase from Ganoderma lucidum (Ling-zhi) in ganoderic acid biosynthesis pathway.

    PubMed

    Ren, Ang; Ouyang, Xiang; Shi, Liang; Jiang, Ai-Liang; Mu, Da-Shuai; Li, Meng-Jiao; Han, Qin; Zhao, Ming-Wen

    2013-03-01

    A hydroxymethylglutaryl-CoA synthase gene, designated as GlHMGS (GenBank accession No. JN391469) involved in ganoderic acid (GA) biosynthesis pathway was cloned from Ganoderma lucidum. The full-length cDNA of GlHMGS (GenBank accession No. JN391468) was found to contain an open reading frame of 1,413 bp encoding a polypeptide of 471 amino acid residues. The deduced amino acid sequence of GlHMGS shared high homology with other known hydroxymethylglutaryl-CoA synthase (HMGS) enzymes. In addition, functional complementation of GlHMGS in a mutant yeast strain YSC1021 lacking HMGS activity demonstrated that the cloned cDNA encodes a functional HMGS. A 1,561 bp promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. GlHMGS expression profile analysis revealed that salicylic acid, abscisic acid and methyl jasmonate up-regulated GlHMGS transcript levels over the control. Further expression analysis revealed that the developmental stage and carbon source had significant effects on GlHMGS transcript levels. GlHMGS expression peaked on day 16 before decreasing with prolonged culture time. The highest mRNA level was observed when the carbon source was maltose. Overexpression of GlHMGS enhanced GA content in G. lucidum. This study provides useful information for further studying this gene and on its function in the ganoderic acid biosynthetic pathway in G. lucidum.

  9. Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

    PubMed Central

    Yu, Yong-Hong; Hu, Zhe; Dong, Hui-Juan; Ma, Jin-Cheng; Wang, Hai-Hong

    2016-01-01

    Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants. PMID:27595587

  10. Control analysis of lipid biosynthesis in tissue cultures from oil crops shows that flux control is shared between fatty acid synthesis and lipid assembly.

    PubMed Central

    Ramli, Umi S; Baker, Darren S; Quant, Patti A; Harwood, John L

    2002-01-01

    Top-Down (Metabolic) Control Analysis (TDCA) was used to examine, quantitatively, lipid biosynthesis in tissue cultures from two commercially important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). A conceptually simplified system was defined comprising two blocks of reactions: fatty acid synthesis (Block A) and lipid assembly (Block B), which produced and consumed, respectively, a common and unique system intermediate, cytosolic acyl-CoA. We manipulated the steady-state levels of the system intermediate by adding exogenous oleic acid and, using two independent assays, measured the effect of the addition on the system fluxes (J(A) and J(B)). These were the rate of incorporation of radioactivity: (i) through Block A from [1-(14)C]acetate into fatty acids and (ii) via Block B from [U-(14)C]glycerol into complex lipids respectively. The data showed that fatty acid formation (Block A) exerted higher control than lipid assembly (Block B) in both tissues with the following group flux control coefficients (C):(i) Oil palm: *C(J(TL))(BlkA)=0.64+/-0.05 and *C(J(TL))(BlkB)=0.36+/-0.05(ii) Olive: *C(J(TL))(BlkA)=0.57+/-0.10 and *C(J(TL))(BlkB)=0.43+/-0.10where *C indicates the group flux control coefficient over the lipid biosynthesis flux (J(TL)) and the subscripts BlkA and BlkB refer to defined blocks of the system, Block A and Block B. Nevertheless, because both parts of the lipid biosynthetic pathway exert significant flux control, we suggest strongly that manipulation of single enzyme steps will not affect product yield appreciably. The present study represents the first use of TDCA to examine the overall lipid biosynthetic pathway in any tissue, and its findings are of immediate academic and economic relevance to the yield and nutritional quality of oil crops. PMID:12023882

  11. Control analysis of lipid biosynthesis in tissue cultures from oil crops shows that flux control is shared between fatty acid synthesis and lipid assembly.

    PubMed

    Ramli, Umi S; Baker, Darren S; Quant, Patti A; Harwood, John L

    2002-06-01

    Top-Down (Metabolic) Control Analysis (TDCA) was used to examine, quantitatively, lipid biosynthesis in tissue cultures from two commercially important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). A conceptually simplified system was defined comprising two blocks of reactions: fatty acid synthesis (Block A) and lipid assembly (Block B), which produced and consumed, respectively, a common and unique system intermediate, cytosolic acyl-CoA. We manipulated the steady-state levels of the system intermediate by adding exogenous oleic acid and, using two independent assays, measured the effect of the addition on the system fluxes (J(A) and J(B)). These were the rate of incorporation of radioactivity: (i) through Block A from [1-(14)C]acetate into fatty acids and (ii) via Block B from [U-(14)C]glycerol into complex lipids respectively. The data showed that fatty acid formation (Block A) exerted higher control than lipid assembly (Block B) in both tissues with the following group flux control coefficients (C):(i) Oil palm: *C(J(TL))(BlkA)=0.64+/-0.05 and *C(J(TL))(BlkB)=0.36+/-0.05(ii) Olive: *C(J(TL))(BlkA)=0.57+/-0.10 and *C(J(TL))(BlkB)=0.43+/-0.10where *C indicates the group flux control coefficient over the lipid biosynthesis flux (J(TL)) and the subscripts BlkA and BlkB refer to defined blocks of the system, Block A and Block B. Nevertheless, because both parts of the lipid biosynthetic pathway exert significant flux control, we suggest strongly that manipulation of single enzyme steps will not affect product yield appreciably. The present study represents the first use of TDCA to examine the overall lipid biosynthetic pathway in any tissue, and its findings are of immediate academic and economic relevance to the yield and nutritional quality of oil crops.

  12. Biosynthesis of l-Ascorbic Acid and Conversion of Carbons 1 and 2 of l-Ascorbic Acid to Oxalic Acid Occurs within Individual Calcium Oxalate Crystal Idioblasts1

    PubMed Central

    Kostman, Todd A.; Tarlyn, Nathan M.; Loewus, Frank A.; Franceschi, Vincent R.

    2001-01-01

    l-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various 14C-labeled compounds and examined by micro-autoradiography for incorporation of 14C into calcium oxalate crystals. [14C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-14C]AsA also gave heavy labeling of crystals, whereas [6-14C]AsA gave no labeling. Labeled precursors of AsA (l-[1-14C]galactose; d-[1-14C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, d-[1-14C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > l-galactose > d-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via d-mannose and l-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments. PMID:11161021

  13. Human Disease Isolates of Serotype M4 and M22 Group A Streptococcus Lack Genes Required for Hyaluronic Acid Capsule Biosynthesis

    PubMed Central

    Flores, Anthony R.; Jewell, Brittany E.; Fittipaldi, Nahuel; Beres, Stephen B.; Musser, James M.

    2012-01-01

    ABSTRACT Group A streptococcus (GAS) causes human pharyngitis and invasive infections and frequently colonizes individuals asymptomatically. Many lines of evidence generated over decades have shown that the hyaluronic acid capsule is a major virulence factor contributing to these infections. While conducting a whole-genome analysis of the in vivo molecular genetic changes that occur in GAS during longitudinal human pharyngeal interaction, we discovered that serotypes M4 and M22 GAS strains lack the hasABC genes necessary for hyaluronic acid capsule biosynthesis. Using targeted PCR, we found that all 491 temporally and geographically diverse disease isolates of these two serotypes studied lack the hasABC genes. Consistent with the lack of capsule synthesis genes, none of the strains produced detectable hyaluronic acid. Despite the lack of a hyaluronic acid capsule, all strains tested multiplied extensively ex vivo in human blood. Thus, counter to the prevailing concept in GAS pathogenesis research, strains of these two serotypes do not require hyaluronic acid to colonize the upper respiratory tract or cause abundant mucosal or invasive human infections. We speculate that serotype M4 and M22 GAS have alternative, compensatory mechanisms that promote virulence. PMID:23131832

  14. Adoptive Transfer of Dendritic Cells Expressing Fas Ligand Modulates Intestinal Inflammation in a Model of Inflammatory Bowel Disease

    PubMed Central

    de Jesus, Edelmarie Rivera; Isidro, Raymond A; Cruz, Myrella L; Marty, Harry; Appleyard, Caroline B

    2016-01-01

    Background Inflammatory bowel diseases (IBD) are chronic relapsing inflammatory conditions of unknown cause and likely result from the loss of immunological tolerance, which leads to over-activation of the gut immune system. Gut macrophages and dendritic cells (DCs) are essential for maintaining tolerance, but can also contribute to the inflammatory response in conditions such as IBD. Current therapies for IBD are limited by high costs and unwanted toxicities and side effects. The possibility of reducing intestinal inflammation with DCs genetically engineered to over-express the apoptosis-inducing FasL (FasL-DCs) has not yet been explored. Objective Investigate the immunomodulatory effect of administering FasL-DCs in the rat trinitrobenzene sulfonic acid (TNBS) model of acute colitis. Methods Expression of FasL on DCs isolated from the mesenteric lymph nodes (MLNs) of normal and TNBS-colitis rats was determined by flow cytometry. Primary rat bone marrow DCs were transfected with rat FasL plasmid (FasL-DCs) or empty vector (EV-DCs). The effect of these DCs on T cell IFNγ secretion and apoptosis was determined by ELISPOT and flow cytometry for Annexin V, respectively. Rats received FasL-DCs or EV-DCs intraperitoneally 96 and 48 hours prior to colitis induction with TNBS. Colonic T cell and neutrophil infiltration was determined by immunohistochemistry for CD3 and myeloperoxidase activity assay, respectively. Macrophage number and phenotype was measured by double immunofluorescence for CD68 and inducible Nitric Oxide Synthase. Results MLN dendritic cells from normal rats expressed more FasL than those from colitic rats. Compared to EV-DCs, FasL-DCs reduced T cell IFNγ secretion and increased T cell apoptosis in vitro. Adoptive transfer of FasL-DCs decreased macroscopic and microscopic damage scores and reduced colonic T cells, neutrophils, and proinflammatory macrophages when compared to EV-DC adoptive transfer. Conclusion FasL-DCs are effective at treating colonic

  15. p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase.

    PubMed

    Chang, Z; Sun, Y; He, J; Vining, L C

    2001-08-01

    Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3.8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.

  16. Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

    PubMed

    Rom, Oren; Jeries, Helana; Hayek, Tony; Aviram, Michael

    2017-01-02

    During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017.

  17. Development of a nuclear transformation system for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis.

    PubMed

    Zorin, Boris; Grundman, Omer; Khozin-Goldberg, Inna; Leu, Stefan; Shapira, Michal; Kaye, Yuval; Tourasse, Nicolas; Vallon, Olivier; Boussiba, Sammy

    2014-01-01

    Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2-5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.

  18. Assessment of a land-locked Atlantic salmon (Salmo salar L.) population as a potential genetic resource with a focus on long-chain polyunsaturated fatty acid biosynthesis.

    PubMed

    Betancor, M B; Olsen, R E; Solstorm, D; Skulstad, O F; Tocher, D R

    2016-03-01

    The natural food for Atlantic salmon (Salmo salar) in freshwater has relatively lower levels of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) than found in prey for post-smolt salmon in seawater. Land-locked salmon such as the Gullspång population feed exclusively on freshwater type lipids during its entire life cycle, a successful adaptation derived from divergent evolution. Studying land-locked populations may provide insights into the molecular and genetic control mechanisms that determine and regulate n-3 LC-PUFA biosynthesis and retention in Atlantic salmon. A two factorial study was performed comparing land-locked and farmed salmon parr fed diets formulated with fish or rapeseed oil for 8 weeks. The land-locked parr had higher capacity to synthesise n-3 LC-PUFA as indicated by higher expression and activity of desaturase and elongase enzymes. The data suggested that the land-locked salmon had reduced sensitivity to dietary fatty acid composition and that dietary docosahexaenoic acid (DHA) did not appear to suppress expression of LC-PUFA biosynthetic genes or activity of the biosynthesis pathway, probably an evolutionary adaptation to a natural diet lower in DHA. Increased biosynthetic activity did not translate to enhanced n-3 LC-PUFA contents in the flesh and diet was the only factor affecting this parameter. Additionally, high lipogenic and glycolytic potentials were found in land-locked salmon, together with decreased lipolysis which in turn could indicate increased use of carbohydrates as an energy source and a sparing of lipid.

  19. Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, ove...

  20. Impact of oxygen level in gaseous phase on gene transcription and ganoderic acid biosynthesis in liquid static cultures of Ganoderma lucidum.

    PubMed

    Zhang, Wen-Xian; Tang, Ya-Jie; Zhong, Jian-Jiang

    2010-08-01

    Liquid static cultivation of Ganoderma lucidum was previously found to be very efficient for improving the production of its valuable antitumor compound ganoderic acid (GA) (Fang and Zhong in Biotechnol Prog 18:51-54, 2002). In this work, effects of oxygen concentration within the range of 21-100% (v/v) in the gaseous phase on the mycelia growth, GA production, and gene transcription of key enzymes for GA biosynthesis in liquid static cultures of G. lucidum were investigated. A high cell density of 29.8 +/- 1.7 g/l DW and total GA production of 1427.2 +/- 74.2 mg/l were obtained under an optimal gaseous O(2) level of 80%. The expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, squalene synthase and lanosterol synthase genes of GA biosynthetic pathway as detected by quantitative real-time PCR was also affected by the gaseous oxygen concentration in the liquid static culture. H(2)O(2) was generated as reactive oxygen species in response to high oxygen concentrations in the gas phase, and it seemed to be involved in the regulation of GA biosynthesis. The information obtained in this study provided an insight into the role of gaseous O(2) in the GA production and it will be helpful for further enhancing its productivity.

  1. Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.

    PubMed

    Niehaus, Thomas D; Nguyen, Thuy N D; Gidda, Satinder K; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A; McCarty, Donald R; Downs, Diana M; Cooper, Arthur J L; Fiehn, Oliver; Mullen, Robert T; Hanson, Andrew D

    2014-07-01

    RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.

  2. Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.

    PubMed Central

    Marin, E; Nussaume, L; Quesada, A; Gonneau, M; Sotta, B; Hugueney, P; Frey, A; Marion-Poll, A

    1996-01-01

    Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location. Images PMID:8665840

  3. De novo Sequencing and Transcriptome Analysis of Pinellia ternata Identify the Candidate Genes Involved in the Biosynthesis of Benzoic Acid and Ephedrine

    PubMed Central

    Zhang, Guang-hui; Jiang, Ni-hao; Song, Wan-ling; Ma, Chun-hua; Yang, Sheng-chao; Chen, Jun-wen

    2016-01-01

    Background: The medicinal herb, Pinellia ternata, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL) and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. Results: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeq™ 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternata, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Also, 14,468 simple sequence repeats (SSRs) were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. Conclusion: RNA-seq data was used for the first time to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant. PMID:27579029

  4. Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids[C][W][OPEN

    PubMed Central

    Niehaus, Thomas D.; Nguyen, Thuy N.D.; Gidda, Satinder K.; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A.; McCarty, Donald R.; Downs, Diana M.; Cooper, Arthur J.L.; Fiehn, Oliver; Mullen, Robert T.; Hanson, Andrew D.

    2014-01-01

    RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase. PMID:25070638

  5. Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Lee, Kyungjin; Lee, Hye-Jung; Back, Kyoungwhan

    2014-11-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis.

  6. D-Glucosone and L-sorbosone, putative intermediates of L-ascorbic acid biosynthesis in detached bean and spinach leaves. [Phaseolus vulgaris L. ; Spinacia oleracea L

    SciTech Connect

    Saito, Kazumi; Nick, J.A.; Loewus, F.A. )

    1990-11-01

    D-(6-{sup 14}C)Glucosone that had been prepared enzymically from D-(6-{sup 14}C)glucose was used to compare relative efficiencies of these two sugars for L-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, {sup 14}C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from (6-{sup 14}C)glucose underwent considerable redistribution during AA formation, whereas {sup 14}C from (6-{sup 14}C)glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, L-(U-{sup 14}C)sorbosone was found to be equivalent to (6-{sup 14}C)glucose as a source of {sup 14}C for AA. In the presence of 0.1% D-glucosone, conversion of (6-{sup 14}C) glucose into labeled AA was greatly repressed. In a comparable experiment with L-sorbosone replacing D-glucosone, the effect was much less. The experiments described here give substance to the proposal that D-glucosone and L-sorbosone are putative intermediates in the conversion of D-glucose to AA in higher plants.

  7. Xanthomonas campestris diffusible factor is 3-hydroxybenzoic acid and is associated with xanthomonadin biosynthesis, cell viability, antioxidant activity, and systemic invasion.

    PubMed

    He, Ya-Wen; Wu, Ji'en; Zhou, Lian; Yang, Fan; He, Yong-Qiang; Jiang, Bo-Le; Bai, Linquan; Xu, Yuquan; Deng, Zixin; Tang, Ji-Liang; Zhang, Lian-Hui

    2011-08-01

    Xanthomonas campestris pv. campestris produces a membrane-bound yellow pigment called xanthomonadin. A diffusible factor (DF) has been reported to regulate xanthomonadin biosynthesis. In this study, DF was purified from bacterial culture supernatants using a combination of solvent extraction, flash chromatography, and high-performance liquid chromatography. Mass spectrometry and nuclear magnetic resonance analyses resolved the DF chemical structure as 3-hydroxybenzoic acid (3-HBA), which was further confirmed by synthetic 3-HBA. Significantly, bioassay and in silico analysis suggest that DF production is widely conserved in a range of bacterial species. Analysis of DF derivatives established the hydroxyl group and its position as the key structural features for the role of DF in xanthomonadin biosynthesis. In addition, we showed that DF is also associated with bacterial survival, H2O2 resistance, and systemic invasion. Furthermore, evidence was also presented that DF and diffusible signaling factor have overlapping functions in modulation of bacterial survival, H2O2 resistance, and virulence. Utilization of different mechanisms to modulate similar virulence traits may provide X. campestris pv. campestris with plasticity in response to various environmental cues.

  8. Fas transduces dual apoptotic and trophic signals in hematopoietic progenitors.

    PubMed

    Pearl-Yafe, Michal; Stein, Jerry; Yolcu, Esma S; Farkas, Daniel L; Shirwan, Haval; Yaniv, Isaac; Askenasy, Nadir

    2007-12-01

    Stem cells and progenitors are often required to realize their differentiation potential in hostile microenvironments. The Fas/Fas ligand (FasL) interaction is a major effector pathway of apoptosis, which negatively regulates the expansion of differentiated hematopoietic cells. The involvement of this molecular interaction in the function of hematopoietic stem and progenitor cells is not well understood. In the murine syngeneic transplant setting, both Fas and FasL are acutely upregulated in bone marrow-homed donor cells; however, the Fas(+) cells are largely insensitive to FasL-induced apoptosis. In heterogeneous populations of lineage-negative (lin(-)) bone marrow cells and progenitors isolated by counterflow centrifugal elutriation, trimerization of the Fas receptor enhanced the clonogenic activity. Inhibition of caspases 3 and 8 did not affect the trophic signals mediated by Fas, yet it efficiently blocked the apoptotic pathways. Fas-mediated tropism appears to be of physiological significance, as pre-exposure of donor cells to FasL improved the radioprotective qualities of hematopoietic progenitors, resulting in superior survival of myeloablated hosts. Under these conditions, the activity of long-term reconstituting cells was not affected, as determined in sequential secondary and tertiary transplants. Dual caspase-independent tropic and caspase-dependent apoptotic signaling place the Fas receptor at an important junction of activation and death. This regulatory mechanism of hematopoietic homeostasis activates progenitors to promote the recovery from aplasia and converts into a negative regulator in distal stages of cell differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

  9. Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

    PubMed Central

    Udhane, Sameer S.; Pandey, Amit V.; Hofer, Gaby; Mullis, Primus E.; Flück, Christa E.

    2015-01-01

    Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells. PMID:25970467

  10. Effect of environmental factors on the biosynthesis of the neuro-excitatory amino acid β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid) in callus tissue of Lathyrus sativus.

    PubMed

    Haque, Rabiul M; Kuo, Yu-Haey; Lambein, Fernand; Hussain, Muhammed

    2011-03-01

    Habituated callus tissues derived from leaf explants of Lathyrus sativus L. (grass pea) were cultured under different environmental conditions such as drought, salinity and deficiency or oversupply of micronutrients. The biosynthesis of the neuro-excitatory β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid) was induced by feeding the precursor BIA, (β-isoxazolin-5-on-2-yl)-alanine, to those calli habituated under different stress conditions. Conversion of BIA into β-ODAP was reduced by Zn(2+) at different levels of Fe(2+) supplements while excess of Fe(2+) enhanced it at different Zn(2+) levels in the media. The biosynthesis of β-ODAP was increased by both oversupply and deficiency of Mn(2+) manganese while B(3+) as well as Co(2+) increased it significantly by oversupply. Al(3+) enhanced the conversion of BIA into β-ODAP significantly in a concentration-dependent way. Cu(2+) also reduced the formation of β-ODAP when increased in the media. Mo(6+) had no apparent effect. NaCl decreased the conversion of BIA into β-ODAP proportionately with the increase in salinity. β-ODAP was increased with increasing mannitol concentration till -0.23MPa while at this osmotic potential created with PEG-20,000 the formation of β-ODAP is completely inhibited in low toxin calli. These experiments demonstrate the importance of environmental factors, especially micronutrients and salinity, on the biosynthesis of β-ODAP.

  11. Signal transducer and oxidative stress mediated modulation of phenylpropanoid pathway to enhance rosmarinic acid biosynthesis in fungi elicited whole plant culture of Solenostemon scutellarioides.

    PubMed

    Dewanjee, Saikat; Gangopadhyay, Moumita; Das, Urmi; Sahu, Ranabir; Samanta, Amalesh; Banerjee, Pamela

    2014-11-01

    This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation with phytopathogenic fungi. Amongst selected fungi, Aternaria alternata caused significant elevation (p<0.05-0.01) in RA accumulation (∼1.3-1.6-fold) between 25 and 100 μg l(-1). However, elicitation at the dose of 50 μg l(-1) has been found to be most effective and intracellular RA content reached almost ∼1.6-fold (p<0.01) higher in day 7. Therefore, A. alternata (50 μg l(-1)) was selected for mechanism evaluation. A significant elevation of intercellular jasmonic acid was observed up to day 6 after elicitation with A. alternata (50 μg l(-1)). A significant increase in tissue H2O2 and lipid peroxidation coupled with depletion of antioxidant enzymes superoxide dismutase and catalase indicated augmented oxidative stress associated with biotic interaction. Preceding the elicitor-induced RA accumulation, a notable alteration in the specific activities of biosynthetic enzymes namely PAL and TAT was recorded, while, no significant change in the activities of RAS was observed. HPPR activity was slightly improved in elicited plant. Therefore, it could be concluded that A. alternata elicited the biosynthesis of rosmarinic acid via signal transduction through jasmonic acid coupled with elicitor induced oxidative stress and associated mechanism.

  12. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    SciTech Connect

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  13. Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumour formation.

    PubMed

    Reineke, Gavin; Heinze, Bernadette; Schirawski, Jan; Buettner, Hermann; Kahmann, Regine; Basse, Christoph W

    2008-05-01

    Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis is characterized by excessive host tumour formation. U. maydis is able to produce indole-3-acetic acid (IAA) efficiently from tryptophan. To assess a possible connection to the induction of host tumours, we investigated the pathways leading to fungal IAA biosynthesis. Besides the previously identified iad1 gene, we identified a second indole-3-acetaldehyde dehydrogenase gene, iad2. Deltaiad1Deltaiad2 mutants were blocked in the conversion of both indole-3-acetaldehyde and tryptamine to IAA, although the reduction in IAA formation from tryptophan was not significantly different from Deltaiad1 mutants. To assess an influence of indole-3-pyruvic acid on IAA formation, we deleted the aromatic amino acid aminotransferase genes tam1 and tam2 in Deltaiad1Deltaiad2 mutants. This revealed a further reduction in IAA levels by five- and tenfold in mutant strains harbouring theDeltatam1 andDeltatam1Deltatam2 deletions, respectively. This illustrates that indole-3-pyruvic acid serves as an efficient precursor for IAA formation in U. maydis. Interestingly, the rise in host IAA levels upon U. maydis infection was significantly reduced in tissue infected with Deltaiad1Deltaiad2Deltatam1 orDeltaiad1Deltaiad2Deltatam1Deltatam2 mutants, whereas induction of tumours was not compromised. Together, these results indicate that fungal IAA production critically contributes to IAA levels in infected tissue, but this is apparently not important for triggering host tumour formation.

  14. Lymphocytes with Aberrant Expression of Fas or Fas-ligand Attenuate Immune Bone Marrow Failure in a Mouse Model

    PubMed Central

    Omokaro, Stephanie O.; Desierto, Marie J.; Eckhaus, Michael A.; Ellison, Felicia M.; Chen, Jichun; Young, Neal S.

    2012-01-01

    Bone marrow (BM) and lymphocyte samples from aplastic anemia patients show up-regulated Fas and Fas-ligand (FasL) expression respectively, supporting a relationship between immune-mediated BM destruction and the Fas apoptotic pathway. Mice with spontaneous lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) mutations exhibit abnormal expression of Fas and FasL; serving as potential models to elucidate underlying mechanisms of BM failure. We examined cellular and functional characteristics of lpr and gld mutants on the C57BL/6 (B6) background. Lymph node (LN) cells from lpr and gld mice produced less apoptosis when co-incubated with C.B10-H2b/LilMcd (C.B10) BM cells in vitro. This functional difference was confirmed by infusing lpr, gld, and B6 LN cells into sub-lethally irradiated CB10 mice; all donor LN cells showed significant T cell expansion and activation but only B6 LN cells caused severe BM destruction. Mice infused with gld LN cells developed mild to moderate BM failure, despite receiving FasL-deficient effectors, thus suggesting the existence of alternative pathways or incomplete penetrance of the mutation. Paradoxically, mice that received Fas-deficient lpr LN cells also had reduced BM failure, likely due to down-regulation of pro-apoptotic genes, an effect that can be overcome by higher doses of lpr LN cells. Our model demonstrates that abnormal Fas or FasL expression interferes with the development of pancytopenia and marrow hypoplasia, validating a major role for the Fas/FasL cytotoxic pathway in immune-mediated BM failure, although disruption of this pathway does not completely abolish marrow destruction. PMID:19265119

  15. Lymphocytes with aberrant expression of Fas or Fas ligand attenuate immune bone marrow failure in a mouse model.

    PubMed

    Omokaro, Stephanie O; Desierto, Marie J; Eckhaus, Michael A; Ellison, Felicia M; Chen, Jichun; Young, Neal S

    2009-03-15

    Bone marrow (BM) and lymphocyte samples from aplastic anemia patients show up-regulated Fas and Fas-ligand (FasL) expression, respectively, supporting a relationship between immune-mediated BM destruction and the Fas apoptotic pathway. Mice with spontaneous lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) mutations exhibit abnormal expression of Fas and FasL, serving as potential models to elucidate underlying mechanisms of BM failure. We examined cellular and functional characteristics of lpr and gld mutants on the C57BL/6 (B6) background. Lymph node (LN) cells from lpr and gld mice produced less apoptosis when coincubated with C.B10-H2(b)/LilMcd (C.B10) BM cells in vitro. This functional difference was confirmed by infusing lpr, gld, and B6 LN cells into sublethally irradiated CB10 mice. All donor LN cells showed significant T cell expansion and activation, but only B6 LN cells caused severe BM destruction. Mice infused with gld LN cells developed mild to moderate BM failure despite receiving FasL-deficient effectors, thus suggesting the existence of alternative pathways or incomplete penetrance of the mutation. Paradoxically, mice that received Fas-deficient lpr LN cells also had reduced BM failure, likely due to down-regulation of proapoptotic genes, an effect that can be overcome by higher doses of lpr LN cells. Our model demonstrates that abnormal Fas or FasL expression interferes with the development of pancytopenia and marrow hypoplasia, validating a major role for the Fas/FasL cytotoxic pathway in immune-mediated BM failure, although disruption of this pathway does not completely abolish marrow destruction.

  16. Physiological management of dietary deficiency in n-3 fatty acids by spawning Gulf killifish (Fundulus grandis).

    PubMed

    Patterson, Joshua T; Green, Christopher C

    2015-08-01

    Lipid dynamics of spawning fish are critical to the production of viable embryos and larvae. The present study utilized manipulation of dietary fatty acid (FA) profiles to examine the ability of spawning Gulf killifish (Fundulus grandis) to mobilize critical lipid components from somatic reserves or synthesize long-chain polyunsaturated FAs (LC-PUFAs) de novo from shorter-chain C18 precursors. An egg and multi-tissue evaluation of changes in FA concentrations across time after fish were switched from LC-PUFA-rich to LC-PUFA-deficient experimental diets was employed. The two experimental diets contained lipid sources which differed drastically in n-3 C18 FA content but had similar levels of n-6 C18 FAs. Discrete effects of dietary n-3 FAs can be analyzed because n-3 and n-6 represent distinct metabolic families which cannot be exchanged in vivo. Results indicate that a combination of mobilization and de novo synthesis is likely utilized to maintain physiologically required FA levels in critical tissues and embryos. Mobilization was supported by decreases in LC-PUFAs in somatic tissues and decreases in intraperitoneal fat content and liver mass. Evidence for biosynthesis was provided by a higher level of n-3 LC-PUFAs in the liver and ova of fish fed diets containing n-3 C18 precursors versus those fed diets with low levels of precursor FAs. The characteristic physiological plasticity of Gulf killifish is exemplified in the nutritional domain by its management of dietary FA deficiency.

  17. Cloning and functional analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits.

    PubMed

    Zhang, Mei; Leng, Ping; Zhang, Guanglian; Li, Xiangxin

    2009-08-15

    Ripening and senescence are generally controlled by ethylene in climacteric fruits like peaches, and the ripening process of grape, a non-climacteric fruit, may have some relationship to abscisic acid (ABA) function. In order to better understand the role of ABA in ripening and senescence of these two types of fruits, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from peaches and grapes using an RT-PCR approach. The NCED gene fragments were cloned from peaches (PpNCED1and PpNCED2, each 740bp) and grapes (VVNCED1, 741bp) using degenerate primers designed based on the conserved amino acids sequence of NCEDs in other plants. PpNCED1 showed 78.54% homology with PpNCED2, 74.90% homology with VVNCED1, and both showed high homology to NCEDs from other plants. The expression patterns of PpNCED1 and VVNCED1 were very similar. Both were highly expressed at the beginning of ripening when ABA content becomes high. The maximum ABA preceded ethylene production in peach fruit. ABA in the grape gradually increased from the beginning of ripening and reached the highest level at 20d before the harvest stage. However, ethylene remained at low levels during the entire process of fruit development, including ripening and senescence. ABA content, and ripening and softening of both types of fruits, were promoted or delayed by exogenous ABA or Fluridone (or NDGA) treatment. The roles of ABA and ethylene in the later ripening of fruit are complex. Based on results obtained in this study, we concluded that PpNCED1 and VVNCED1 initiate ABA biosynthesis at the beginning of fruit ripening, and that ABA accumulation might play a key role in the regulation of ripeness and senescence of both peach and grape fruits.

  18. Cysteine proteinases Fas1 and Fas2 are diagnostic markers for Fasciola hepatica infection in alpacas (Lama pacos).

    PubMed

    Neyra, Victor; Chavarry, Elizabeth; Espinoza, Jose R

    2002-04-19

    Circulating antibody against Fasciola hepatica antigens was determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis in alpacas naturally exposed to F. hepatica. Serological assay parameters were established by using sera from eight infected animals and seven controls with no record of this parasitic infection. Excretory--secretory (ES-) products, Fas1- and Fas2-ELISA were used to survey 307 alpacas from a F. hepatica endemic area in the Peruvian Andes. Seroprevalence of F. hepatica infection varied from 56.7, 64.8 and 66.8% measured by Fas1-, Fas2- and ES-ELISA, respectively. The sensitivity for ES-ELISA was 95%, corresponding Fas1- and Fas2-ELISA sensitivity values were 90 and 95%. In this population, 7% of animals were positive for F. hepatica eggs in faeces, other parasites detected were Trichuris sp. (40%), Nematodirus sp. (34.6%), Lamanema sp. (12.8%) and Eimeria sp. (11.8%). The results show that F. hepatica infected animals elicit circulating antibodies against ES, Fas1 and Fas2. Fas2-ELISA may be proposed as a sensitive assay for the immunodiagnosis of fasciolosis in alpacas.