Science.gov

Sample records for acid ra biosynthesis

  1. 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. PMID:19560175

  2. 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.

  3. Biosynthesis of pulcherriminic acid

    PubMed Central

    MacDonald, J. C.

    1965-01-01

    1. Candida pulcherrima was grown on a complex medium to which various compounds had been added to determine their effect on the biosynthesis of pulcherriminic acid. Most of the pulcherriminic acid synthesized by C. pulcherrima PRL2019 was derived from the l-[1-14C]leucine added to the medium. 2. The cyclic dipeptide of l-leucine (cyclo-l-leucyl-l-leucyl) was shown, by trapping experiments involving cycloleucyl-leucyl isomers, to be synthesized by strain PRL2019. Cyclo-l-leucyl-l-leucyl was derived from l-leucine and was converted into pulcherriminic acid. Cyclo-l-leucyl-l-leucyl was a precursor of pulcherriminic acid in strain PRL2007 also. 3. The results supported the hypothesis that pulcherriminic acid is derived from l-leucine and that cyclo-l-leucyl-l-leucyl is an intermediate in the biosynthesis. PMID:5837792

  4. Physiological insights into all-trans-retinoic acid biosynthesis

    PubMed Central

    Napoli, Joseph L.

    2011-01-01

    All-trans-retinoic acid (atRA) provides essential support to diverse biological systems and physiological processes. Epithelial differentiation and its relationship to cancer and embryogenesis have typified intense areas of interest into atRA function. Recently, however, interest in atRA action in the nervous system, the immune system, energy balance and obesity has increased considerably, especially concerning postnatal function. atRA action depends on atRA biosynthesis: defects in retinoid-dependent processes increasingly relate to defects in atRA biogenesis. Considerable evidence indicates that physiological atRA biosynthesis occurs via a regulated process, consisting of a complex interaction of retinoid binding-proteins and retinoid recognizing enzymes. An accrual of biochemical, physiological and genetic data have identified specific functional outcomes for the retinol dehydrogenases, RDH1, RDH10, and DHRS9, as physiological catalysts of the first step in atRA biosynthesis, and for the retinal dehydrogenases RALDH1, RALDH2, and RALDH3, as catalysts of the second and irreversible step. Each of these enzymes associates with explicit biological processes mediated by atRA. Redundancy occurs, but seems limited. Cumulative data supports a model of interactions among these enzymes with retinoid binding-proteins, with feedback regulation and/or control by atRA via modulating gene expression of multiple participants. The ratio apo-CRBP1/holo-CRBP1 participates by influencing retinol flux into and out of storage as retinyl esters, thereby modulating substrate to support atRA biosynthesis. atRA biosynthesis requires presence of both an RDH and an RALDH: conversely, absence of one isozyme of either step does not indicate lack of atRA biosynthesis at the site. PMID:21621639

  5. 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

  6. Oleic acid biosynthesis in cyanobacteria

    SciTech Connect

    VanDusen, W.J.; Jaworski, J.G.

    1986-05-01

    The biosynthesis of fatty acids in cyanobacteria is very similar to the well characterized system found in green plants. However, the initial desaturation of stearic acid in cyanobacteria appears to represent a significant departure from plant systems in which stearoyl-ACP is the exclusive substrate for desaturation. In Anabaena variabilis, the substrate appears to be monoglucosyldiacylglycerol, a lipid not found in plants. The authors examined five different cyanobacteria to determine if the pathway in A. variabilis was generally present in other cyanobacteria. The cyanobacteria studied were A. variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis marina, and Anacystis nidulans. Each were grown in liquid culture, harvested, and examined for stearoyl-ACP desaturase activity or incubated with /sup 14/CO/sub 2/. None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating /sup 14/CO/sub 2/ into monoglucosyldiacylglycerol and results from incubations of 20 min, 1 hr, 1 hr + 10 hr chase were consistent with monoglucosyldiacylglycerol serving as precursor for monogalctosyldiacylglycerol. Thus, initial evidence is consistent with oleic acid biosynthesis occurring by desaturation of stearoyl-monoglucosyldiacylglycerol in all cyanobacteria.

  7. 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

  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. MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress

    PubMed Central

    Sharma, Rishabh; Keshari, Deepa; Singh, Kumar Sachin; Yadav, Shailendra; Singh, Sudheer Kumar

    2016-01-01

    Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile. PMID:27353854

  10. MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress.

    PubMed

    Sharma, Rishabh; Keshari, Deepa; Singh, Kumar Sachin; Yadav, Shailendra; Singh, Sudheer Kumar

    2016-01-01

    Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile.

  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. The Molecular Genetics of Mycolic Acid Biosynthesis.

    PubMed

    Pawełczyk, Jakub; Kremer, Laurent

    2014-08-01

    Mycolic acids are major and specific long-chain fatty acids that represent essential components of the Mycobacterium tuberculosis cell envelope. They play a crucial role in the cell wall architecture and impermeability, hence the natural resistance of mycobacteria to most antibiotics, and represent key factors in mycobacterial virulence. Biosynthesis of mycolic acid precursors requires two types of fatty acid synthases (FASs), the eukaryotic-like multifunctional enzyme FAS I and the acyl carrier protein (ACP)-dependent FAS II systems, which consists of a series of discrete mono-functional proteins, each catalyzing one reaction in the pathway. Unlike FAS II synthases of other bacteria, the mycobacterial FAS II is incapable of de novo fatty acid synthesis from acetyl-coenzyme A, but instead elongates medium-chain-length fatty acids previously synthesized by FAS I, leading to meromycolic acids. In addition, mycolic acid subspecies with defined biological properties can be distinguished according to the chemical modifications decorating the meromycolate. Nearly all the genetic components involved in both elongation and functionalization of the meromycolic acid have been identified and are generally clustered in distinct transcriptional units. A large body of information has been generated on the enzymology of the mycolic acid biosynthetic pathway and on their genetic and biochemical/structural characterization as targets of several antitubercular drugs. This chapter is a comprehensive overview of mycolic acid structure, function, and biosynthesis. Special emphasis is given to recent work addressing the regulation of mycolic acid biosynthesis, adding new insights to our understanding of how pathogenic mycobacteria adapt their cell wall composition in response to environmental changes.

  13. The Role of Retinoic Acid (RA) in Spermatogonial Differentiation.

    PubMed

    Busada, Jonathan T; Geyer, Christopher B

    2016-01-01

    Retinoic acid (RA) directs the sequential, but distinct, programs of spermatogonial differentiation and meiotic differentiation that are both essential for the generation of functional spermatozoa. These processes are functionally and temporally decoupled, as they occur in distinct cell types that arise over a week apart, both in the neonatal and adult testis. However, our understanding is limited in terms of what cellular and molecular changes occur downstream of RA exposure that prepare differentiating spermatogonia for meiotic initiation. In this review, we describe the process of spermatogonial differentiation and summarize the current state of knowledge regarding RA signaling in spermatogonia. PMID:26559678

  14. 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.

  15. Construction of a chimeric biosynthetic pathway for the de novo biosynthesis of rosmarinic acid in Escherichia coli.

    PubMed

    Bloch, Sarah E; Schmidt-Dannert, Claudia

    2014-11-01

    Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA), the most well-known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, microbial production of HCEs could be a sustainable, controlled means of production. Through the overexpression of a six-enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof-of-concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non-natural HCEs.

  16. 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.

  17. Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?

    PubMed

    Cook, Sam D; Nichols, David S; Smith, Jason; Chourey, Prem S; McAdam, Erin L; Quittenden, Laura; Ross, John J

    2016-06-01

    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

  18. Biosynthesis of the Aromatic Amino Acids.

    PubMed

    Pittard, James; Yang, Ji

    2008-09-01

    This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and changing from a monomer to a dimer or a hexamer are all described. There is also an analysis of the various strategies which allow TyrR in conjunction with particular amino acids to differentially affect the expression of individual genes of the TyrR regulon. PMID:26443741

  19. 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. PMID:10506831

  20. Clavulanic acid biosynthesis and genetic manipulation for its overproduction.

    PubMed

    Song, Ju Yeon; Jensen, Susan E; Lee, Kye Joon

    2010-10-01

    Clavulanic acid, a β-lactamase inhibitor, is used together with β-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other β-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.

  1. 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

  2. Total Biosynthesis of Legionaminic Acid, a Bacterial Sialic Acid Analogue.

    PubMed

    Hassan, Mohamed I; Lundgren, Benjamin R; Chaumun, Michael; Whitfield, Dennis M; Clark, Brady; Schoenhofen, Ian C; Boddy, Christopher N

    2016-09-19

    Legionaminic acid, Leg5,7Ac2 , a nonulosonic acid like 5-acetamido neuraminic acid (Neu5Ac, sialic acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac2 has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac2 biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over-expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac2 (≈120 mg L(-1) of culture broth) to be produced. Pure Leg5,7Ac2 could be isolated and converted into CMP-activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac2 enabling study of its role in prokaryotic and eukaryotic glycobiology. PMID:27538580

  3. A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

    PubMed Central

    Sun, Xinxiao; Lin, Yuheng; Huang, Qin; Yuan, Qipeng

    2013-01-01

    Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid. PMID:23603682

  4. Inhibitors of amino acids biosynthesis as antifungal agents.

    PubMed

    Jastrzębowska, Kamila; Gabriel, Iwona

    2015-02-01

    Fungal microorganisms, including the human pathogenic yeast and filamentous fungi, are able to synthesize all proteinogenic amino acids, including nine that are essential for humans. A number of enzymes catalyzing particular steps of human-essential amino acid biosynthesis are fungi specific. Numerous studies have shown that auxotrophic mutants of human pathogenic fungi impaired in biosynthesis of particular amino acids exhibit growth defect or at least reduced virulence under in vivo conditions. Several chemical compounds inhibiting activity of one of these enzymes exhibit good antifungal in vitro activity in minimal growth media, which is not always confirmed under in vivo conditions. This article provides a comprehensive overview of the present knowledge on pathways of amino acids biosynthesis in fungi, with a special emphasis put on enzymes catalyzing particular steps of these pathways as potential targets for antifungal chemotherapy.

  5. Biosynthesis of resorcylic acid lactone lasiodiplodin in Lasiodiplodia theobromae.

    PubMed

    Kashima, Takasumi; Takahashi, Kosaku; Matsuura, Hideyuki; Nabeta, Kensuke

    2009-05-01

    The biosynthesis of lasiodiplodin (1) and its (5S)-5-hydroxylated derivative (2) were investigated by the administration of (13)C-labeled acetates to Lasiodiplodia theobromae. The labeling patterns of biosynthetically (13)C-labeled 1 and 2 were determined by (13)C-NMR and INADEQUATE spectra, demonstrating the octaketide origins of 1 and 2. Taking into account the biosynthetic study of resorcylic acid lactones, the involvement of highly reduced acyl intermediates in the biosynthesis of lasiodiplodins was presumed; thus, we synthesized (2)H-labeled hypothetical acyl intermediates of 1, 9-hydroxydecanoic acid (4) and its N-acetylcysteamine thioester (SNAC, 5). When L. theobromae was incubated with 5 mM of a (2)H-labeled intermediate, the (2)H-label from the intermediate was incorporated at the expected position of 1. These incorporation studies revealed that 1 was produced via a pathway which closely resembles that of resorcylic acid lactone biosynthesis. PMID:19420710

  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. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

    PubMed Central

    Chang, Perng-Kuang; Ehrlich, Kenneth C.; Fujii, Isao

    2009-01-01

    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 what is currently known about the toxicity of CPA to animals and humans, both by itself or in combination with other mycotoxins. The review also discusses CPA biosynthesis and the genetic diversity of CPA production in A. flavus/oryzae populations. PMID:22069533

  8. Protein biosynthesis with conformationally restricted amino acids

    SciTech Connect

    Mendel, D. Lawrence Berkeley Lab., CA ); Ellman, J.; Schultz, P.G. )

    1993-05-19

    The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. The ability to site-specifically introduce constrained amino acids into large polypeptide chains would provide a similar opportunity to probe the flexibility, conformation, folding and stability of proteins. To this end, we have examined the competence of the Escherichia coli protein biosynthetic machinery to incorporate a number of these unnatural amino acids into the 164 residue protein T4 lysozyme (T4L). Results clearly demonstrate that the protein biosynthetic machinery can accommodate a wide variety of conformationally constrained amino acids. The expansion of structural motifs that can be biosynthetically incorporated into proteins to include a large number of conformationally constrained amino acids significantly increases the power of mutagenesis methods as probes of protein structure and function and provides additional insights into the steric requirements of the translational machinery. 13 refs., 2 figs.

  9. 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

  10. Regulation of fatty acid biosynthesis in Escherichia coli.

    PubMed Central

    Magnuson, K; Jackowski, S; Rock, C O; Cronan, J E

    1993-01-01

    Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth. PMID:8246839

  11. Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke.

    PubMed

    Moglia, Andrea; Lanteri, Sergio; Comino, Cinzia; Acquadro, Alberto; de Vos, Ric; Beekwilder, Jules

    2008-09-24

    Leaf extracts from globe artichoke ( Cynara cardunculus L. var. scolymus) have been widely used in medicine as hepatoprotectant and choleretic agents. Globe artichoke leaves represent a natural source of phenolic acids with dicaffeoylquinic acids, such as cynarin (1,3-dicaffeoylquinic acid), along with its biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the most abundant molecules. This paper reports the development of an experimental system to induce caffeoylquinic acids. This system may serve to study the regulation of the biosynthesis of (poly)phenolic compounds in globe artichoke and the genetic basis of this metabolic regulation. By means of HPLC-PDA and accurate mass LC-QTOF MS and MS/MS analyses, the major phenolic compounds in globe artichoke leaves were identified: four isomers of dicaffeoylquinic acid, three isomers of caffeoylquinic acid, and the flavone luteolin 7-glucoside. Next, plant material was identified in which the concentration of phenolic compounds was comparable in the absence of particular treatments, with the aim to use this material to test the effect of stress application on the regulation of biosynthesis of caffeoylquinic acids. Using this material, the effect of UV-C, methyl jasmonate, and salicylic acid treatments on (poly)phenolic compounds was tested in different globe artichoke genotypes. UV-C exposure consistently increased the levels of dicaffeoylquinic acids in all genotypes, whereas the effect on compounds from the same biosynthetic pathway, for example, chlorogenic acid and luteolin-7-glucoside, was much less pronounced and was not statistically significant. No effect of methyl jasmonate or salicylic acid was found. Time-response experiments indicated that the level of dicaffeoylquinic acids reached a maximum at 24 h after UV radiation. On the basis of these results a role of dicaffeoylquinic acids in UV protection in globe artichoke is hypothesized.

  12. Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke.

    PubMed

    Moglia, Andrea; Lanteri, Sergio; Comino, Cinzia; Acquadro, Alberto; de Vos, Ric; Beekwilder, Jules

    2008-09-24

    Leaf extracts from globe artichoke ( Cynara cardunculus L. var. scolymus) have been widely used in medicine as hepatoprotectant and choleretic agents. Globe artichoke leaves represent a natural source of phenolic acids with dicaffeoylquinic acids, such as cynarin (1,3-dicaffeoylquinic acid), along with its biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the most abundant molecules. This paper reports the development of an experimental system to induce caffeoylquinic acids. This system may serve to study the regulation of the biosynthesis of (poly)phenolic compounds in globe artichoke and the genetic basis of this metabolic regulation. By means of HPLC-PDA and accurate mass LC-QTOF MS and MS/MS analyses, the major phenolic compounds in globe artichoke leaves were identified: four isomers of dicaffeoylquinic acid, three isomers of caffeoylquinic acid, and the flavone luteolin 7-glucoside. Next, plant material was identified in which the concentration of phenolic compounds was comparable in the absence of particular treatments, with the aim to use this material to test the effect of stress application on the regulation of biosynthesis of caffeoylquinic acids. Using this material, the effect of UV-C, methyl jasmonate, and salicylic acid treatments on (poly)phenolic compounds was tested in different globe artichoke genotypes. UV-C exposure consistently increased the levels of dicaffeoylquinic acids in all genotypes, whereas the effect on compounds from the same biosynthetic pathway, for example, chlorogenic acid and luteolin-7-glucoside, was much less pronounced and was not statistically significant. No effect of methyl jasmonate or salicylic acid was found. Time-response experiments indicated that the level of dicaffeoylquinic acids reached a maximum at 24 h after UV radiation. On the basis of these results a role of dicaffeoylquinic acids in UV protection in globe artichoke is hypothesized. PMID:18710252

  13. Ribonucleic acid (RNA) biosynthesis in human cancer.

    PubMed

    Hajjawi, Omar S

    2015-01-01

    In many respects, the most remarkable chemical substances within the genome of eukaryotic cells are remarkable proteins which are the critical structural and functional units of living cells. The specifications for everything that goes in the cell are natural digital-to-digital decoding process in an archive sequence by deoxyribonucleic acid (DNA) and an articulate construction by ribonucleic acid (RNA). The products of DNA transcription are long polymers of ribonucleotides rather than deoxyribonucleotides and are termed ribonucleic acids. Certain deoxyribonucleotide sequences, or genes, give rise to transfer RNA (tRNA) and other ribosomal RNA (rRNA) when transcribed. The ribonucleotide sequences fold extensively and rRNA is associated with specific proteins to yield the essential cell components, ribosomes. Transcription of other special sequences yields messenger RNAs (mRNAs) that contain ribonucleotide sequences that will be ultimately translated into new types of amino acid sequences of functional cellular protein molecules. This switch to a different variety of cellular molecular sequences is complex, but each sequence of the three ribonucleotides specifies the insertion of one particular amino acid into the polypeptide chain under production. Whilst mRNA is considered the vehicle by which genetic information is transmitted from the genome and allocated in the appropriate cytoplasmic sites for translation into protein via cap-dependent mechanism, the actual translation depends also on the presence of other so-called household and luxury protein molecules. Recent evidence suggests RNA species are required at initiation, because treatment of cells with antibiotics or drugs that inhibit RNA synthesis cause a decrease in protein synthesis. The rRNA is necessary as a structural constituent of the ribosomes upon which translation takes place, whereas tRNA is necessary as an adaptor in amino acid activation and elongation protein chains to ribosomes. In this article

  14. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    PubMed

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

  15. A blueprint of the amino acid biosynthesis network of hemiascomycetes.

    PubMed

    Förster, Jan; Halbfeld, Christoph; Zimmermann, Martin; Blank, Lars M

    2014-11-01

    The structure and regulation of biosynthesis pathways in Saccharomyces cerevisiae have been detailed extensively. For other hemiascomycetes, genomic sequences are primarily available, whereas biochemical information on them is scarce. The resulting biochemical networks that are used for research in basic science and biotechnology are often biased by data from S. cerevisiae, assuming that there are often implicitly conserved structures between species. We examined the structure of the amino acid biosynthesis network in nine hemiascomycetes, spanning the phylogenetic clade. Differences in the genetic inventory included the presence and absence of isoenzymes and compartmentation of the pathways. Notably, no two hemiascomycetes had identical genetic inventories. For example, the lack of the mitochondrial αIPMS isoenzyme and presence of only one copy of the BCAA aminotransferase in Pichia pastoris indicate a disparately compartmented leucine biosynthesis pathway. Our findings suggest that αIPMS and BCAA aminotransferase are solely located in the cytosol of P. pastoris, requiring correction of the leucine biosynthesis pathway layout in this species. Our results argue for careful use of information from S. cerevisiae and for joint efforts to fill the knowledge gaps in other species. Such analysis will lead to contributions in biotechnology disciplines, such as protein production and compartment engineering. PMID:25187056

  16. 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. PMID:24484956

  17. 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.

  18. Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

    PubMed

    Tivendale, Nathan D; Davidson, Sandra E; Davies, Noel W; Smith, Jason A; Dalmais, Marion; Bendahmane, Abdelhafid I; Quittenden, Laura J; Sutton, Lily; Bala, Raj K; Le Signor, Christine; Thompson, Richard; Horne, James; Reid, James B; Ross, John J

    2012-07-01

    Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea. PMID:22573801

  19. 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.

  20. Farnesylation mediates brassinosteroid biosynthesis to regulate abscisic acid responses.

    PubMed

    Northey, Julian G B; Liang, Siyu; Jamshed, Muhammad; Deb, Srijani; Foo, Eloise; Reid, James B; McCourt, Peter; Samuel, Marcus A

    2016-01-01

    Protein farnesylation is a post-translational modification involving the addition of a 15-carbon farnesyl isoprenoid to the carboxy terminus of select proteins(1-3). Although the roles of this lipid modification are clear in both fungal and animal signalling, many of the mechanistic functions of farnesylation in plant signalling are still unknown. Here, we show that CYP85A2, the cytochrome P450 enzyme that performs the last step in brassinosteroid biosynthesis (conversion of castasterone to brassinolide)(4), must be farnesylated to function in Arabidopsis. Loss of either CYP85A2 or CYP85A2 farnesylation results in reduced brassinolide accumulation and increased plant responsiveness to the hormone abscisic acid (ABA) and overall drought tolerance, explaining previous observations(5). This result not only directly links farnesylation to brassinosteroid biosynthesis but also suggests new strategies to maintain crop yield under challenging climatic conditions. PMID:27455172

  1. 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

  2. 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.

  3. Abscisic acid inhibits root growth in Arabidopsis through ethylene biosynthesis.

    PubMed

    Luo, Xingju; Chen, Zhizhong; Gao, Junping; Gong, Zhizhong

    2014-07-01

    When first discovered in 1963, abscisic acid (ABA) was called abscisin II because it promotes abscission. Later, researchers found that ABA accelerates abscission via ethylene. In Arabidopsis, previous studies have shown that high concentrations of ABA inhibit root growth through ethylene signaling but not ethylene production. In the present study in Arabidopsis, we found that ABA inhibits root growth by promoting ethylene biosynthesis. The ethylene biosynthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine reduces ABA inhibition of root growth, and multiple mutants of ACS (1-aminocyclopropane-1-carboxylate synthase) are more resistant to ABA in terms of root growth than the wild-type is. Two ABA-activated calcium-dependent protein kinases, CPK4 and CPK11, phosphorylate the C-terminus of ACS6 and increase the stability of ACS6 in ethylene biosynthesis. Plants expressing an ACS6 mutant that mimics the phosphorylated form of ACS6 produce more ethylene than the wild-type. Our results reveal an important mechanism by which ABA promotes ethylene production. This mechanism may be highly conserved among higher plants.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. Inhibitors of fatty acid biosynthesis in sunflower seeds.

    PubMed

    Pleite, Rafael; Martínez-Force, Enrique; Garcés, Rafael

    2006-09-01

    During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds. PMID:16500723

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

    PubMed Central

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

    2015-01-01

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

  10. Intermediates of Salicylic Acid Biosynthesis in Tobacco1

    PubMed Central

    Ribnicky, David M.; Shulaev, Vladimir; Raskin, Ilya

    1998-01-01

    Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis. PMID:9765542

  11. Fatty acid biosynthesis during the life cycle of Debaryomyces etchellsii.

    PubMed

    Arous, Fatma; Mechichi, Tahar; Nasri, Moncef; Aggelis, George

    2016-07-01

    Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased. PMID:27129978

  12. Biosynthesis of polyunsaturated fatty acids in lower eukaryotes.

    PubMed

    Uttaro, Antonio D

    2006-10-01

    Polyunsaturated fatty acids have important structural roles in cell membranes. They are also intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediate fever, inflammation, blood pressure and neurotransmission. Arachidonic and docosahexaenoic acids are essential components of brain tissues and, through their involvement in the development of neural and retinal functions, important dietary nutrients for neonatal babies. Lower eukaryotes are particularly rich in C20-22 polyunsaturated fatty acids. Fungi and marine microalgae are currently used to produce nutraceutic oils. Other protists and algae are being studied because of the variability in their enzymes involved in polyunsaturated fatty acid biosynthesis. Such enzymes could be used as source for the production of transgenic organisms able to synthesize designed oils for human diet or, in the case of parasitic protozoa, they might be identified as putative chemotherapeutic targets. Polyunsaturated fatty acids can be synthesized by two different pathways: an anaerobic one, by using polyketide synthase related enzymes, and an aerobic one, which involves the action of elongases and oxygen dependent desaturases. Desaturases can be classified into three main types, depending on which of the consecutive steps of polyunsaturated fatty acid synthesis they are involved with. The enzymes may be specialized to act on: saturated substrates (type I); mono- and di-unsaturated fatty acids by introducing additional double bonds at the methyl-end site of the existing double bonds (type II); or the carboxy half ('front-end') of polyunsaturated ones (type III). Type III desaturases require the alternating action of elongases. A description of the enzymes that have been isolated and functionally characterized is provided, in order to highlight the different pathways found in lower eukaryotes.

  13. Carnosic acid biosynthesis elucidated by a synthetic biology platform.

    PubMed

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

    2016-03-29

    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

  14. 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

  15. 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

  16. 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

  17. Biosynthesis of alkyl lysophosphatidic acid by diacylglycerol kinases.

    PubMed

    Gellett, Amanda M; Kharel, Yugesh; Sunkara, Manjula; Morris, Andrew J; Lynch, Kevin R

    2012-06-15

    Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways.

  18. Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth.

    PubMed

    Saxena, Bhawna; Subramaniyan, Mayavan; Malhotra, Karan; Bhavesh, Neel Sarovar; Potlakayala, Shobha Devi; Kumar, Shashi

    2014-03-01

    Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of 13C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.

  19. Flusilazole induces spatio-temporal expression patterns of retinoic acid-, differentiation- and sterol biosynthesis-related genes in the rat Whole Embryo Culture.

    PubMed

    Dimopoulou, Myrto; Verhoef, Aart; van Ravenzwaay, Bennard; Rietjens, Ivonne M C M; Piersma, Aldert H

    2016-09-01

    Embryotoxic responses are critically dependent on the timing of exposure during embryo development. Here, we examined the time- dependent developmental effects in rat embryos exposed to flusilazole (FLU), and their link to retinoic acid (RA) mediated pathways. To this end, we assessed the effects of 4h exposure of rat embryos in vitro to 300μM FLU during four developmental time windows (0-4, 4-8, 24-28 and 44-48h), evaluating morphological parameters, expression and localization of five genes directly or indirectly linked with the RA pathway. These were RA- (Cyp26a1 and Dhrs3), differentiation- (Gbx2 and Cdx1) and sterol biosynthesis- (Cyp51) related genes. Extended exposure for 48h to 300μM FLU resulted in morphological changes, typical for triazoles and RA, while the 4h exposure times did not. Time dependent significant upregulation of the five selected genes was observed. These results corroborate that the embryotoxic responses to FLU are correlated with the regulation of the RA pathway. Thus, these gene expression markers can be considered early biomarkers of FLU-induced potential developmental toxicity later in the development. PMID:27094377

  20. 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. PMID:27004948

  1. 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.

  2. 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.

  3. 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.

  4. Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in Escherichia coli

    PubMed Central

    Rehm, Bernd H. A.; Mitsky, Timothy A.; Steinbüchel, Alexander

    2001-01-01

    Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the β-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight. PMID:11425728

  5. 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

  6. Proteomic Signature of Fatty Acid Biosynthesis Inhibition Available for In Vivo Mechanism-of-Action Studies▿

    PubMed Central

    Wenzel, Michaela; Patra, Malay; Albrecht, Dirk; Chen, David Y.-K.; Nicolaou, K. C.; Metzler-Nolte, Nils; Bandow, Julia E.

    2011-01-01

    Fatty acid biosynthesis is a promising novel antibiotic target. Two inhibitors of fatty acid biosynthesis, platencin and platensimycin, were recently discovered and their molecular targets identified. Numerous structure-activity relationship studies for both platencin and platensimycin are currently being undertaken. We established a proteomic signature for fatty acid biosynthesis inhibition in Bacillus subtilis using platencin, platensimycin, cerulenin, and triclosan. The induced proteins, FabHA, FabHB, FabF, FabI, PlsX, and PanB, are enzymes involved in fatty acid biosynthesis and thus linked directly to the target pathway. The proteomic signature can now be used to assess the in vivo mechanisms of action of compounds derived from structure-activity relationship programs, as demonstrated for the platensimycin-inspired chromium bioorganometallic PM47. It will further serve as a reference signature for structurally novel natural and synthetic antimicrobial compounds with unknown mechanisms of action. In summary, we described a proteomic signature in B. subtilis consisting of six upregulated proteins that is diagnostic of fatty acid biosynthesis inhibition and thus can be applied to advance antibacterial drug discovery programs. PMID:21383089

  7. 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...

  8. 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.

  9. A model of proteolysis and amino acid biosynthesis for Lactobacillus delbrueckii subsp. bulgaricus in whey.

    PubMed

    Liu, Enuo; Zheng, Huajun; Hao, Pei; Konno, Tomonobu; Yu, Yao; Kume, Hisae; Oda, Munehiro; Ji, Zai-Si

    2012-12-01

    Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038) is a bacterium that is used as a starter for dairy products by Meiji Co., Ltd of Japan. Culturing L. bulgaricus 2038 with whey as the sole nitrogen source results in a shorter lag phase than other milk proteins under the same conditions (carbon source, minerals, and vitamins). Microarray results of gene expression revealed characteristics of amino acid anabolism with whey as the nitrogen source and established a model of proteolysis and amino acid biosynthesis for L. bulgaricus. Whey peptides and free amino acids are readily metabolized, enabling rapid entry into the logarithmic growth phase. The oligopeptide transport system is the primary pathway for obtaining amino acids. Amino acid biosynthesis maintains the balance between amino acids required for cell growth and the amount obtained from environment. The interconversion of amino acids is also important for L. bulgaricus 2038 growth.

  10. 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.

  11. Five Additional Genes Are Involved in Clavulanic Acid Biosynthesis in Streptomyces clavuligerus

    PubMed Central

    Jensen, S. E.; Paradkar, A. S.; Mosher, R. H.; Anders, C.; Beatty, P. H.; Brumlik, M. J.; Griffin, A.; Barton, B.

    2004-01-01

    An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for β-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster. PMID:14693539

  12. Polyunsaturated fatty acids influence differential biosynthesis of oxylipids and other lipid mediators during bovine coliform mastitis.

    PubMed

    Mavangira, Vengai; Gandy, Jeffery C; Zhang, Chen; Ryman, Valerie E; Daniel Jones, A; Sordillo, Lorraine M

    2015-09-01

    Coliform mastitis is a severe and sometimes fatal disease characterized by an unregulated inflammatory response. The initiation, progression, and resolution of inflammatory responses are regulated, in part, by potent oxylipid metabolites derived from polyunsaturated fatty acids. The purpose of this study was to characterize the biosynthesis and diversity of oxylipid metabolites during acute bovine coliform mastitis. Eleven cows diagnosed with naturally occurring acute systemic coliform mastitis and 13 healthy control cows, matched for lactation number and days in milk, were selected for comparison of oxylipid and free fatty acid concentrations in both milk and plasma. Oxylipids and free fatty acids were quantified using liquid chromatography-tandem mass spectrometry. All polyunsaturated fatty acids quantified in milk were elevated during coliform mastitis with linoleic acid being the most abundant. Oxylipids synthesized through the lipoxygenase and cytochrome P450 pathways accounted for the majority of the oxylipid biosynthesis. This study demonstrated a complex and diverse oxylipid network, most pronounced at the level of the mammary gland. Substrate availability, biosynthetic pathways, and degree of metabolism influence the biosynthesis of oxylipids during bovine coliform mastitis. Further studies are required to identify targets for novel interventions that modulate oxylipid biosynthesis during coliform mastitis to optimize inflammation.

  13. 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.

  14. 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.

  15. Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

    PubMed Central

    Guo, Chenglin; Qiu, Juanping

    2015-01-01

    Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab), L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes. PMID:26078961

  16. The biosynthesis of alginic acid by Azotobacter vinelandii.

    PubMed Central

    Pindar, D F; Bucke, C

    1975-01-01

    The sequence of reactions by which alginic acid is biosynthesized from sucrose in Azotobacter vinelandii was determined both by feeding radioactive individual enzymes involved. Results indicate that the first polymeric substance formed in the synthesis is polymannuronic acid and that mannuronic acid units are epimerized to guluronic acid at the polymer level. Guluronic acid does not appear to be formed at the monomer level, either free or in combination with GDP. PMID:179528

  17. The biosynthesis of alginic acid by Azotobacter vinelandii.

    PubMed

    Pindar, D F; Bucke, C

    1975-12-01

    The sequence of reactions by which alginic acid is biosynthesized from sucrose in Azotobacter vinelandii was determined both by feeding radioactive individual enzymes involved. Results indicate that the first polymeric substance formed in the synthesis is polymannuronic acid and that mannuronic acid units are epimerized to guluronic acid at the polymer level. Guluronic acid does not appear to be formed at the monomer level, either free or in combination with GDP.

  18. The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids.

    PubMed Central

    Qi, Q.; Kleppinger-Sparace, K. F.; Sparace, S. A.

    1995-01-01

    Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum sativum L.) root plastids. At the highest concentrations tested (3-5mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99 and 57 nmol h-1 mg-1 protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, in the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis. Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 10 and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95%) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating in pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required PMID:12228367

  19. 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.

  20. 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.

  1. 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

  2. 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. PMID:25360565

  3. Biosynthesis of 'essential' amino acids by scleractinian corals.

    PubMed Central

    Fitzgerald, L M; Szmant, A M

    1997-01-01

    Animals rely on their diet for amino acids that they are incapable either of synthesizing or of synthesizing in sufficient quantities to meet metabolic needs. These are the so-called 'essential amino acids'. This set of amino acids is similar among the vertebrates and many of the invertebrates. Previously, no information was available for amino acid synthesis by the most primitive invertebrates, the Cnidaria. The purpose of this study was to examine amino acid synthesis by representative cnidarians within the Order Scleractinia. Three species of zooxanthellate reef coral, Montastraea faveolata, Acropora cervicornis and Porites divaricata, and two species of non-zooxanthellate coral, Tubastrea coccinea and Astrangia poculata, were incubated with 14C-labelled glucose or with the 14C-labelled amino acids glutamic acid, lysine or valine. Radiolabel tracer was followed into protein amino acids. A total of 17 amino acids, including hydroxyproline, were distinguishable by the techniques used. Of these, only threonine was not found radiolabelled in any of the samples. We could not detect tryptophan or cysteine, nor distinguish between the amino acid pairs glutamic acid and glutamine, or aspartic acid and asparagine. Eight amino acids normally considered essential for animals were made by the five corals tested, although some of them were made only in small quantities. These eight amino acids are valine, isoleucine, leucine, tyrosine, phenylalanine histidine, methionine and lysine. The ability of cnidarians to synthesize these amino acids could be yet another indicator of a separate evolutionary history of the cnidarians from the rest of the Metazoa. PMID:9078264

  4. Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants.

    PubMed

    Rogalski, Marcelo; Carrer, Helaine

    2011-06-01

    The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.

  5. l-Ascorbic Acid Biosynthesis in Ochromonas danica1

    PubMed Central

    Helsper, Johannes P.; Kagan, Lea; Hilby, Coral L.; Maynard, Tracy M.; Loewus, Frank A.

    1982-01-01

    Ochromonas danica Pringsheim, a freshwater chrysomonad, converts d-glucose into l-ascorbic acid over a metabolic pathway that `inverts' the carbon chain of the sugar. In this respect, l-ascorbic acid formation resembles that found in ascorbic acid-synthesizing animals. It differs from this process in that d-galacturonate and l-galactono-1,4-lactone, rather than d-glucuronate and l-gulono-1,4-lactone, enhance production of ascorbic acid and repress the incorporation of 14C from d-[1-14C]glucose into ascorbic acid. PMID:16662230

  6. Precursor directed biosynthesis of odd-numbered fatty acids by different yeasts.

    PubMed

    Řezanka, Tomáš; Kolouchová, Irena; Sigler, Karel

    2015-09-01

    Precursor-directed biosynthesis was used for directed preparation of positional isomers of heptadecanoic acid (17:1), which have convenient pharmacological properties. Cultivation of Candida sp., Kluyveromyces polysporus, Rhodotorula glutinis, Saccharomyces cerevisiae, Torulaspora delbrueckii, Trichosporon cutaneum, and Yarrowia lipolytica on 20 g/L glucose, 4 g/L acetic, or 4 g/L propionic acids yielded different proportions of 17:1. Cultivation on carbon sources with even numbers of carbon atoms (glucose and acetic acid) produced preferentially 8Z- and 10Z-heptadecenoic acids in about equal amounts, in agreement with the proposed biosynthesis of fatty acids, whereas cultivation on propionic acid as the only carbon source produced over 90 % of total fatty acids of 9-17:1 out of all possible positional isomers. The structures of positional isomers of 17:1 acid were determined using dimethyl disulfides of fatty acid methyl esters. In cultivation of Candida sp. on propionic acid, the yield of heptadecenoic acid reached 111 mg/L cultivation medium. Principal component analysis was used for identifying the effect of cultivation conditions on the production of the 17:1 acid by individual yeast strains. PMID:25813199

  7. Biosynthesis of vanillin via ferulic acid in Vanilla planifolia.

    PubMed

    Negishi, Osamu; Sugiura, Kenji; Negishi, Yukiko

    2009-11-11

    (14)C-Labeled phenylalanine, 4-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, ferulic acid, and methionine were applied to disks of green vanilla pods 3 and 6 months after pollination (immature and mature pods), and the conversion of these compounds to vanillin or glucovanillin was investigated. In mature green vanilla pods, radioactivities of 11, 15, 29, and 24% from (14)C-labeled phenylalanine, 4-coumaric acid, ferulic acid, and methionine, respectively, were incorporated into glucovanillin within 24 h. In the incorporation processes of methionine and phenylalanine into glucovanillin, some of the (14)C labels were also trapped by the unlabeled ferulic acid. However, (14)C-labeled 4-hydroxybenzaldehyde and 4-hydroxybenzyl alcohol were not converted to glucovanillin. On the other hand, in immature green vanilla pods radioactivities of the above six compounds were not incorporated into glucovanillin. Although 4-coumaric acid, ferulic acid, 4-hydroxybenzaldehyde, and 4-hydroxybenzyl alcohol were converted to the respective glucose esters or glucosides and vanillin was converted to glucovanillin, their conversions were believed to be from the detoxication of the aglycones. These results suggest that the biosynthetic pathway for vanillin is 4-coumaric acid --> --> ferulic acid --> --> vanillin --> glucovanillin in mature vanilla pods.

  8. Mechanism of lysergic acid diethylamide interference with rabbit antibody biosynthesis.

    PubMed

    Voss, E W; Winkelhake, J L

    1974-04-01

    Lymphoid cells from hyperimmune rabbits producing antibodies to a hapten, incubated in the presence of d-lysergic acid diethylamide, continued to synthesize protein at a normal rate. Isoelectric focusing analysis of the low-molecular-weight protein secreted by the cells incubated with lysergic acid diethylamide indicated two components, with pI's of 4.9 and 5.2. Immune cells not exposed to lysergic acid diethylamide secreted only 7S IgG molecules with an average pI of approximately 7.0.

  9. Ursolic acid from Plantago major, a selective inhibitor of cyclooxygenase-2 catalyzed prostaglandin biosynthesis.

    PubMed

    Ringbom, T; Segura, L; Noreen, Y; Perera, P; Bohlin, L

    1998-10-01

    A hexane extract of Plantago major was investigated by bioactivity-directed fractionation, using an in vitro cyclooxygenase-2 (COX-2) catalyzed prostaglandin biosynthesis inhibition assay, and resulted in the isolation of ursolic acid (1). This triterpenoid showed a significant COX-2 inhibitory effect, directly on the enzyme activity, with an IC50 value of 130 microM and a COX-2/COX-1 selectivity ratio of 0.6. The structural isomer oleanolic acid (2) was found to be less active than 1, with an IC50 value of 295 microM, but showed a similar selectivity ratio (0.8). Furthermore, no significant inhibition on COX-2 or COX-1 was observed by the triterpenoid, 18beta-glycyrrhetinic acid (3). The direct inhibitory effect of 1 and 2 on COX-2 catalyzed prostaglandin biosynthesis increased with preincubation, indicating a time-dependent inhibition, while the effect on COX-1 was found to be independent of preincubation time.

  10. 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

  11. 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.

  12. The biosynthesis of erucic acid in developing embryos of brassica rapa

    PubMed

    Bao; Pollard; Ohlrogge

    1998-09-01

    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-pyridinyloxyphenoxy] 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-pyridinyloxyphenoxy]-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.

  13. 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.

  14. Coordination of glycerol utilization and clavulanic acid biosynthesis to improve clavulanic acid production in Streptomyces clavuligerus.

    PubMed

    Guo, Dekun; Zhao, Youbao; Yang, Keqian

    2013-07-01

    The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyl operon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaR expressed from Pgyl (the gyl promoter) was integrated into the chromosome of S. clavuligerus NRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyl operon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaR controlled by its own promoter (PccaR). Consistent with enhanced CA production, the transcription levels of ccaR, ceas2 and claR were significantly up-regulated in the transformants containing Pgyl-controlled ccaR.

  15. Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

    PubMed Central

    McDonald, Roderick A.; Satyanarayana, T.; Kaplan, J. G.

    1973-01-01

    The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (Ki = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system. PMID:4698210

  16. Biosynthesis and Elongation of Short- and Medium-Chain-Length Fatty Acids

    PubMed Central

    van der Hoeven, Rutger S.; Steffens, John C.

    2000-01-01

    Short- and medium-chain-length fatty acids (FAs) are important constituents of a wide array of natural products. Branched and straight short-chain-length FAs originate from branched chain amino acid metabolism, and serve as primers for elongation in FA synthase-like reactions. However, a recent model proposes that the one-carbon extension reactions that utilize 2-oxo-3-methylbutyric acid in leucine biosynthesis also catalyze a repetitive one-carbon elongation of short-chain primers to medium-chain-length FAs. The existence of such a mechanism would require a novel form of regulation to control carbon flux between amino acid and FA biosynthesis. A critical re-analysis of the data used to support this pathway fails to support the hypothesis for FA elongation by one-carbon extension cycles of α-ketoacids. Therefore, we tested the hypothesis experimentally using criteria that distinguish between one- and two-carbon elongation mechanisms: (a) isotopomer patterns in terminal carbon atom pairs of branched and straight FAs resulting from differential labeling with [13C]acetate; (b) [13C]threonine labeling patterns in odd- and even chain length FAs; and (c) differential sensitivity of elongation reactions to inhibition by cerulenin. All three criteria indicated that biosynthesis of medium-chain length FAs is mediated primarily by FA synthase-like reactions. PMID:10631271

  17. Biosynthesis of jasmonic acid in a plant pathogenic fungus, Lasiodiplodia theobromae.

    PubMed

    Tsukada, Kohei; Takahashi, Kosaku; Nabeta, Kensuke

    2010-12-01

    Jasmonic acid (JA) is a plant hormone that plays an important role in a wide variety of plant physiological processes. The plant pathogenic fungus, Lasiodiplodia theobromae also produces JA; however, its biosynthesis in this fungus has yet to be explored. Administration of [1-(13)C] and [2-(13)C] NaOAc into L. theobromae established that JA in this fungus originates from a fatty acid synthetic pathway. The methyl ester of 12-oxo-phytodienoic acid (OPDA) was detected in the culture extracts of L. theobromae by GC-MS analysis. This finding indicates the presence of OPDA (a known intermediate of JA biosynthesis in plants) in L. theobromae. (2)H NMR spectroscopic data of JA produced by L. theobromae with the incorporation of [9,10,12,13,15,16-(2)H(6)] linolenic acid showed that five deuterium atoms remained intact. In plants, this is speculated to arise from JA being produced by the octadecanoid pathway. However, the observed stereoselectivity of the cyclopentenone olefin reduction in L. theobromae was opposite to that observed in plants. These data suggest that JA biosynthesis in L. theobromae is similar to that in plants, but differing in the facial selectivity of the enone reduction. PMID:20952041

  18. Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters 1

    PubMed Central

    Walters, Donald S.; Steffens, John C.

    1990-01-01

    Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O-acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C4 and C5 acids, and branched and straight chain C10, C11, and C12 acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [14C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C4 and C5 acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C4 and C5 branched acids were elongated by two carbon units to produce the branched C10-C12 groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters. PMID:16667654

  19. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions.

    PubMed

    Muñoz-Espinoza, Valeria A; López-Climent, María F; Casaretto, José A; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  20. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions

    PubMed Central

    Muñoz-Espinoza, Valeria A.; López-Climent, María F.; Casaretto, José A.; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  1. 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.

  2. 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.

  3. Acquisition and biosynthesis of saturated and unsaturated fatty acids by trypanosomatids.

    PubMed

    Uttaro, Antonio D

    2014-08-01

    As components of phospholipids and glycosylphosphatidylinositol anchors, fatty acids are responsible for forming the core of biological membranes and the correct localization of proteins within membranes. They also contribute to anchoring proteins by direct acylation of specific amino acids. Fatty acids can be used as energy sources and serve as signaling molecules or precursors for their synthesis. All these processes highlight the important role of fatty acids in cell physiology, justifying the diverse strategies for their acquisition evolved by different organisms. This review describes several recent findings in the salvage and biosynthesis of fatty acids by parasitic protists belonging to the class Kinetoplastea. They include two biosynthetic routes, the mitochondrial one and a peculiar membrane-associated pathway, the synthesis of polyunsaturated fatty acids, and the scavenging of lysophospholipids and lipoproteins from host plasma. These different processes are also explored as putative targets for chemotherapy.

  4. 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.

  5. Traumatic Acid Reduces Oxidative Stress and Enhances Collagen Biosynthesis in Cultured Human Skin Fibroblasts.

    PubMed

    Jabłońska-Trypuć, Agata; Pankiewicz, Walentyn; Czerpak, Romuald

    2016-09-01

    Traumatic acid (TA) is a plant hormone (cytokinin) that in terms of chemical structure belongs to the group of fatty acids derivatives. It was isolated from Phaseolus vulgaris. TA activity and its influence on human cells and organism has not previously been the subject of research. The aim of this study was to examine the effects of TA on collagen content and basic oxidative stress parameters, such as antioxidative enzyme activity, reduced glutathione, thiol group content, and lipid peroxidation in physiological conditions. The results show a stimulatory effect of TA on tested parameters. TA caused a decrease in membrane phospholipid peroxidation and exhibited protective properties against ROS production. It also increases protein and collagen biosynthesis and its secretion into the culture medium. The present findings reveal that TA exhibits multiple and complex activity in fibroblast cells in vitro. TA, with its activity similar to unsaturated fatty acids, shows antioxidant and stimulatory effects on collagen biosynthesis. It is a potentially powerful agent with applications in the treatment of many skin diseases connected with oxidative stress and collagen biosynthesis disorders. PMID:27423205

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

    PubMed

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

    2011-12-27

    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 (V(max)/k(m)) 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.

  7. 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.

  8. Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in Streptomyces clavuligerus

    PubMed Central

    Jensen, Susan E.; Elder, Kenneth J.; Aidoo, Kwamena A.; Paradkar, Ashish S.

    2000-01-01

    Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a “supercluster” in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway. PMID:10681345

  9. 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).

  10. Gene-Enzyme Relationships of Aromatic Amino Acid Biosynthesis in Higher Plants

    SciTech Connect

    2002-08-12

    Inhibition studies of amino acids in Nicotiana silvestris suspension cells gave clues to the difficulties for obtaining mutants deficient in post prephenate pathway proteins of aromatic amino acid biosynthesis (prephenate aminotransferase, arogenate dehydrogenase and arogenate dehydratase). Such mutants, if successfully obtained, would allow gene-enzyme relationships of aromatic amino acid proteins to be studied. We found that amino acids were inhibitory toward plant cell growth, and thus were unable to rescue analog resistant mutants. Toxicity of all amino acids toward exponentially dividing Nicotiana silvestris suspension cultured cells was monitored by following growth rates. Except for L-glutamine, all 19 protein amino acids inhibited cell growth. Inhibition of growth progressed to cell deterioration. Electron microscopy showed that amino acids triggered a state of cell shrinkage that eventually degenerated to total cellular disorganization. L-glutamine was not only an effective agent for prevention of amino acid toxicity, but enhanced the final growth yield. L-glutamine also was able to completely reverse inhibition effects in cells that had been in the slowed exponential phase. Two types of inhibition occurred and we have proposed that any amino acid inhibition that can be completely antagonized by L-glutamine be called ''general amino acid inhibition''. ''Specific amino acid inhibition'' resulting from particular pathway imbalances caused by certain exogenous amino acids, can be recognized and studied in the presence of L-glutamine which can abolishes the complication effects of general amino acid inhibition.

  11. Regulation of polyunsaturated fatty acid biosynthesis by seaweed fucoxanthin and its metabolite in cultured hepatocytes.

    PubMed

    Aki, Tsunehiro; Yamamoto, Masaya; Takahashi, Toshiaki; Tomita, Kohki; Toyoura, Rieko; Iwashita, Kazuhiro; Kawamoto, Seiji; Hosokawa, Masashi; Miyashita, Kazuo; Ono, Kazuhisa

    2014-02-01

    The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on ∆6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin. PMID:24174374

  12. Regulation of polyunsaturated fatty acid biosynthesis by seaweed fucoxanthin and its metabolite in cultured hepatocytes.

    PubMed

    Aki, Tsunehiro; Yamamoto, Masaya; Takahashi, Toshiaki; Tomita, Kohki; Toyoura, Rieko; Iwashita, Kazuhiro; Kawamoto, Seiji; Hosokawa, Masashi; Miyashita, Kazuo; Ono, Kazuhisa

    2014-02-01

    The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on ∆6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin.

  13. Sulfuric acid vapor in the atmosphere of Venus as observed by the Venus Express Radio Science experiment VeRa

    NASA Astrophysics Data System (ADS)

    Oschlisniok, Janusz; Pätzold, Martin; Häusler, Bernd; Tellmann, Silvia; Bird, Mike; Andert, Tom

    2016-04-01

    The cloud deck within Venus' atmosphere, which covers the entire planet between approx. 50 and 70 km altitude, consists mostly of liquid and gaseous sulfuric acid. The gaseous part increases strongly just below the main clouds and builds an approx. 15 km thick haze layer of H2SO4. This region is responsible for a strong absorption of radio waves as seen in VeRa radio science observations. The amount of the absorption, which is used to derive the abundance of gaseous sulfuric acid, depends on the signal frequency. VeRa probed the atmosphere of Venus between 2006 and 2015 with radio signals at 13 cm (S-band) and 3.6 cm (X-band) wavelengths. We present H2SO4 profiles derived from S-band and X-band absorption during the first occultation season in 2006. The comparison of the H2SO4 profiles derived from both frequency bands provides a reliable picture of the H2SO4 abundance. Distinct differences in the S- and X-band profiles may give a clue to increased SO2 abundances. The derived VeRa results shall be compared with results provided by other experiments onboard Venus Express as well as with previous missions.

  14. Ferulic acid, an efficient inhibitor of type B trichothecene biosynthesis and Tri gene expression in Fusarium liquid cultures.

    PubMed

    Boutigny, Anne-Laure; Barreau, Christian; Atanasova-Penichon, Vessela; Verdal-Bonnin, Marie-Noëlle; Pinson-Gadais, Laëtitia; Richard-Forget, Florence

    2009-01-01

    The effect of ferulic acid, the most abundant phenolic acid in wheat bran, was studied in vitro on type B trichothecene biosynthesis by Fusarium. It was demonstrated that ferulic acid is an efficient inhibitor of mycotoxin production by all strains of Fusarium tested, including different chemotypes and species. To analyse the mechanism of toxin biosynthesis inhibition by ferulic acid, expression of representative Tri genes, involved in the trichothecene biosynthesis pathway, was monitored by real-time RT-PCR. A decrease in the level of Tri gene expression was measured, suggesting that inhibition of toxin synthesis by ferulic acid could be regulated at the transcriptional level. Moreover, toxin production was shown to be reduced proportionally to the initial amount of ferulic acid added in the culture medium. Addition of ferulic acid either at the spore germination step or to a mycelial culture resulted in the same final inhibitory effect on mycotoxin accumulation. A cumulative inhibitory effect on trichothecene biosynthesis was even observed with successive supplementation of ferulic acid. Ferulic acid, which content varies among wheat varieties, could then play an important role in modulating trichothecene biosynthesis by Fusarium in some wheat varieties.

  15. Biosynthesis of the Halogenated Auxin, 4-Chloroindole-3-Acetic Acid1[W][OA

    PubMed Central

    Tivendale, Nathan D.; Davidson, Sandra E.; Davies, Noel W.; Smith, Jason A.; Dalmais, Marion; Bendahmane, Abdelhafid I.; Quittenden, Laura J.; Sutton, Lily; Bala, Raj K.; Le Signor, Christine; Thompson, Richard; Horne, James; Reid, James B.; Ross, John J.

    2012-01-01

    Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea. PMID:22573801

  16. Abscisic acid biosynthesis in water-stressed leaves

    SciTech Connect

    Li, Yi.

    1989-01-01

    Although abscisic acid (ABA) was discovered 30 years ago, very little is known about its biosynthetic pathway in higher plants. Two hypotheses have been proposed: (i) a direct pathway involving only C-15 intermediates like farnesyl pyrophosphate, (ii) an indirect pathway involving C-40 intermediates like the xanthophylls. When {sup 14}CO{sub 2} was fed into greened bean plants, the {sup 14}C specific activity of ABA was always lower than those in xanthophylls, such as violaxanthin and lutein, regardless of {sup 12}CO{sub 2} chase periods. The ABA accumulation in green leaves was not affected by fluridone when plants were stressed once, but the {sup 14}C incorporation into ABA was inhibited to the same extent as those of xanthophylls. The incorporation of {sup 18}O into the ABA ring when violaxanthin was labeled by {sup 18}O in vivo via the violaxanthin cycle indicates that at least a portion of ABA was derived from {sup 18}O-labeled violaxanthin during water stress.

  17. D-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation.

    PubMed

    Zhang, Yixing; Vadlani, Praveen V

    2013-12-01

    Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly L-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-L and D-lactic acid and has a higher melting temperature. To date, several studies have explored the production of L-lactic acid, but information on biosynthesis of D-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of D-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to D-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L(-1) of D-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g(-1) and 1.01 g L(-1) h(-1), respectively. Luedeking-Piret model described the mixed growth-associated production of D-lactic acid with a maximum specific growth rate 0.2 h(-1) and product formation rate 0.026 h(-1), obtained for this strain. The efficient synthesis of D-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.

  18. 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. PMID:25339514

  19. 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.

  20. Suppression of Spermatogenesis by Bisdichloroacetyldiamines Is Mediated by Inhibition of Testicular Retinoic Acid Biosynthesis

    PubMed Central

    Amory, John K.; Muller, Charles H.; Shimshoni, Jakob A.; Isoherranen, Nina; Paik, Jisun; Moreb, Jan S.; Amory, David W.; Evanoff, Ryan; Goldstein, Alex S.; Griswold, Michael D.

    2012-01-01

    The bisdichloroacetyldiamine WIN 18,446 reversibly inhibits spermatogenesis in many species, including humans; however, the mechanism by which WIN 18,446 functions is unknown. As retinoic acid is essential for spermatogenesis, we hypothesized that WIN 18,446 might inhibit retinoic acid biosynthesis from retinol (vitamin A) within the testes by inhibiting the enzyme aldehyde dehydrogenase 1a2 (ALDH1a2). We studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro, and on spermatogenesis and fertility in vivo, in mature male rabbits for 16 weeks. WIN 18,446 markedly inhibited ALDH1a2 enzyme activity in vitro with an IC50 of 0.3 μM. In vivo, the oral administration of 200 mg/kg WIN 18,446 to male rabbits for 16 weeks significantly reduced intratesticular concentrations of retinoic acid, severely impaired spermatogenesis, and caused infertility. Reduced concentrations of intratesticular retinoic acid were apparent after only 4 weeks of treatment and preceded the decrease in sperm counts and the loss of mature germ cells in tissue samples. Sperm counts and fertility recovered after treatment was discontinued. These findings demonstrate that bisdichloroacetyldiamines such as WIN 18,446 reversibly suppress spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2. These findings suggest that ALDH1a2 is a promising target for the development of a reversible, nonhormonal male contraceptive. PMID:20705791

  1. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

    PubMed

    Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J; Ivison, David; Généreux, Catherine; Dubus, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J; Lloyd, Adrian J; Roper, David I; Schofield, Christopher J; Andersson, Inger; McDonough, Michael A

    2013-08-01

    Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

  2. [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.

  3. [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. PMID:26087546

  4. Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a psychrotrophic bacterium

    SciTech Connect

    Wada, M.; Fukunaga, N.; Sasaki, S. )

    1989-08-01

    Biosynthesis of palmitic, palmitoleic, and cis-vaccenic acids in Pseudomonas sp. strain E-3 was investigated with in vitro and in vivo systems. (1-{sup 14}C)palmitic acid was aerobically converted to palmitoleate and cis-vaccenate, and the radioactivities on their carboxyl carbons were 100 and 43%, respectively, of the total radioactivity in the fatty acids. Palmitoyl coenzyme A desaturase activity was found in the membrane fraction. (1-{sup 14}C)stearic acid was converted to octadecenoate and C16 fatty acids. The octadecenoate contained oleate and cis-vaccenate, but only oleate was produced in the presence of cerulenin. (1-{sup 14}C)lauric acid was aerobically converted to palmitate, palmitoleate, and cis-vaccenate. Under anaerobic conditions, palmitate (62%), palmitoleate (4%), and cis-vaccenate (34%) were produced from (1-{sup 14}C)acetic acid, while they amounted to 48, 39, and 14%, respectively, under aerobic conditions. In these incorporation experiments, 3 to 19% of the added radioactivity was detected in released {sup 14}CO{sub 2}, indicating that part of the added fatty acids were oxidatively decomposed. Partially purified fatty acid synthetase produced saturated and unsaturated fatty acids with chain lengths of C10 to C18. These results indicated that both aerobic and anaerobic mechanisms for the synthesis of unsaturated fatty acid are operating in this bacterium.

  5. [Effect of organic acids on the biosynthesis of macrotetralide antibiotics by an Actinomyces chrysomallus var. carotenoides strain].

    PubMed

    Nefelova, M V; Sverdlova, A N; Silaev, A B

    1978-07-01

    The biosynthesis of macrotetrolides by Actinomyces chrysomalus var. carotenoides was stimulated by acetic, succinic, propionic, oxalic, malic, tartaric, citric, pyruvic, alpha-ketoglutaric and fumaric acids. Incorporation of 14C-acetate into the molecule of the antibiotic and the data on dependence of the stimulating effect upon the quantitative ratio and time of the organic acid addition were indicative of the role of acetic, succinic and propionic acids as precursors of macrotetrolides. The other organic acids increased the biosynthesis of macrotetolides when added to the culture within wide time ranges of the culture development and prolonged the period of the mycelium productive state.

  6. 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. PMID:25447786

  7. Role of pipecolic acid in the biosynthesis of lysine in Rhodotorula glutinis.

    PubMed

    Kinzel, J J; Bhattacharjee, J K

    1979-05-01

    The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, lys3, and lys5; alpha-aminoadipic acid supported the growth of lys5; but neither alpha-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intracellular pool. In tracer experiments, radioactivity from [(14)C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. l-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lys5 enzyme in the presence of glutamate and reduced nicotin-amide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of alpha-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as alpha-aminoadipic-semialdehyde, which in solution remains in equilibrium with Delta(1)-piperideine-6-carboxylic acid. Since alpha-aminoadipic-semialdehyde is a known intermediate of the alpha-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic

  8. 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

  9. 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.

  10. Regulation of Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: Isolation of Regulatory Mutants

    PubMed Central

    Calvo, J. M.; Freundlich, M.; Umbarger, H. E.

    1969-01-01

    5′,5′,5′-Trifluoro-dl-leucine inhibited the activity of α-isopropylmalate synthetase (the initial enzyme unique to leucine biosynthesis) as well as the growth of Salmonella typhimurium. Mutants of S. typhimurium resistant to the analogue were isolated and characterized. In most cases, they overproduced and excreted leucine or leucine, valine, and isoleucine as a result of an alteration in the regulation of branched-chain amino acid biosynthesis. Biochemical and genetic tests allowed the mutants to be grouped into three classes: I, a moderately large group (13%) which had high, constitutive leucine biosynthetic enzyme levels and mutant sites linked to the leucine operon (operator constitutive); II, a single mutant in which the mutant site was linked to the leucine operon and in which α-isopropylmalate synthetase was not inhibited by leucine (feedback negative); III, a majority type which had constitutive levels of leucine, valine, and isoleucine biosynthetic enzymes and mutant sites unlinked to the leucine operon. Mutants of class I provide important evidence for the concept of an operon organization of genes involved in leucine biosynthesis. The properties of class III mutants indicate that there is some element involved in regulation which is common to the three pathways. Images PMID:4887507

  11. Nitric oxide metabolism and indole acetic acid biosynthesis cross-talk in Azospirillum brasilense SM.

    PubMed

    Koul, Vatsala; Tripathi, Chandrakant; Adholeya, Alok; Kochar, Mandira

    2015-04-01

    Production of nitric oxide (NO) and the presence of NO metabolism genes, nitrous oxide reductase (nosZ), nitrous oxide reductase regulator (nosR) and nitric oxide reductase (norB) were identified in the plant-associated bacterium (PAB) Azospirillum brasilense SM. NO presence was confirmed in all overexpressing strains, while improvement in the plant growth response of these strains was mediated by increased NO and indole-3-acetic acid (IAA) levels in the strains. Electron microscopy showed random distribution to biofilm, with surface colonization of pleiomorphic Azospirilla. Quantitative IAA estimation highlighted a crucial role of nosR and norBC in regulating IAA biosynthesis. The NO quencher and donor reduced/blocked IAA biosynthesis by all strains, indicating their common regulatory role in IAA biosynthesis. Tryptophan (Trp) and l-Arginine (Arg) showed higher expression of NO genes tested, while in the case of ipdC, only Trp and IAA increased expression, while Arg had no significant effect. The highest nosR expression in SMnosR in the presence of IAA and Trp, along with its 2-fold IAA level, confirmed the relationship of nosR overexpression with Trp in increasing IAA. These results indicate a strong correlation between IAA and NO in A. brasilense SM and suggest the existence of cross-talk or shared signaling mechanisms in these two growth regulators.

  12. 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

  13. 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

  14. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

    PubMed Central

    Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

    2015-01-01

    ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many

  15. Biosynthesis of Polyunsaturated Fatty Acids in the Oleaginous Marine Diatom Fistulifera sp. Strain JPCC DA0580

    PubMed Central

    Liang, Yue; Maeda, Yoshiaki; Sunaga, Yoshihiko; Muto, Masaki; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

    2013-01-01

    Studies of polyunsaturated fatty acid (PUFA) biosynthesis in microalgae are of great importance for many reasons, including the production of biofuel and variable omega 3-long chain PUFAs. The elucidation of the PUFA biosynthesis pathway is necessary for bioengineering to increase or decrease PUFA content in certain microalgae. In this study, we identified the PUFA synthesis pathway in the oleaginous marine diatom, Fistulifera sp. strain JPCC DA0580, a promising candidate for biodiesel production. The data revealed not only the presence of the desaturases and elongases involved in eicosapentaenoic acid (EPA) synthesis, but also the unexpected localization of ω3-desaturase expression in the chloroplast. This suggests that this microalga might perform the final step of EPA synthesis in the chloroplast and not in the endoplasmic reticulum (ER) like other diatoms. The detailed fatty acid profile suggests that the EPA was synthesized only through the ω6-pathway in this strain, which was also different from other diatoms. Finally, the transcriptome analysis demonstrated an overall down-regulation of desaturases and elongases over incubation time. These genetic features might explain the decrease of PUFA percentage over incubation time in this strain. The important insights into metabolite synthesis acquired here will be useful for future metabolic engineering to control PUFA content in this diatom. PMID:24335525

  16. 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

  17. 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.

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

    PubMed

    Kandra, L; Wagner, G J

    1990-11-01

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

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

    PubMed Central

    Kandra, Lili; Wagner, George J.

    1990-01-01

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

  20. 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

    PubMed

    Salim, Vonny; Wiens, Brent; Masada-Atsumi, Sayaka; Yu, Fang; De Luca, Vincenzo

    2014-05-01

    Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds.

  1. 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

    PubMed

    Salim, Vonny; Wiens, Brent; Masada-Atsumi, Sayaka; Yu, Fang; De Luca, Vincenzo

    2014-05-01

    Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds. PMID:24594312

  2. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

    PubMed Central

    Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

    2015-01-01

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

  3. 40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new...

  4. 40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new...

  5. Metazoan remaining genes for essential amino acid biosynthesis: sequence conservation and evolutionary analyses.

    PubMed

    Costa, Igor R; Thompson, Julie D; Ortega, José Miguel; Prosdocimi, Francisco

    2014-12-24

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  6. 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

  7. Conservation of the 2-keto-3-deoxymanno-octulosonic acid (Kdo) biosynthesis pathway between plants and bacteria.

    PubMed

    Smyth, Kevin M; Marchant, Alan

    2013-10-18

    The increasing prevalence of multi-drug resistant bacteria is driving efforts in the development of new antibacterial agents. This includes a resurgence of interest in the Gram-negative bacteria lipopolysaccharide (LPS) biosynthesis enzymes as drug targets. The six carbon acidic sugar 2-keto-3-deoxymanno-octulosonic acid (Kdo) is a component of the lipid A moiety of the LPS in Gram-negative bacteria. In most cases the lipid A substituted by Kdo is the minimum requirement for cell growth, thus presenting the possibility of targeting either the synthesis or incorporation of Kdo for the development of antibacterial agents. Indeed, potent in vitro inhibitors of Kdo biosynthesis enzymes have been reported but have so far failed to show sufficient in vivo action against Gram-negative bacteria. As part of an effort to design more potent antibacterial agents targeting Kdo biosynthesis, the crystal structures of the key Kdo biosynthesis enzymes from Escherichia coli have been solved and their structure based mechanisms characterized. In eukaryotes, Kdo is found as a component of the pectic polysaccharide rhamnogalacturonan II in the plant primary cell wall. Interestingly, despite incorporating Kdo into very different macromolecules the Kdo biosynthesis and activation pathway is almost completely conserved between plants and bacteria. This raises the possibility for plant research to exploit the increasingly detailed knowledge and resources being generated by the microbiology community. Likewise, insights into Kdo biosynthesis in plants will be potentially useful in efforts to produce new antimicrobial compounds.

  8. All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

    PubMed Central

    Varani, J; Perone, P; Griffiths, C E; Inman, D R; Fligiel, S E; Voorhees, J J

    1994-01-01

    Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status. Images PMID:7962521

  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. Biosynthesis of indole-3-acetic acid via the indole-3-acetamide pathway in Streptomyces spp.

    PubMed

    Manulis, S; Shafrir, H; Epstein, E; Lichter, A; Barash, I

    1994-05-01

    Various Streptomyces spp. including S. violaceus, S. scabies, S. griseus, S. exfoliatus, S. coelicolor and S. lividans secrete indole-3-acetic acid (IAA) when fed with L-tryptophan (Trp). Production of IAA was detected in Streptomyces strains causing potato scab as well as in non-pathogenic strains. The pathways for IAA synthesis from Trp were investigated in S. violaceus and S. exfoliatus. Indole-3-acetamide (IAM), indole-3-lactic acid (ILA), indole-3-ethanol (IEt) and IAA were identified by HPLC and GC-MS. Streptomyces cells were capable of catabolizing IAM, ILA, IEt and indole-3-acetaldehyde (IAAId) into IAA. Incorporation of radioactivity into IAM, IAA and ILA but not IEt was detected when cells were fed with L-[3-14C]tryptophan. Results indicate the presence of the IAM pathway (Trp-->IAM-->IAA) and the possible presence of additional pathways for IAA biosynthesis in Streptomyces. PMID:8025670

  11. 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.

  12. Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from Jeotgalicoccus Species ▿ †

    PubMed Central

    Rude, Mathew A.; Baron, Tarah S.; Brubaker, Shane; Alibhai, Murtaza; Del Cardayre, Stephen B.; Schirmer, Andreas

    2011-01-01

    Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleTJE), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleTJE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family. PMID:21216900

  13. Heme biosynthesis modulation via δ-aminolevulinic acid administration attenuates chronic hypoxia-induced pulmonary hypertension

    PubMed Central

    Alhawaj, Raed; Patel, Dhara; Kelly, Melissa R.; Sun, Dong

    2015-01-01

    This study examines how heme biosynthesis modulation with δ-aminolevulinic acid (ALA) potentially functions to prevent 21-day hypoxia (10% oxygen)-induced pulmonary hypertension in mice and the effects of 24-h organoid culture with bovine pulmonary arteries (BPA) with the hypoxia and pulmonary hypertension mediator endothelin-1 (ET-1), with a focus on changes in superoxide and regulation of micro-RNA 204 (miR204) expression by src kinase phosphorylation of signal transducer and activator of transcription-3 (STAT3). The treatment of mice with ALA attenuated pulmonary hypertension (assessed through echo Doppler flow of the pulmonary valve, and direct measurements of right ventricular systolic pressure and right ventricular hypertrophy), increases in pulmonary arterial superoxide (detected by lucigenin), and decreases in lung miR204 and mitochondrial superoxide dismutase (SOD2) expression. ALA treatment of BPA attenuated ET-1-induced increases in mitochondrial superoxide (detected by MitoSox), STAT3 phosphorylation, and decreases in miR204 and SOD2 expression. Because ALA increases BPA protoporphyrin IX (a stimulator of guanylate cyclase) and cGMP-mediated protein kinase G (PKG) activity, the effects of the PKG activator 8-bromo-cGMP were examined and found to also attenuate the ET-1-induced increase in superoxide. ET-1 increased superoxide production and the detection of protoporphyrin IX fluorescence, suggesting oxidant conditions might impair heme biosynthesis by ferrochelatase. However, chronic hypoxia actually increased ferrochelatase activity in mouse pulmonary arteries. Thus, a reversal of factors increasing mitochondrial superoxide and oxidant effects that potentially influence remodeling signaling related to miR204 expression and perhaps iron availability needed for the biosynthesis of heme by the ferrochelatase reaction could be factors in the beneficial actions of ALA in pulmonary hypertension. PMID:25659899

  14. Insights into the Biosynthesis of 12-Membered Resorcylic Acid Lactones from Heterologous Production in Saccharomyces cerevisiae

    PubMed Central

    2015-01-01

    The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed “stuttering” by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions. PMID:24597618

  15. Insights into the biosynthesis of 12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae.

    PubMed

    Xu, Yuquan; Zhou, Tong; Espinosa-Artiles, Patricia; Tang, Ying; Zhan, Jixun; Molnár, István

    2014-05-16

    The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed "stuttering" by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions. PMID:24597618

  16. Azospirillum brasilense Produces the Auxin-Like Phenylacetic Acid by Using the Key Enzyme for Indole-3-Acetic Acid Biosynthesis

    PubMed Central

    Somers, E.; Ptacek, D.; Gysegom, P.; Srinivasan, M.; Vanderleyden, J.

    2005-01-01

    An antimicrobial compound was isolated from Azospirillum brasilense culture extracts by high-performance liquid chromatography and further identified by gas chromatography-mass spectrometry as the auxin-like molecule, phenylacetic acid (PAA). PAA synthesis was found to be mediated by the indole-3-pyruvate decarboxylase, previously identified as a key enzyme in indole-3-acetic acid (IAA) production in A. brasilense. In minimal growth medium, PAA biosynthesis by A. brasilense was only observed in the presence of phenylalanine (or precursors thereof). This observation suggests deamination of phenylalanine, decarboxylation of phenylpyruvate, and subsequent oxidation of phenylacetaldehyde as the most likely pathway for PAA synthesis. Expression analysis revealed that transcription of the ipdC gene is upregulated by PAA, as was previously described for IAA and synthetic auxins, indicating a positive feedback regulation. The synthesis of PAA by A. brasilense is discussed in relation to previously reported biocontrol properties of A. brasilense. PMID:15812004

  17. Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge.

    PubMed

    Ma, Pengda; Liu, Jingling; Zhang, Chenlu; Liang, Zongsuo

    2013-07-01

    Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of various diseases. Phenolic acids in S. miltiorrhiza, as important effective compounds, have become a new research focus in plant secondary metabolism in recent years. This review summarizes the recent advances in the regulation of water-soluble phenolic acid biosynthesis in S. miltiorrhiza via regulators at molecular level, such as the phenylalanine ammonia-lyase gene (PAL), cinnamic acid 4-hydroxylase gene (C4H), 4-coumarate-CoA ligase gene (4CL), tyrosine aminotransferase gene (TAT), 4-hydroxyphenylpyruvate reductase gene (HPPR), 4-hydroxyphenylpyruvated dioxygenase gene (HPPD), hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase-like gene (RAS-like), and v-myb avian myeloblastosis viral oncogene homolog 4 gene (MYB4), and production of anthocyanin pigmentation 1 gene (AtPAP1), and via regulators at cell level, such as methyl jasmonate, salicylic acid, abscisic acid, polyamines, metal ions, hydrogen peroxide (H₂O₂), ultraviolet-B radiation, and yeast elicitor.

  18. Developmental changes in aspartate-family amino acid biosynthesis in pea chloroplasts

    SciTech Connect

    Mills, W.R.; Cato, L.W.; Stephens, B.W.; Reeves, M. )

    1990-05-01

    Isolated chloroplasts are known to synthesize the asp-derived amino acids (ile, hse, lys and thr) from ({sup 14}C)asp (Mills et al, 1980, Plant Physiol. 65, 1166). Now, we have studied the influence of tissue age on essential amino acid biosynthesis in pea (Pisum sativum) plastids. Chloroplasts from the younger (third and fourth) leaves of 12 day old plants, were 2-3 times more active in synthesizing lys and thr from ({sup 14}C)asp than those from older (first or second) leaves. We also examined two key pathway enzymes (aspartate kinase and homoserine dehydrogenase); with each enzyme,a activity in younger leaves was about 2 times that in plastids from older tissue. Both lys- and thr-sensitive forms of aspartate kinase are known in plants; in agreement with earlier work, we found that lys-sensitive activity was about 4 times higher in the younger tissues, while the thr-sensitive activity changed little during development (Davies and Miflin, 1977, Plant Sci. Lett. 9, 323). Recently the role of aspartate kinase and homoserine dehydrogenase in controlling asp-family amino acid synthesis has been questioned (Giovanelli et al, 1989, Plant Physiol. 90, 1584); we hope that measurements of amino acid levels in chloroplasts as well as further enzyme studies will help us to better understand the regulation of asp-family amino acid synthesis.

  19. 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.

  20. Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants.

    PubMed Central

    Meuwly, P.; Molders, W.; Buchala, A.; Metraux, J. P.

    1995-01-01

    Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance. PMID:12228656

  1. Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production.

    PubMed

    Ratledge, Colin

    2004-11-01

    Single cell oils (SCOs) are now produced by various microorganisms as commercial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA). These oils are now used extensively as dietary supplements in infant formulas. An understanding of the underlying biochemistry and genetics of oil accumulation in such microorganisms is therefore essential if lipid yields are to be improved. Also an understanding of the biosynthetic pathways involved in the production of these polyunsaturated fatty acids (PUFAs) is also highly desirable as a prerequisite to increasing their content in the oils. An account is provided of the biosynthetic machinery that is necessary to achieve oil accumulation in an oleaginous species where it can account for lipid build up in excess of 70% of the cell biomass. Whilst PUFA production in most microorganisms uses a conventional fatty acid synthase (FAS) system followed by a series of desaturases and elongases, in Schizochytrium sp., and probably related thraustochytrid marine protists, PUFA synthesis now appears to be via a polyketide synthase (PKS) route. This route is discussed. It clearly represents a major departure from conventional fatty acid biosynthesis, possibly as a means of decreasing the amount of NADPH that is needed in the overall process.

  2. 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

  3. 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

  4. The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

    SciTech Connect

    Marzinke, Mark A.; Clagett-Dame, Margaret

    2012-01-01

    The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21{sup Cip1}, a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G{sub 1}/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer Calmin is a retinoic acid-responsive gene. Black-Right-Pointing-Pointer Calmin promotes cell cycle exit in N2A cells. Black-Right-Pointing-Pointer Calmin overexpression increases p21Cip1 and decreases cyclin D1. Black-Right-Pointing-Pointer Calmin is required for RA-induced growth inhibition and neurite outgrowth.

  5. 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

  6. 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

  7. Improvement of clavulanic acid production in Streptomyces clavuligerus by genetic manipulation of structural biosynthesis genes.

    PubMed

    Jnawali, Hum Nath; Yoo, Jin Cheol; Sohng, Jae Kyung

    2011-06-01

    To enhance clavulanic acid production, four structural clavulanic acid biosynthesis genes, carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2) and proclavaminate amidinohydrolase (pah2), were amplified from Streptomyces clavuligerus genomic DNA. They were cloned in the pSET152 integration and pIBR25 expression vectors containing the strong ermE* promoter to generate pHN18 and pHN19, respectively, and both plasmids were introduced into S. clavuligerus by protoplast transformation. Clavulanic acid production was increased by 8.7-fold (to ~310 mg/l) in integrative pHN18 transformants and by 5.1-fold in pHN19 transformants compared to controls. Transcriptional analyses showed that the expression levels of ceas2, bls2, cas2 and pah2 were markedly increased in both transformants as compared with wild-type. The elevation of the ceas2, bls2, cas2 and pah2 transcripts was consistent with the enhanced production of clavulanic acid.

  8. Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids.

    PubMed

    Yu, Jian; Si, Yingtao

    2004-01-01

    Short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (PHAs) by Ralstonia eutropha. Metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of PHA bioplastics. Based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in R. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on utilization of the short chain fatty acids. In fed-batch cultures with constant feeding of acid media, carbon metabolism and distribution in R. eutropha were measured involving CO2, PHA biopolymers, and residual cell mass. As the cells underwent unsteady state metabolism and PHA biosynthesis under nitrogen-limited conditions, accumulative carbon balance was applied for pseudo-steady-state analysis of the metabolic carbon fluxes. Cofactor NADP/NADPH balanced between PHA synthesis and the C3/C4 pathway provided an independent constraint for solution of the underdetermined metabolic network. A major portion of propionyl-CoA was directed to pyruvate via the 2-methylcitrate cycle and further decarboxylated to acetyl-CoA. Only a small amount of propionate carbon (<15% carbon) was directly condensed with acetyl-CoA for 3-hydroxyvalerate. The ratio of glyoxylate shunt to TCA cycle varies from 0 to 0.25, depending on the intracellular acetyl-CoA level and acetic acid in the medium. Malate is the node of the C3/C4 pathway and TCA cycle and its decarboxylation to dehydrogenation ranges from 0.33 to 1.28 in response to the demands on NADPH and oxaloacetate for short chain fatty acids utilization. PMID:15296425

  9. Reinvestigation of the biosynthesis of 2-aminoethylphosphonic acid in Tetrahymena pyriformis

    SciTech Connect

    Barry, R.J.; Dunaway-Mariano, D.; Mariano, P.S.

    1986-05-01

    The initial step in the proposed biosynthetic pathway of 2-aminoethylphosphonate involves an intramolecular rearrangement of phosphoenolpyruvate to form the C-P compound, 3-phosphonopyruvate. A radioisotopic assay with authentic phosphonopyruvate as a cold carrier results in the formation of material chromatographically identical to phosphonopyruvate. NMR and degradation studies reveal that the assayed product is not p-pyr, but rather phosphoglyceric acid. Herein they report results from a reinvestigation of the biosynthesis of AEP in T. pyriformis. Recent study indicates that AEP is formed when radiolabeled Pi or PEP are used as precursors. P-pyr formation has yet to be demonstrated. Results from studies aimed at the direct verification or exclusion of p-pyr as an intermediate in the biosynthetic pathway leading to AEP formation is presented.

  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. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci.

    PubMed

    Lee, Sang Ho; Wang, Hao; Labroli, Marc; Koseoglu, Sandra; Zuck, Paul; Mayhood, Todd; Gill, Charles; Mann, Paul; Sher, Xinwei; Ha, Sookhee; Yang, Shu-Wei; Mandal, Mihir; Yang, Christine; Liang, Lianzhu; Tan, Zheng; Tawa, Paul; Hou, Yan; Kuvelkar, Reshma; DeVito, Kristine; Wen, Xiujuan; Xiao, Jing; Batchlett, Michelle; Balibar, Carl J; Liu, Jenny; Xiao, Jianying; Murgolo, Nicholas; Garlisi, Charles G; Sheth, Payal R; Flattery, Amy; Su, Jing; Tan, Christopher; Roemer, Terry

    2016-03-01

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

  12. Stereo and region-selective biosynthesis of two new dihydroartemisinic acid glycosides by suspension-cultured cells of Artemisia annua

    PubMed Central

    Zhu, Jianhua; Zeng, Zihan; Song, Liyan; Hu, Yanshan; Wen, Wei; Yu, Rongming

    2014-01-01

    Background: The system of plant-cultured cells is one of the optimal systems to investigate biosynthesis pathway and their bioactive intermediates. Objective: To study the biosynthesis of dihydroartemisinic acid (1) by suspension-cultured cells of Artemisia annua. Materials and Methods: Substrate (compound 1) was administered into the suspension-cultured cells of A. annua and co-cultured for 2 days. The methanol extract was separated on various column chromatography methods and the structures of two biosynthesis products were elucidated based on the analysis of 1H NMR, 13C NMR, 2D NMR, and ESI-MS. Time-course curve was also established. Furthermore, in vitro antitumor activities of compounds 1-3 against HepG2, K562, and A549 cell lines were evaluated by MTT assay. Results: Two new compounds were obtained, namely 3α-hydroxy-dihydroartemisinic acid-α-D-glucopyranosyl ester (2) and 15-hydroxy-cadin-4-en-12-oic acid-β-d-glucopyranosyl ester (3). The results demonstrated that the cultured cells of A. annua possessed the abilities to stereo-selective hydroxylate and region-selective glycosylate sesquiterpene compounds in a highly efficient manner. Inhibitory effects of compounds 1-3 on proliferation of HepG2, K562, and A549 cell lines in vitro were also investigated. Conclusion: Two new dihydroartemisinic acid glycosides were obtained by stereo- and region-selective biosynthesis with cultured cells of A. annua. PMID:24914289

  13. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci.

    PubMed

    Lee, Sang Ho; Wang, Hao; Labroli, Marc; Koseoglu, Sandra; Zuck, Paul; Mayhood, Todd; Gill, Charles; Mann, Paul; Sher, Xinwei; Ha, Sookhee; Yang, Shu-Wei; Mandal, Mihir; Yang, Christine; Liang, Lianzhu; Tan, Zheng; Tawa, Paul; Hou, Yan; Kuvelkar, Reshma; DeVito, Kristine; Wen, Xiujuan; Xiao, Jing; Batchlett, Michelle; Balibar, Carl J; Liu, Jenny; Xiao, Jianying; Murgolo, Nicholas; Garlisi, Charles G; Sheth, Payal R; Flattery, Amy; Su, Jing; Tan, Christopher; Roemer, Terry

    2016-03-01

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci. PMID:26962156

  14. 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. PMID:27214242

  15. 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.

  16. 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

  17. 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.

  18. Discovery of a new family of Dieckmann cyclases essential to tetramic acid and pyridone-based natural products biosynthesis.

    PubMed

    Gui, Chun; Li, Qinglian; Mo, Xuhua; Qin, Xiangjing; Ma, Junying; Ju, Jianhua

    2015-02-01

    Bioinformatic analyses indicate that TrdC, SlgL, LipX2, KirHI, and FacHI belong to a group of highly homologous proteins involved in biosynthesis of actinomycete-derived tirandamycin B, streptolydigin, α-lipomycin, kirromycin, and factumycin, respectively. However, assignment of their biosynthetic roles has remained elusive. Gene inactivation and complementation, in vitro biochemical assays with synthetic analogues, point mutations, and phylogenetic tree analyses reveal that these proteins represent a new family of Dieckmann cyclases that drive tetramic acid and pyridone scaffold biosynthesis.

  19. 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. PMID:19053387

  20. 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.

  1. Salicylic Acid sans Aspirin in Animals and Man: Persistence in Fasting and Biosynthesis from Benzoic Acid

    PubMed Central

    2008-01-01

    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 13C6 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. PMID:19053387

  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. PMID:25921651

  4. 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.

  5. 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

  6. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid

    PubMed Central

    Zheng, Xiao-yu; Zhou, Mian; Yoo, Heejin; Pruneda-Paz, Jose L.; Spivey, Natalie Weaver; Kay, Steve A.; Dong, Xinnian

    2015-01-01

    The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-LIKE 9 (NTL9) and CCA1 HIKING EXPEDITION (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, PHYTOALEXIN-DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators CALMODULIN BINDING PROTEIN 60g (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production. PMID:26139525

  7. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid.

    PubMed

    Zheng, Xiao-Yu; Zhou, Mian; Yoo, Heejin; Pruneda-Paz, Jose L; Spivey, Natalie Weaver; Kay, Steve A; Dong, Xinnian

    2015-07-28

    The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-like 9 (NTL9) and CCA1 hiking expedition (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, phytoalexin-deficient 4 (PAD4) and enhanced disease susceptibility 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators calmodulin binding protein 60g (CBP60g) and systemic acquired resistance deficient 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production.

  8. Retinoic Acid Biosynthesis Is Impaired in Human and Murine Endometriosis1

    PubMed Central

    Pierzchalski, Keely; Taylor, Robert N.; Nezhat, Ceana; Jones, Jace W.; Napoli, Joseph L.; Yang, Guixiang; Kane, Maureen A.; Sidell, Neil

    2014-01-01

    ABSTRACT Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1−/− mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1−/− mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an “endometriosis phenotype” that enables cells to implant and grow at ectopic sites. PMID:25143356

  9. 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.

  10. Studies on the site of biosynthesis of acidic glycoproteins of guinea-pig serum

    PubMed Central

    Simkin, J. L.; Jamieson, J. C.

    1967-01-01

    1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an α-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-14C]Leucine or -valine and d-[1-14C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea

  11. Fatty acid biosynthesis from glutamate and glutamine is specifically induced in neuronal cells under hypoxia.

    PubMed

    Brose, Stephen A; Marquardt, Amanda L; Golovko, Mikhail Y

    2014-05-01

    Hypoxia is involved in many neuronal and non-neuronal diseases, and defining the mechanisms for tissue adaptation to hypoxia is critical for the understanding and treatment of these diseases. One mechanism for tissue adaptation to hypoxia is increased glutamine and/or glutamate (Gln/Glu) utilization. To address this mechanism, we determined incorporation of Gln/Glu and other lipogenic substrates into lipids and fatty acids in both primary neurons and a neuronal cell line under normoxic and hypoxic conditions and compared this to non-neuronal primary cells and non-neuronal cell lines. Incorporation of Gln/Glu into total lipids was dramatically and specifically increased under hypoxia in neuronal cells including both primary (2.0- and 3.0-fold for Gln and Glu, respectively) and immortalized cultures (3.5- and 8.0-fold for Gln and Glu, respectively), and 90% to 97% of this increase was accounted for by incorporation into fatty acids (FA) depending upon substrate and cell type. All other non-neuronal cells tested demonstrated decreased or unchanged FA synthesis from Gln/Glu under hypoxia. Consistent with these data, total FA mass was also increased in neuronal cells under hypoxia that was mainly accounted for by the increase in saturated and monounsaturated FA with carbon length from 14 to 24. Incorporation of FA synthesized from Gln/Glu was increased in all major lipid classes including cholesteryl esters, triacylglycerols, diacylglycerols, free FA, and phospholipids, with the highest rate of incorporation into triacylglycerols. These results indicate that increased FA biosynthesis from Gln/Glu followed by esterification may be a neuronal specific pathway for adaptation to hypoxia. We identified a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid biosynthesis from glutamine and glutamate (Gln/Glu) followed by esterification into lipids. All other non-neuronal cells tested demonstrated decreased or unchanged lipid synthesis from Gln

  12. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis.

    PubMed

    Holt, Jason A; Luo, Guizhen; Billin, Andrew N; Bisi, John; McNeill, Y Yvette; Kozarsky, Karen F; Donahee, Mary; Wang, Da Yuan; Mansfield, Traci A; Kliewer, Steven A; Goodwin, Bryan; Jones, Stacey A

    2003-07-01

    The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression of CYP7A1 in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.

  13. 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.

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

    PubMed

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

    2016-08-01

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

  15. Melatonin biosynthesis requires N-acetylserotonin methyltransferase activity of caffeic acid O-methyltransferase in rice

    PubMed Central

    Byeon, Yeong; Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

    2015-01-01

    Caffeic acid O-methyltransferase (COMT) methylates N-acetylserotonin into melatonin; that is, it has N-acetylserotonin O-methyltransferase (ASMT) activity. The ASMT activity of COMT was first detected in Arabidopsis thaliana COMT (AtCOMT). To confirm the involvement of COMT on melatonin synthesis in other plant species, the ASMT activity of a COMT from rice (Oryza sativa) (OsCOMT) was evaluated. Purified recombinant OsCOMT protein from Escherichia coli was used to validate the high ASMT activity of OsCOMT, similar to that of AtCOMT. The K m and V max values for the ASMT activity of OsCOMT were 243 µM and 2400 pmol min−1 mg protein−1, which were similar to those of AtCOMT. Similar to AtCOMT, OsCOMT was localized in the cytoplasm. In vitro ASMT activity was significantly inhibited by either caffeic acid or quercetin in a dose-dependent manner. Analogously, in vivo production of melatonin was significantly inhibited by quercetin in 4-week-old detached rice leaves. Lastly, the transgenic rice plants overexpressing rice COMT showed an increase in melatonin levels whereas transgenic rice plants suppressing the rice COMT had a significant decrease on melatonin levels, suggestive of the direct role of COMT in melatonin biosynthesis in plants. PMID:26276868

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

    PubMed

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

    2016-08-01

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

  17. Melatonin biosynthesis requires N-acetylserotonin methyltransferase activity of caffeic acid O-methyltransferase in rice.

    PubMed

    Byeon, Yeong; Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

    2015-11-01

    Caffeic acid O-methyltransferase (COMT) methylates N-acetylserotonin into melatonin; that is, it has N-acetylserotonin O-methyltransferase (ASMT) activity. The ASMT activity of COMT was first detected in Arabidopsis thaliana COMT (AtCOMT). To confirm the involvement of COMT on melatonin synthesis in other plant species, the ASMT activity of a COMT from rice (Oryza sativa) (OsCOMT) was evaluated. Purified recombinant OsCOMT protein from Escherichia coli was used to validate the high ASMT activity of OsCOMT, similar to that of AtCOMT. The K m and V max values for the ASMT activity of OsCOMT were 243 µM and 2400 pmol min(-1) mg protein(-1), which were similar to those of AtCOMT. Similar to AtCOMT, OsCOMT was localized in the cytoplasm. In vitro ASMT activity was significantly inhibited by either caffeic acid or quercetin in a dose-dependent manner. Analogously, in vivo production of melatonin was significantly inhibited by quercetin in 4-week-old detached rice leaves. Lastly, the transgenic rice plants overexpressing rice COMT showed an increase in melatonin levels whereas transgenic rice plants suppressing the rice COMT had a significant decrease on melatonin levels, suggestive of the direct role of COMT in melatonin biosynthesis in plants.

  18. Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants.

    PubMed

    Qualley, Anthony V; Widhalm, Joshua R; Adebesin, Funmilayo; Kish, Christine M; Dudareva, Natalia

    2012-10-01

    Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C(3) side chain by two carbon units, which can occur by a non-β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

  19. A structural basis for the biosynthesis of the major chlorogenic acids found in coffee.

    PubMed

    Lallemand, Laura A; Zubieta, Chloe; Lee, Soon Goo; Wang, Yechun; Acajjaoui, Samira; Timmins, Joanna; McSweeney, Sean; Jez, Joseph M; McCarthy, James G; McCarthy, Andrew A

    2012-09-01

    Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.

  20. 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

  1. Metabolic engineering of a novel muconic acid biosynthesis pathway via 4-hydroxybenzoic acid in Escherichia coli.

    PubMed

    Sengupta, Sudeshna; Jonnalagadda, Sudhakar; Goonewardena, Lakshani; Juturu, Veeresh

    2015-12-01

    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, aroF(FBR), 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

  2. 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.

  3. Genomic Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185.

    PubMed

    Zhao, Xianming; Dauenpen, Meesapyodsuk; Qu, Cunmin; Qiu, Xiao

    2016-09-01

    Thraustochytrium sp. 26185 is a marine protist that can produce a large amount of docosahexaenoic acid (DHA, 22:6n-3), an ω3 very long chain polyunsaturated fatty acid (VLCPUFA) of nutritional importance. However, the mechanism of how this fatty acid is synthesized and assembled into the storage lipid triacylglycerol is unclear. Here we report sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Genome sequencing produced a total of 2,418,734,139 bp clean sequences with about 62 fold genome coverage. Annotation of the genome sequences revealed 10,797 coding genes. Among them, 10,216 genes could be assigned into 25 KOG classes where 451 genes were specifically assigned to the group of lipid transport and metabolism. Detailed analysis of these genes revealed co-existence of both aerobic pathway and anaerobic pathways for the biosynthesis of DHA in this species. However, in the aerobic pathway, a key gene encoding stearate Δ9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. Genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine:diacylglycerol cholinephosphotransferase, an important enzyme in bridging two types of glycerolipids, diacylglycerols (DAG) and phosphatidylcholines (PtdCho). These results shed new insight on the biosynthesis and assembly of VLCPUFA in the Thraustochytrium. PMID:27514858

  4. 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

  5. IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro

    PubMed Central

    2012-01-01

    Introduction Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1β) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model. Methods IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1β and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student's t-tests. Results IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1β as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days. Conclusions In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1β on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation. PMID:22863285

  6. Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the “pathway redundancy” in metabolic networks

    PubMed Central

    2013-01-01

    Background Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the “pathway redundancy” of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer. Results Here we report the genetic characterization of CPOAA biosynthesis with revealing a “pathway redundancy” in metabolic networks. Independent mutation of the four genes (polL-N and polP) directly resulted in the accumulation of polyoxin I, suggesting their positive roles for CPOAA biosynthesis. Moreover, the individual mutant of polN and polP also partially retains polyoxin production, suggesting the existence of the alternative homologs substituting their functional roles. Conclusions It is unveiled that argA and argB in L-arginine biosynthetic pathway contributed to the “pathway redundancy”, more interestingly, argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide an example for the research on the “pathway redundancy” in metabolic networks, and lay a solid foundation for targeted enhancement of polyoxin production with synthetic biology strategies. PMID:24314013

  7. 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

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

    PubMed

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

    2015-09-01

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

  9. 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. PMID:27610104

  10. 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

  11. 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.

  12. Cloning and transcriptional analysis of Crepis alpina fatty acid desaturases affecting the biosynthesis of crepenynic acid.

    PubMed

    Nam, Jeong-Won; Kappock, T Joseph

    2007-01-01

    Crepis alpina acetylenase is a variant FAD2 desaturase that catalyses the insertion of a triple bond at the Delta12 position of linoleic acid, forming crepenynic acid in developing seeds. Seeds contain a high level of crepenynic acid but other tissues contain none. Using reverse transcriptase-coupled PCR (RT-PCR), acetylenase transcripts were identified in non-seed C. alpina tissues, which were highest in flower heads. To understand why functional expression of the acetylenase is limited to seeds, genes that affect acetylenase activity by providing substrate (FAD2) or electrons (cytochrome b5), or that compete for substrate (FAD3), were cloned. RT-PCR analysis indicated that the availability of a preferred cytochrome b5 isoform is not a limiting factor. Developing seeds co-express acetylenase and FAD2 isoform 2 (FAD2-2) at high levels. Flower heads co-express FAD2-3 and FAD3 at high levels, and FAD2-2 and acetylenase at moderate levels. FAD2-3 was not expressed in developing seed. Real-time RT-PCR absolute transcript quantitation showed 10(4)-fold higher acetylenase expression in developing seeds than in flower heads. Collectively, the results show that both the acetylenase expression level and the co-expression of other desaturases may contribute to the tissue specificity of crepenynate production. Helianthus annuus contains a Delta12 acetylenase in a polyacetylene biosynthetic pathway, so does not accumulate crepenynate. Real-time RT-PCR analysis showed relatively strong acetylenase expression in young sunflowers. Acetylenase transcription is observed in both species without accumulation of the enzymatic product, crepenynate. Functional expression of acetylenase appears to be affected by competition and collaboration with other enzymes. PMID:17329262

  13. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms.

    PubMed

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms.

  14. A Revised Pathway Proposed for Staphylococcus aureus Wall Teichoic Acid Biosynthesis Based on In Vitro Reconstitution of the Intracellular Steps

    PubMed Central

    Brown, Stephanie; Zhang, Yu-Hui; Walker*, Suzanne

    2008-01-01

    Summary Resistance has emerged to every family of clinically used antibiotics, and there is a pressing need to explore novel antibacterial targets. Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of many Gram-positive bacteria. Because WTAs play an essential role in Staphylococcus aureus colonization and infection, the enzymes involved in WTA biosynthesis are proposed to be targets for antibiotic development. To facilitate the discovery of WTA inhibitors, we have reconstituted the intracellular steps of S. aureus WTA biosynthesis. We show that two intracellular steps in the biosynthetic pathway are different from what was proposed. The work reported here lays the foundation for the discovery and characterization of inhibitors of wall teichoic acid biosynthetic enzymes to assess their potential for treating bacterial infections. PMID:18215769

  15. 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.

  16. 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.

  17. Promotion by gibberellic Acid of polyamine biosynthesis in internodes of light-grown dwarf peas.

    PubMed

    Dai, Y R; Kaur-Sawhney, R; Galston, A W

    1982-01-01

    When gibberellic acid (GA(3); 5-35 micrograms per milliliter) is sprayed on 9-day-old light-grown dwarf Progress pea (Pisum sativum) seedlings, it causes a marked increase in the activity of arginine decarboxylase (ADC; EC 4.1.1.9) in the fourth internodes. The titer of putrescine and spermidine, polyamines produced indirectly as a result of ADC action, also rises markedly, paralleling the effect of GA(3) on internode growth. Ammonium (5-hydroxycarvacryl) trimethyl chloride piperidine carboxylate (AMO-1618; 100-200 micrograms per milliliter) causes changes in the reverse direction for enzyme activity, polyamine content, and growth. GA(3) also reverses the red-light-induced inhibition of ADC activity in etiolated Alaska pea epicotyls; this is additional evidence for gibberellin-light interaction in the control of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17), an alternate source of putrescine arising from arginine, is not increased by GA(3) or by AMO-1618.The results support the hypothesis that ADC and polyamine content are important regulators of plant growth. PMID:16662137

  18. [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. PMID:6524887

  19. X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster.

    PubMed

    Elkins, Jonathan M; Kershaw, Nadia J; Schofield, Christopher J

    2005-01-15

    The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates. PMID:15352873

  20. Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV-tolerance.

    PubMed

    Clé, Carla; Hill, Lionel M; Niggeweg, Ricarda; Martin, Cathie R; Guisez, Yves; Prinsen, Els; Jansen, Marcel A K

    2008-08-01

    Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in tomato (Solanum lycopersicum). Hydroxycinnamoyl CoA quinate transferase (HQT) is the key enzyme catalysing CGA biosynthesis in tomato. We have studied the relationship between phenolic accumulation and UV-susceptibility in transgenic tomato plants with altered HQT expression. Overall, increased CGA accumulation was associated with increased UV-protection. However, the genetic manipulation of HQT expression also resulted in more complex alterations in the profiles of phenolics. Levels of rutin were relatively high in both HQT gene-silenced and HQT-overexpressing plants raised in plant growth tunnels. This suggests plasticity in the flux along different branches of phenylpropanoid metabolism and the existence of regulatory mechanisms that direct the flow of phenolic precursors in response to both metabolic parameters and environmental conditions. These changes in composition of the phenolic pool affected the relative levels of UV-tolerance. We conclude that the capability of the phenolic compounds to protect against potentially harmful UV radiation is determined both by the total levels of phenolics that accumulate in leaves as well as by the specific composition of the phenolic profile.

  1. 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

  2. Biosynthesis of the pyrimidinyl amino acid lathyrine by Lathyrus tingitanus L.

    PubMed Central

    Brown, E G; Al-Baldowi, N F

    1977-01-01

    The biosynthesis of the pyrimidinyl amino acid lathyrine by seedlings of Lathyrus tingitanus L. was shown to be stimulated by uracil. [6(-14)C]Orotate, [2(-14)C]uracil and [3(-14)C]serine were incorporated into lathyrine; the incorporation of [6(-14)C]orotate was substantially decreased in the presence of uracil. Chemical degradation to locate the 14C incorporated from labelled precursors showed that 90% of the radioactivity incorporated into lathyrine from [3(-14)C]serine could be recovered in the alanine side chain. Over 80% of the radioactivity incorporated from [2(-14)C]uracil was shown to be located in C-2 of lathyrine. It is concluded that under the conditions studied, lathyrine arises from a preformed pyrimidine arising via the orotate pathway. Paradoxically, it was also possible to confirm previous reports that radioactivity from L-[guanidino-14C]homoarginine is incorporated into lathyrine and gamma-hydroxyhomoarginine. However, as homoarginine and gamma-hydroxyhomoarginine are also both labelled by [2(-14)C]uracil, it is suggested that they are products of the ring-opening of lathyrine and that reversibility of this process accounts, at least in part, for their observed experimental incorporation into lathyrine. PMID:883953

  3. 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. PMID:25956452

  4. The potential role of juvenile hormone acid methyltransferase in methyl farnesoate (MF) biosynthesis in the swimming crab, Portunus trituberculatus.

    PubMed

    Xie, Xi; Tao, Tian; Liu, Mingxin; Zhou, Yanqi; Liu, Zhiye; Zhu, Dongfa

    2016-05-01

    Juvenile hormone (JH) and methyl farnesoate (MF) play essential roles in the development and reproduction of insects and crustaceans respectively. Juvenile hormone acid methyltransferase (JHAMT) catalyzes the methyl esterification in insect JH biosynthesis, while the corresponding step in crustacean MF biosynthesis was long thought to be catalyzed by farnesoic acid O-methyltransferase (FAMeT). However, the new discovery of JHAMT orthologs in crustaceans indicates that JHAMT may also play essential role in the MF biosynthesis in crustaceans. Here we cloned and characterized the full-length cDNA encoding JHAMT in the swimming crab Portunus trituberculatus (PtJHAMT). Sequence and structure analysis of PtJHAMT revealed that it was composed of a 6-stranded β sheet with 9 α helices, and contained a signature Sadenosyl-L-methionine (SAM) binding motif, which is the hallmark in all SAM dependent methyltransferases (SAM-MTs). Several active sites that are critical for the interaction of SAM and JH/FA substrate were also conserved in PtJHAMT. The gene expression of PtJHAMT was highly specific to the mandibular organ, which is the sole site of MF synthesis. PtJHAMT expression significantly increased in the late-vitellogenic stage and mature stage, which suggests a possible role of PtJHAMT in modulating ovarian development. The role of PtJHAMT and PtFAMeT in MF biosynthesis was further investigated by RNA interfering (RNAi). Injection of PtJHAMT and PtFAMeT dsRNA both led to a decrease in hemolymph MF titers. Injection of PtHMGR dsRNA caused the decrease in PtJHAMT expression, but had no effect on mRNA level of PtFAMeT. Together these results suggested that JHAMT and FAMeT are both involved in the MF biosynthesis in crustaceans, while the JHAMT is highly specific to FA substrate, and FAMeT may have more catalytic functions. PMID:26952760

  5. A natural protecting group strategy to carry an amino acid starter unit in the biosynthesis of macrolactam polyketide antibiotics.

    PubMed

    Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2011-11-16

    Macrolactam antibiotics are an important class of macrocyclic polyketides that contain a unique nitrogen-containing starter unit. In the present study, a set of starter biosynthetic enzymes in the macrolactam antibiotic vicenistatin was characterized. We found that the protection-deprotection strategy of the aminoacyl-ACP intermediate was critical in this system. On the basis of bioinformatics, the described pathway is also proposed as a common method for carrying amino acids in the biosynthesis of other macrolactam antibiotics. PMID:22010945

  6. Fatty acid biosynthesis from glutamate and glutamine is specifically induced in neuronal cells under hypoxia

    PubMed Central

    Brose, Stephen A.; Marquardt, Amanda L.; Golovko, Mikhail Y.

    2014-01-01

    Hypoxia is involved in many neuronal and non-neuronal diseases, and defining the mechanisms for tissue adaptation to hypoxia is critical for the understanding and treatment of these diseases. One mechanism for tissue adaptation to hypoxia is increased glutamine and/or glutamate (Gln/Glu) utilization. To address this mechanism, we determined total Gln/Glu incorporation into lipids and fatty acids in both primary neurons and a neuronal cell line under normoxic and hypoxic conditions and compared this to non-neuronal primary cells and non-neuronal cell lines. Incorporation of Gln/Glu into total lipids was dramatically and specifically increased under hypoxia in neuronal cells including both primary (2.0- and 3.0- fold for Gln and Glu, respectively) and immortalized cultures (3.5- and 8.0- fold for Gln and Glu, respectively), and 90% to 97% of this increase was accounted for by incorporation into fatty acids (FA) depending upon substrate and cell type. All other non-neuronal cells tested demonstrated decreased or unchanged FA synthesis from Gln/Glu under hypoxia. Consistent with these data, total FA mass was also increased in neuronal cells under hypoxia that was mainly accounted for by the increase in saturated and monounsaturated FA with carbon length from 14 to 24. Incorporation of FA synthesized from Gln/Glu was increased in all major lipid classes including cholesteryl esters, TAGs, DAGs, free FA, and phospholipids, with the highest rate of incorporation into TAGs. These results indicate that increased FA biosynthesis from Gln/Glu followed by esterification may be a neuronal specific pathway for adaptation to hypoxia. PMID:24266789

  7. Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase.

    PubMed

    Austin, Michael B; Saito, Tamao; Bowman, Marianne E; Haydock, Stephen; Kato, Atsushi; Moore, Bradley S; Kay, Robert R; Noel, Joseph P

    2006-09-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis. PMID:16906151

  8. 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. PMID:26055979

  9. Wound-Inducible Biosynthesis of Phytoalexin Hydroxycinnamic Acid Amides of Tyramine in Tryptophan and Tyrosine Decarboxylase Transgenic Tobacco Lines1

    PubMed Central

    Guillet, Gabriel; De Luca, Vincenzo

    2005-01-01

    The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC ×TYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine. PMID:15665252

  10. Biosynthesis and metabolism of retinoic acid: roles of CRBP and CRABP in retinoic acid: roles of CRBP and CRABP in retinoic acid homeostasis.

    PubMed

    Napoli, J L

    1993-02-01

    The enzymes that constitute the pathway of retinoic acid biosynthesis and metabolism may recognize retinoid binding proteins as effectors and substrates. Apocellular retinol-binding protein (CRBP) stimulates a bile-salt independent membrane-bound retinyl ester hydrolase resulting in the hydrolysis of endogenous retinyl esters and the formation of holoCRBP. HoloCRBP delivers retinol to a microsomal nicotin-amide-adenine dinucleotide phosphate-dependent dehydrogenase, protects it from artifactual oxidation and denies enzymes that cannot recognize the binding protein access to retinol. The retinal synthesized may be transferred from the microsomes to the cytosol by CRBP. A cytosolic retinal dehydrogenase has been purified that produces retinoic acid from retinal generated by microsomes in the presence of CRBP and from the complex CRBP-retinal itself. Thus, CRBP(type I) seems to channel retinoids through the reactions of retinoic acid synthesis via a series of protein-protein interactions. Cellular retinoic acid-binding protein (type I) facilitates retinoic acid metabolism by sequestering it and by acting as a low Km substrate, thereby also modulating the steady-state concentrations of retinoic acid. PMID:8381481

  11. Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors).

    PubMed

    Khozin, I.; Adlerstein, D.; Bigongo, C.; Heimer, Y. M.; Cohen, Z.

    1997-05-01

    In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, [omega]6 and [omega]3, which involve cytoplasmic and chloroplastic lipids. In the [omega]6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4[omega]6-PC by a sequence that includes a [delta]6 desaturase, an elongation step, and a [delta]5 desaturase. In the minor [omega]3 pathway, 18:2-PC is presumably desaturated to 18:3[omega]3, which is sequentially converted by the enzymatic sequence of the [omega]6 pathway to 20:5[omega]3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4[omega]6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic [delta]17 ([omega]3) desaturase.

  12. Unsaturated macrocyclic dihydroxamic acid siderophores produced by Shewanella putrefaciens using precursor-directed biosynthesis.

    PubMed

    Soe, Cho Z; Codd, Rachel

    2014-04-18

    To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; [M + H(+)](+), m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4-diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of l-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, (15)N2-1,4-diaminobutane ((15)N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of (15)N-labeled pbH2 ((15)N4-pbH2; [M + H(+)](+), m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; [M + H(+)](+), m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; [M + H(+)](+), m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 ([Fe(pb)](+); [M](+), m/zcalc 426.1, m/zobs 426.2), (15)N4-pbH2 ([Fe((15)N4-pb)](+); [M](+), m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 ([Fe(E,E-pbe)](+); [M](+), m/zcalc 422.1, m/zobs 422.0), or E-pbxH2 ([Fe(E-pbx)](+); [M](+), m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical

  13. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels.

    PubMed

    Mandal, Shantanu; Upadhyay, Shivangi; Wajid, Saima; Ram, Mauji; Jain, Dharam Chand; Singh, Ved Pal; Abdin, Malik Zainul; Kapoor, Rupam

    2015-07-01

    It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

  14. Novel Type II Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents

    PubMed Central

    Schrader, Florian C.; Glinca, Serghei; Sattler, Julia M.; Dahse, Hans-Martin; Afanador, Gustavo A.; Prigge, Sean T.; Lanzer, Michael; Mueller, Ann-Kristin; Klebe, Gerhard; Schlitzer, Martin

    2013-01-01

    Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC50 values of 1.7 and 3.0 µm and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine. PMID:23341167

  15. Global effect of indole-3-acetic acid biosynthesis on multiple virulence factors of Erwinia chrysanthemi 3937.

    PubMed

    Yang, Shihui; Zhang, Qiu; Guo, Jianhua; Charkowski, Amy O; Glick, Bernard R; Ibekwe, A Mark; Cooksey, Donald A; Yang, Ching-Hong

    2007-02-01

    Production of the plant hormone indole-3-acetic acid (IAA) is widespread among plant-associated microorganisms. The non-gall-forming phytopathogen Erwinia chrysanthemi 3937 (strain Ech3937) possesses iaaM (ASAP16562) and iaaH (ASAP16563) gene homologues. In this work, the null knockout iaaM mutant strain Ech138 was constructed. The IAA production by Ech138 was reduced in M9 minimal medium supplemented with l-tryptophan. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected. The pectate lyase production of Ech138 was diminished. Compared with wild-type Ech3937, the expression levels of an oligogalacturonate lyase gene, ogl, and three endopectate lyase genes, pelD, pelI, and pelL, were reduced in Ech138 as determined by a green fluorescent protein-based fluorescence-activated cell sorting promoter activity assay. In addition, the transcription of type III secretion system (T3SS) genes, dspE (a putative T3SS effector) and hrpN (T3SS harpin), was found to be diminished in the iaaM mutant Ech138. Compared with Ech3937, reduced expression of hrpL (a T3SS alternative sigma factor) and gacA but increased expression of rsmA in Ech138 was also observed, suggesting that the regulation of T3SS and pectate lyase genes by IAA biosynthesis might be partially due to the posttranscriptional regulation of the Gac-Rsm regulatory pathway. PMID:17189441

  16. X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster

    PubMed Central

    2004-01-01

    The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 Å (1 Å=0.1 nm) resolution. The larger domain of the structure consists of an αββα sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the αββα sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an α2β2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme–substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates. PMID:15352873

  17. Putrescine biosynthesis in Lactococcus lactis is transcriptionally activated at acidic pH and counteracts acidification of the cytosol.

    PubMed

    Del Rio, Beatriz; Linares, Daniel; Ladero, Victor; Redruello, Begoña; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

    2016-11-01

    Lactococcus lactis subsp. cremoris CECT 8666 is a lactic acid bacterium that synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The AGDI genes cluster includes aguR. This encodes a transmembrane protein that functions as a one-component signal transduction system, the job of which is to sense the agmatine concentration of the medium and accordingly regulate the transcription of the catabolic operon aguBDAC. The latter encodes the proteins necessary for agmatine uptake and its conversion into putrescine. This work reports the effect of extracellular pH on putrescine biosynthesis and on the genetic regulation of the AGDI pathway. Increased putrescine biosynthesis was detected at acidic pH (pH5) compared to neutral pH. Acidic pH induced the transcription of the catabolic operon via the activation of the aguBDAC promoter PaguB. However, the external pH had no significant effect on the activity of the aguR promoter PaguR, or on the transcription of the aguR gene. The transcriptional activation of the AGDI pathway was also found to require a lower agmatine concentration at pH5 than at neutral pH. Finally, the following of the AGDI pathway counteracted the acidification of the cytoplasm under acidic external conditions, suggesting it to provide protection against acid stress.

  18. Gluconic acid-producing Pseudomonas sp. prevent γ-actinorhodin biosynthesis by Streptomyces coelicolor A3(2).

    PubMed

    Galet, Justine; Deveau, Aurélie; Hôtel, Laurence; Leblond, Pierre; Frey-Klett, Pascale; Aigle, Bertrand

    2014-09-01

    Streptomyces are ubiquitous soil bacteria well known for their ability to produce a wide range of secondary metabolites including antibiotics. In their natural environments, they co-exist and interact with complex microbial communities and their natural products are assumed to play a major role in mediating these interactions. Reciprocally, their secondary metabolism can be influenced by the surrounding microbial communities. Little is known about these complex interactions and the underlying molecular mechanisms. During pairwise co-culture experiments, a fluorescent Pseudomonas, Pseudomonas fluorescens BBc6R8, was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 without altering the biosynthesis of the intracellular actinorhodin. A mutant of the BBc6R8 strain defective in the production of gluconic acid from glucose and consequently unable to acidify the culture medium did not show any effect on the γ-actinorhodin biosynthesis in contrast to the wild-type strain and the mutant complemented with the wild-type allele. In addition, when glucose was substituted by mannitol in the culture medium, P. fluorescens BBc6R8 was unable to acidify the medium and to prevent the biosynthesis of the antibiotic. All together, the results show that P. fluorescens BBc6R8 impairs the biosynthesis of the lactone form of actinorhodin in S. coelicolor by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. We propose some hypotheses on the ecological significance of such interaction.

  19. The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis.

    PubMed

    Milborrow, B V

    2001-06-01

    The pathway of biosynthesis of abscisic acid (ABA) can be considered to comprise three stages: (i) early reactions in which small phosphorylated intermediates are assembled as precursors of (ii) intermediate reactions which begin with the formation of the uncyclized C40 carotenoid phytoene and end with the cleavage of 9'-cis-neoxanthin (iii) to form xanthoxal, the C15 skeleton of ABA. The final phase comprising C15 intermediates is not yet completely defined, but the evidence suggests that xanthoxal is first oxidized to xanthoxic acid by a molybdenum-containing aldehyde oxidase and this is defective in the aba3 mutant of Arabidopsis and present in a 1-fold acetone precipitate of bean leaf proteins. This oxidation precludes the involvement of AB-aldehyde as an intermediate. The oxidation of the 4'-hydroxyl group to the ketone and the isomerization of the 1',2'-epoxy group to the 1'-hydroxy-2'-ene may be brought about by one enzyme which is defective in the aba2 mutant and is present in the 3-fold acetone fraction of bean leaves. Isopentenyl diphosphate (IPP) is now known to be derived by the pyruvate-triose (Methyl Erythritol Phosphate, MEP) pathway in chloroplasts. (14C)IPP is incorporated into ABA by washed, intact chloroplasts of spinach leaves, but (14C)mevalonate is not, consequently, all three phases of biosynthesis of ABA occur within chloroplasts. The incorporation of labelled mevalonate into ABA by avocado fruit and orange peel is interpreted as uptake of IPP made in the cytoplasm, where it is the normal precursor of sterols, and incorporated into carotenoids after uptake by a carrier in the chloroplast envelope. An alternative bypass pathway becomes more important in aldehyde oxidase mutants, which may explain why so many wilty mutants have been found with this defect. The C-1 alcohol group is oxidized, possibly by a mono-oxygenase, to give the C-1 carboxyl of ABA. The 2-cis double bond of ABA is essential for its biological activity but it is not known

  20. Identification, Biosynthesis, and Function of 1,3,4,6-Hexanetetracarboxylic Acid in Methanobacterium thermoautotrophicum ΔH

    PubMed Central

    Gorkovenko, Alexander; Roberts, Mary F.; White, Robert H.

    1994-01-01

    An unusual compound, 1,3,4,6-hexanetetracarboxylic acid, was identified by 1H and 13C two-dimensional nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as one of the major components of the small-molecule pool in Methanobacterium thermoautotrophicum ΔH under optimal conditions of cell growth. Incorporation of 13C- and 2H-labeled acetates was consistent with the biosynthesis of this tetracarboxylic acid from α-ketoglutarate, two molecules of acetyl-coenzyme A, and one molecule of CO2, as established for the tetracarboxylic acid moiety of methanofuran. 13CO2 pulse- 12CO2 chase methodology was used to establish the turnover rate for this compound. In contrast to the two other major solutes in this bacterium, cyclic 2,3-diphosphoglycerate and glutamate, which are key metabolic intermediates, this free tetracarboxylic acid was metabolically inactive, with a half-life that exceeded the cell doubling time. Hence, this molecular pool cannot serve as a metabolic intermediate in cell biosynthesis. The functional role of free tetracarboxylate as a conservative part of a system that maintains high positive internal osmotic pressure in this bacterium is proposed. PMID:16349232

  1. 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.

  2. The biosynthesis of N-arachidonoyl dopamine (NADA), a putative endocannabinoid and endovanilloid, via conjugation of arachidonic acid with dopamine.

    PubMed

    Hu, Sherry Shu-Jung; Bradshaw, Heather B; Benton, Valery M; Chen, Jay Shih-Chieh; Huang, Susan M; Minassi, Alberto; Bisogno, Tiziana; Masuda, Kim; Tan, Bo; Roskoski, Robert; Cravatt, Benjamin F; Di Marzo, Vincenzo; Walker, J Michael

    2009-10-01

    N-arachidonoyl dopamine (NADA) is an endogenous ligand that activates the cannabinoid type 1 receptor and the transient receptor potential vanilloid type 1 channel. Two potential biosynthetic pathways for NADA have been proposed, though no conclusive evidence exists for either. The first is the direct conjugation of arachidonic acid with dopamine and the other is via metabolism of a putative N-arachidonoyl tyrosine (NA-tyrosine). In the present study we investigated these biosynthetic mechanisms and report that NADA synthesis requires TH in dopaminergic terminals; however, NA-tyrosine, which we identify here as an endogenous lipid, is not an intermediate. We show that NADA biosynthesis primarily occurs through an enzyme-mediated conjugation of arachidonic acid with dopamine. While this conjugation likely involves a complex of enzymes, our data suggest a direct involvement of fatty acid amide hydrolase in NADA biosynthesis either as a rate-limiting enzyme that liberates arachidonic acid from AEA, or as a conjugation enzyme, or both.

  3. Polyunsaturated fatty acid biosynthesis is involved in phenylephrine-mediated calcium release in vascular smooth muscle cells.

    PubMed

    Irvine, Nicola A; Lillycrop, Karen A; Fielding, Barbara; Torrens, Christopher; Hanson, Mark A; Burdge, Graham C

    2015-10-01

    Stimulation of vascular smooth muscle (VSM) α1-adrenoceptors induces myosin phosphorylation and vasoconstriction via mobilisation of intracellular calcium and production of specific eicosanoids. Polyunsaturated fatty acid (PUFA) biosynthesis in VSM cells is involved, although the precise mechanism is not known. To address this, we characterised PUFA biosynthesis in VSM cells and determined its role in intracellular calcium release and eicosanoid production. Murine VSM cells converted 18:2n-6 to longer chain PUFA including 22:5n-6. Δ6 (D6d) and Δ5 (D5d) desaturase, and elongase (Elovl) 5 were expressed. Elovl2 was not detected in human, mouse or rat VSM cells, or in rat or mouse aortae, but tit was not associated with hypermethylation of its promoter. D6d or D5d inhibition reduced 18:3n-6 and 20:4n-6 synthesis, respectively, and induced concentration-related decrease in phenylephrine-mediated calcium release, and in PGE2 and PGF2α secretion. Together these findings suggest that PUFA biosynthesis in VSM cells is involved in calcium release associated with vasoconstriction.

  4. Polyunsaturated fatty acid biosynthesis is involved in phenylephrine-mediated calcium release in vascular smooth muscle cells.

    PubMed

    Irvine, Nicola A; Lillycrop, Karen A; Fielding, Barbara; Torrens, Christopher; Hanson, Mark A; Burdge, Graham C

    2015-10-01

    Stimulation of vascular smooth muscle (VSM) α1-adrenoceptors induces myosin phosphorylation and vasoconstriction via mobilisation of intracellular calcium and production of specific eicosanoids. Polyunsaturated fatty acid (PUFA) biosynthesis in VSM cells is involved, although the precise mechanism is not known. To address this, we characterised PUFA biosynthesis in VSM cells and determined its role in intracellular calcium release and eicosanoid production. Murine VSM cells converted 18:2n-6 to longer chain PUFA including 22:5n-6. Δ6 (D6d) and Δ5 (D5d) desaturase, and elongase (Elovl) 5 were expressed. Elovl2 was not detected in human, mouse or rat VSM cells, or in rat or mouse aortae, but tit was not associated with hypermethylation of its promoter. D6d or D5d inhibition reduced 18:3n-6 and 20:4n-6 synthesis, respectively, and induced concentration-related decrease in phenylephrine-mediated calcium release, and in PGE2 and PGF2α secretion. Together these findings suggest that PUFA biosynthesis in VSM cells is involved in calcium release associated with vasoconstriction. PMID:26324193

  5. Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus▿†

    PubMed Central

    Gómez, Cristina; Horna, Dina H.; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J.; Braña, Alfredo F.; Méndez, Carmen; Salas, José A.

    2011-01-01

    Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin. PMID:21665968

  6. The tomato mutation nxd1 reveals a gene necessary for neoxanthin biosynthesis and demonstrates that violaxanthin is a sufficient precursor for abscisic acid biosynthesis.

    PubMed

    Neuman, Hadar; Galpaz, Navot; Cunningham, Francis X; Zamir, Dani; Hirschberg, Joseph

    2014-04-01

    Carotenoid pigments are indispensable for plant life. They are synthesized within plastids where they provide essential functions in photosynthesis. Carotenoids serve as precursors for the synthesis of the strigolactone phytohormones, which are made from β-carotene, and of abscisic acid (ABA), which is produced from certain xanthophylls. Despite the significant progress that has been made in our understanding of the carotenoid biosynthesis pathway, the synthesis of the xanthophyll neoxanthin has remained unknown. We report here on the isolation of a tomato (Solanum lycopersicum) mutant, neoxanthin-deficient 1 (nxd1), which lacks neoxanthin, and on the cloning of a gene that is necessary for neoxanthin synthesis in both tomato and Arabidopsis. The locus nxd1 encodes a gene of unknown function that is conserved in all higher plants. The activity of NXD1 is essential but cannot solely support neoxanthin synthesis. Lack of neoxanthin does not significantly reduce the fitness of tomato plants in cultivated field conditions and does not impair the synthesis of ABA, suggesting that in tomato violaxanthin is a sufficient precursor for ABA production in vivo.

  7. 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".

  8. 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

  9. 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.

  10. Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis

    PubMed Central

    Chirgadze, Nickolay Y.; Briggs, Steven L.; McAllister, Kelly A.; Fischl, Anthony S.; Zhao, Genshi

    2000-01-01

    Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3′5′-ADP, a product of AcpS, at 2.0 and 1.9 Å resolution, respectively. The crystal structure reveals an α/β fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity. PMID:11032795

  11. 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

  12. 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

  13. The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development.

    PubMed

    Bhatt, Apoorva; Molle, Virginie; Besra, Gurdyal S; Jacobs, William R; Kremer, Laurent

    2007-06-01

    Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development. PMID:17555433

  14. Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

    PubMed Central

    Li, Rongfeng; Khaleeli, Nusrat; Townsend, Craig A.

    2000-01-01

    Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed. PMID:10869089

  15. 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

  16. 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.

  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. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis

    PubMed Central

    Ye, Jie; Hu, Tixu; Yang, Congmei; Li, Hanxia; Yang, Mingze; Ijaz, Raina; Ye, Zhibiao; Zhang, Yuyang

    2015-01-01

    Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism. PMID:26133783

  19. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis.

    PubMed

    Ye, Jie; Hu, Tixu; Yang, Congmei; Li, Hanxia; Yang, Mingze; Ijaz, Raina; Ye, Zhibiao; Zhang, Yuyang

    2015-01-01

    Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.

  20. Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots.

    PubMed

    Yamauchi, Takaki; Shiono, Katsuhiro; Nagano, Minoru; Fukazawa, Aya; Ando, Miho; Takamure, Itsuro; Mori, Hitoshi; Nishizawa, Naoko K; Kawai-Yamada, Maki; Tsutsumi, Nobuhiro; Kato, Kiyoaki; Nakazono, Mikio

    2015-09-01

    In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.

  1. Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots1

    PubMed Central

    Yamauchi, Takaki; Shiono, Katsuhiro; Nagano, Minoru; Fukazawa, Aya; Ando, Miho; Takamure, Itsuro; Mori, Hitoshi; Nishizawa, Naoko K.; Kawai-Yamada, Maki; Tsutsumi, Nobuhiro; Kato, Kiyoaki; Nakazono, Mikio

    2015-01-01

    In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex. PMID:26036614

  2. 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

  3. TaOPR2 encodes a 12-oxo-phytodienoic acid reductase involved in the biosynthesis of jasmonic acid in wheat (Triticum aestivum L.).

    PubMed

    Wang, Yukun; Yuan, Guoliang; Yuan, Shaohua; Duan, Wenjing; Wang, Peng; Bai, Jianfang; Zhang, Fengting; Gao, Shiqing; Zhang, Liping; Zhao, Changping

    2016-01-29

    The 12-oxo-phytodienoic acid reductases (OPRs) are involved in the various processes of growth and development in plants, and classified into the OPRⅠ and OPRⅡ subgroups. In higher plants, only OPRⅡ subgroup genes take part in the biosynthesis of endogenous jasmonic acid. In this study, we isolated a novel OPRⅡ subgroup gene named TaOPR2 (GeneBank accession: KM216389) from the thermo-sensitive genic male sterile (TGMS) wheat cultivar BS366. TaOPR2 was predicted to encode a protein with 390 amino acids. The encoded protein contained the typical oxidored_FMN domain, the C-terminus peroxisomal-targeting signal peptide, and conserved FMN-binding sites. TaOPR2 was mapped to wheat chromosome 7B and located on peroxisome. Protein evolution analysis revealed that TaOPR2 belongs to the OPRⅡ subgroup and shares a high degree of identity with other higher plant OPR proteins. The quantitative real-time PCR results indicated that the expression of TaOPR2 is inhibited by abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), low temperatures and high salinity. In contrast, the expression of TaOPR2 can be induced by wounding, drought and methyl jasmonate (MeJA). Furthermore, the transcription level of TaOPR2 increased after infection with Puccinia striiformis f. sp. tritici and Puccinia recondite f. sp. tritici. TaOPR2 has NADPH-dependent oxidoreductase activity. In addition, the constitutive expression of TaOPR2 can rescue the male sterility phenotype of Arabidopsis mutant opr3. These results suggest that TaOPR2 is involved in the biosynthesis of jasmonic acid (JA) in wheat.

  4. Dietary n-3 polyunsaturated fatty acids alter the expression of genes involved in prostaglandin biosynthesis in the bovine uterus.

    PubMed

    Coyne, G S; Kenny, D A; Childs, S; Sreenan, J M; Waters, S M

    2008-09-15

    Nutrition plays a critical role in the regulation of cow fertility. There is emerging evidence that dietary long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) may act as specific regulators of some reproductive processes. In vitro studies suggest that the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may play pivotal roles by suppressing the synthesis of uterine prostaglandin F(2alpha) (PGF(2alpha)) which is centrally involved in the control of the bovine oestrous cycle and in early embryo survival. The objective of the current study was to determine the effect of dietary inclusion of n-3 PUFA on uterine endometrial mRNA expression of key genes regulating PGF(2alpha) biosynthesis. Beef heifers were fed either a low (CON; n=10) or high (HIGH PUFA; n=10) n-3 PUFA diet for 45 days and endometrial tissues were harvested following slaughter. Following analysis, tissues within each dietary group were ranked on the basis of their PUFA concentrations and the highest (n=7) and lowest (n=7) within each of HIGH PUFA and CON, respectively, were used in gene expression studies. Endometrial n-3 PUFA concentrations were more than two-fold higher (P<0.05) and EPA concentrations alone more than seven-fold higher (P<0.01) in the HIGH PUFA than the CON group. Endometrial concentrations of arachidonic acid, were lower (P<0.001) in the tissues from HIGH PUFA than those from the CON group. Total RNA was isolated from all endometrial tissues and real-time reverse transcription (RT) PCR conducted to compare the relative expression of 11 genes with known involvement in uterine biosynthesis of 2-series prostaglandins. Expression of mRNA for prostaglandin E synthase (PGES) and peroxisome proliferator-activated receptors, PPAR alpha and delta was increased (P<0.05) while mRNA expression of phospholipase A(2) (PLA(2)) was decreased (P=0.06) in the HIGH PUFA endometrial tissues. Expression of genes coding for the oxytocin receptor (OTR), phospholipase C (PLC

  5. Fasting Induces IL-1 Resistance and Free-Fatty Acid-Mediated Up-Regulation of IL-1R2 and IL-1RA

    PubMed Central

    Joesting, Jennifer J.; Moon, Morgan L.; Gainey, Stephen J.; Tisza, Brittany L.; Blevins, Neil A.; Freund, Gregory G.

    2014-01-01

    Objective: Weight-loss is a near societal obsession and many diet programs use significant calorie restriction including fasting/short term starvation to generate rapid effects. Fasting is also a well-recognized cause of immunosuppression especially within the innate immune system. In this study, we sought to determine if the IL-1 arm of the neuroimmune system was down-regulated by a 24 h fast and how fasting might generate this effect. Design: Mice were allowed ad libitum access to food or had food withheld for 24 h. Expression of the endogenous IL-1 antagonists, IL-1 receptor type 2 (IL-1R2), and IL-1 receptor antagonist (IL-1RA) was determined as were sickness behaviors before and after IL-1β administration. Results: Fasting markedly increased gene expression of IL-1R2 (83-fold in adipose tissue, 9.5-fold in liver) and IL-1RA (68-fold in liver). Fasted mice were protected from IL-1β-induced weight-loss, hypoglycemia, loss of locomotor, and social anxiety. These protections were coupled to a large positive interaction of fasting and IL-1β on IL-1R2 gene expression in adipose tissue and liver (2.6- and 1.6-fold, respectively). Fasting not only increased IL-1RA and IL-1R2 protein 2.5- and 3.2-fold, respectively, in liver but also increased IL-1R2 1.8-fold in adipose tissue. Fasting, in turn, triggered a 2.4-fold increase in plasma free-fatty acids (FFAs) and a 2.1-fold increase in plasma corticosterone. Inhibition, of glucocorticoid action with mifepristone did not impact fasting-dependent IL-1R2 or IL-1RA gene expression. Administration of the FFA, palmitate, to mice increased liver IL-1R2 and IL-1RA gene expression by 14- and 11-fold, respectively. Conclusion: These findings indicate that fasting augments expression of endogenous IL-1 antagonists inducing IL-1 resistance. Fasting-induced increases in plasma FFAs appears to be a signal that drives immunosuppression during fasting/short term starvation. PMID:25071776

  6. Two New Alleles of the abscisic aldehyde oxidase 3 Gene Reveal Its Role in Abscisic Acid Biosynthesis in Seeds1

    PubMed Central

    González-Guzmán, Miguel; Abia, David; Salinas, Julio; Serrano, Ramón; Rodríguez, Pedro L.

    2004-01-01

    The abscisic aldehyde oxidase 3 (AAO3) gene product of Arabidopsis catalyzes the final step in abscisic acid (ABA) biosynthesis. An aao3-1 mutant in a Landsberg erecta genetic background exhibited a wilty phenotype in rosette leaves, whereas seed dormancy was not affected (Seo et al., 2000a). Therefore, it was speculated that a different aldehyde oxidase would be the major contributor to ABA biosynthesis in seeds (Seo et al., 2000a). Through a screening based on germination under high-salt concentration, we isolated two mutants in a Columbia genetic background, initially named sre2-1 and sre2-2 (for salt resistant). Complementation tests with different ABA-deficient mutants indicated that sre2-1 and sre2-2 mutants were allelic to aao3-1, and therefore they were renamed as aao3-2 and aao3-3, respectively. Indeed, molecular characterization of the aao3-2 mutant revealed a T-DNA insertional mutation that abolished the transcription of AAO3 gene, while sequence analysis of AAO3 in aao3-3 mutant revealed a deletion of three nucleotides and several missense mutations. Physiological characterization of aao3-2 and aao3-3 mutants revealed a wilty phenotype and osmotolerance in germination assays. In contrast to aao3-1, both aao3-2 and aao3-3 mutants showed a reduced dormancy. Accordingly, ABA levels were reduced in dry seeds and rosette leaves of both aao3-2 and aao3-3. Taken together, these results indicate that AAO3 gene product plays a major role in seed ABA biosynthesis. PMID:15122034

  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. Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

    PubMed

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

    2006-02-01

    Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

  9. Neutral Lipid Biosynthesis in Engineered Escherichia coli: Jojoba Oil-Like Wax Esters and Fatty Acid Butyl Esters

    PubMed Central

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

    2006-01-01

    Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms. PMID:16461689

  10. Dietary ɛ-Polylysine Decreased Serum and Liver Lipid Contents by Enhancing Fecal Lipid Excretion Irrespective of Increased Hepatic Fatty Acid Biosynthesis-Related Enzymes Activities in Rats

    PubMed Central

    Hosomi, Ryota; Yamamoto, Daiki; Otsuka, Ren; Nishiyama, Toshimasa; Yoshida, Munehiro; Fukunaga, Kenji

    2015-01-01

    ɛ-Polylysine (EPL) is used as a natural preservative in food. However, few studies have been conducted to assess the beneficial functions of dietary EPL. The purpose of this study was to elucidate the mechanism underlying the inhibition of neutral and acidic sterol absorption and hepatic enzyme activity-related fatty acid biosynthesis following EPL intake. EPL digest prepared using an in vitro digestion model had lower lipase activity and micellar lipid solubility and higher bile acid binding capacity than casein digest. Male Wistar rats were fed an AIN-93G diet containing 1% (wt/wt) EPL or l-lysine. After 4 weeks of feeding these diets, the marked decrease in serum and liver triacylglycerol contents by the EPL diet was partly attributed to increased fecal fatty acid excretion. The activities of hepatic acetyl-coenzyme A carboxylase and glucose-6-phosphate dehydrogenase, which are key enzymes of fatty acid biosynthesis, were enhanced in rats fed EPL diet. The increased fatty acid biosynthesis activity due to dietary EPL may be prevented by the enhancement of fecal fatty acid excretion. The hypocholesterolemic effect of EPL was mediated by increased fecal neutral and acidic sterol excretions due to the EPL digest suppressing micellar lipid solubility and high bile acid binding capacity. These results show that dietary EPL has beneficial effects that could help prevent lifestyle-related diseases such as hyperlipidemia and atherosclerosis. PMID:25866749

  11. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  12. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  13. Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica

    PubMed Central

    2014-01-01

    Background Sterols are vital structural and regulatory components in eukaryotic cells; however, their biosynthetic pathways and functional roles in microalgae remain poorly understood. Results In the oleaginous microalga Nannochloropsis oceanica, the sterol biosynthetic pathway produces phytosterols as minor products and cholesterol as the major product. The evidence together with their deduced biosynthetic pathways suggests that N. oceanica exhibits features of both higher plants and mammals. Temporal tracking of sterol profiles and sterol-biosynthetic transcripts in response to changes in light intensity and nitrogen supply reveal that sterols play roles in cell proliferation, chloroplast differentiation, and photosynthesis. Furthermore, the dynamics of fatty acid (FA) and FA-biosynthetic transcripts upon chemical inhibitor-induced sterol depletion reveal possible co-regulation of sterol production and FA synthesis, in that the squalene epoxidase inhibitor terbinafine reduces sterol content yet significantly elevates free FA production. Thus, a feedback regulation of sterol and FA homeostasis is proposed, with the 1-deoxy-D-xylulose 5-phosphate synthase (DXS, the committed enzyme in isoprenoid and sterol biosynthesis) gene potentially subject to feedback regulation by sterols. Conclusion These findings reveal features of sterol function and biosynthesis in microalgae and suggest new genetic engineering or chemical biology approaches for enhanced oil production in microalgae. PMID:24920959

  14. 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

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

    PubMed

    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.

  16. 4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP).

    PubMed

    Banoglu, Erden; Çelikoğlu, Erşan; Völker, Susanna; Olgaç, Abdurrahman; Gerstmeier, Jana; Garscha, Ulrike; Çalışkan, Burcu; Schubert, Ulrich S; Carotti, Andrea; Macchiarulo, Antonio; Werz, Oliver

    2016-05-01

    In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis (IC50 = 0.24 μM, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC50 ≥ 8 μM). Docking studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis. PMID:26922224

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

    PubMed

    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

  18. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans. PMID:26806391

  19. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.

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

    PubMed

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

    2016-05-10

    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.

  2. 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.

  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. On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein.

    PubMed

    van der Meer, R A; Groen, B W; Duine, J A

    1989-03-27

    Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.

  5. [Effect of organic acids on the biosynthesis of carotenes by an Actinomyces chrysomallus strain].

    PubMed

    Nefelova, M V; Sverdlova, A N; Alekseeva, L N

    1978-01-01

    Synthesis of carotenes by Actinomyces chrysomallus var. carotenoides was stimulated by citric, acetic, oxalacetic, fumaric, succinic, malic, alpha-ketoglutaric, tartaric, pyruvic, and propionic acids. Acetic acid acts as a precursor of carotene synthesis and also has another stimulating mechanism of action on carotenogenesis of the actinomycete. Acetic, furmaric, malic, succinic, and alpha-ketoglutaric acids stimulate cyclization of lycopene yielding beta-carotene.

  6. Synergistic activation of retinoic acid (RA)-responsive genes and induction of embryonal carcinoma cell differentiation by an RA receptor {alpha} (RAR{alpha})-, RAR{beta}-, or RAR{gamma}-selective ligand in combination with retinoid Z receptor-specific ligand

    SciTech Connect

    Roy, B.; Taneja, R.; Chambon, P.

    1995-12-01

    This research indicates thatn retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers activate transcription of RA-responsive genes and induce cell differentiation of P19 and F9 cells in a ligand-dependent manner. 43 refs., 4 figs., 2 tabs.

  7. A Peroxygenase Pathway Involved in the Biosynthesis of Epoxy Fatty Acids in Oat[W][OA

    PubMed Central

    Meesapyodsuk, Dauenpen; Qiu, Xiao

    2011-01-01

    While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation. PMID:21784965

  8. Biosynthesis of fatty acid derived aldehydes is induced upon mechanical wounding and its products show fungicidal activities in cucumber.

    PubMed

    Matsui, Kenji; Minami, Akari; Hornung, Ellen; Shibata, Hidetoshi; Kishimoto, Kyutaro; Ahnert, Volker; Kindl, Helmut; Kajiwara, Tadahiko; Feussner, Ivo

    2006-04-01

    Fatty acid 9/13-hydroperoxide lyase (9/13-HPL) in cucumber is an enzyme that can cleave either 9- or 13-hydroperoxides of polyunsaturated fatty acids to form C9- or C6-aldehydes, respectively, as products. In order to reveal the physiological function of 9/13-HPL, its expression profiles were analyzed, and it was found that 9/13-HPL expression was developmentally regulated and high in the hypocotyls, female flowers and mature fruits. However, its transcript as well as its activity was only induced by mechanical wounding in mature leaves. To analyze the biosynthesis of HPL-derived aldehydes in more detail we isolated and characterized the yet missing 9-lipoxygenase (LOX) that is mainly expressed in hypocotyls, cotyledons and flowers and that may provide HPL with fatty acid 9-hydroperoxides as substrates. As in the case with C6-aldehydes in most plant species, C9-aldehydes were also formed rapidly after disruption of the tissues. C9-aldehydes had fungicidal activities against fungal pathogens, Botrytis cinerea and Fusarium oxysporum. Because the concentration needed to cause toxic effect on the pathogens was almost equivalent to that found in disrupted tissues, the C9-aldehydes thus formed could be helpful to sterilize the wounds since they are less volatile in comparison to C6-aldehydes. PMID:16497344

  9. Surfactants, aromatic and isoprenoid compounds, and fatty acid biosynthesis inhibitors suppress Staphylococcus aureus production of toxic shock syndrome toxin 1.

    PubMed

    McNamara, Peter J; Syverson, Rae Ellen; Milligan-Myhre, Kathy; Frolova, Olga; Schroeder, Sarah; Kidder, Joshua; Hoang, Thanh; Proctor, Richard A

    2009-05-01

    Menstrual toxic shock syndrome is a rare but potentially life-threatening illness manifest through the actions of Staphylococcus aureus toxic shock syndrome toxin 1 (TSST-1). Previous studies have shown that tampon additives can influence staphylococcal TSST-1 production. We report here on the TSST-1-suppressing activity of 34 compounds that are commonly used additives in the pharmaceutical, food, and perfume industries. Many of the tested chemicals had a minimal impact on the growth of S. aureus and yet were potent inhibitors of TSST-1 production. The TSST-1-reducing compounds included surfactants with an ether, amide, or amine linkage to their fatty acid moiety (e.g., myreth-3-myristate, Laureth-3, disodium lauroamphodiacetate, disodium lauramido monoethanolamido, sodium lauriminodipropionic acid, and triethanolamine laureth sulfate); aromatic compounds (e.g. phenylethyl and benzyl alcohols); and several isoprenoids and related compounds (e.g., terpineol and menthol). The membrane-targeting and -altering effects of the TSST-1-suppressing compounds led us to assess the activity of molecules that are known to inhibit fatty acid biosynthesis (e.g., cerulenin, triclosan, and hexachlorophene). These compounds also reduced S. aureus TSST-1 production. This study suggests that more additives than previously recognized inhibit the production of TSST-1.

  10. Phosphatidylcholine biosynthesis during neuronal differentiation and its role in cell fate determination.

    PubMed

    Marcucci, Hebe; Paoletti, Luciana; Jackowski, Suzanne; Banchio, Claudia

    2010-08-13

    Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase alpha (CK(alpha)) and choline cytidylyltransferase alpha (CCT(alpha)). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT(alpha). Later, the transcription of CK(alpha)- and CCT(alpha)-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK(alpha) or CCTalpha increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT(alpha) or CK(alpha) was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration. PMID:20525991

  11. (+)-Abscisic Acid Metabolism, 3-Ketoacyl-Coenzyme A Synthase Gene Expression, and Very-Long-Chain Monounsaturated Fatty Acid Biosynthesis in Brassica napus Embryos1

    PubMed Central

    Qi, Qungang; Rose, Patricia A.; Abrams, Garth D.; Taylor, David C.; Abrams, Suzanne R.; Cutler, Adrian J.

    1998-01-01

    Microspore-derived embryos of Brassica napus cv Reston were used to examine the effects of exogenous (+)-abscisic acid (ABA) and related compounds on the accumulation of very-long-chain monounsaturated fatty acids (VLCMFAs), VLCMFA elongase complex activity, and induction of the 3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing enzyme of the VLCMFA elongation system. Of the concentrations tested, (+)-ABA at 10 μm showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 μm (+)-ABA treatment, was 60% higher than that of the untreated embryos at 24 h. The transcript of the KCS gene was induced by 10 μm (+)-ABA within 1 h and further increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with (+)-ABA for 72 h. Also, (+)-8′-methylene ABA, which is metabolized more slowly than ABA, had a stronger ABA-like effect on the KCS gene transcription, elongase complex activity (28% higher), and level of VLCMFAs (25–30% higher) than ABA. After 24 h approximately 60% of the added (+)-[3H]ABA (10 μm) was metabolized, yielding labeled phaseic and dihydrophaseic acid. This study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via increased expression of the KCS gene and that reducing ABA catabolism would increase VLCMFAs in microspore-derived embryos. PMID:9662540

  12. Isolation and chemical structure of aklanonic acid, an early intermediate in the biosynthesis of anthracyclines.

    PubMed

    Eckardt, K; Tresselt, D; Schumann, G; Ihn, W; Wagner, C

    1985-08-01

    The fermentation, isolation and structure elucidation of aklanonic acid are described. The compound was isolated from fermentations of Streptomyces strain ZIMET 43,717. Aklanonic acid is a yellow-orange crystalline substance, melting at 203-204 degrees C (dec), having the molecular formula C21H16O8, and possessing UV maxima at 258, 282 (sh) and 438 nm (CHCl3). In dimethyl sulfoxide or pyridine aklanonic acid is unstable and a new compound (aklanone) is formed as a conversion product. The elucidation of the structures has shown that aklanonic acid and aklanone are derivatives of 1,8-dihydroxyanthraquinone. PMID:3862658

  13. 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

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

    PubMed

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

    2015-12-16

    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.

  15. 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

  16. Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

    PubMed

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Ishida, Haruka; Tomida, Kensuke; Sakai, Miwa; Hara, Masakazu; Watanabe, Naoharu

    2012-03-15

    In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe. PMID:22236980

  17. Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

    PubMed

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Ishida, Haruka; Tomida, Kensuke; Sakai, Miwa; Hara, Masakazu; Watanabe, Naoharu

    2012-03-15

    In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe.

  18. ω-Azido fatty acids as probes to detect fatty acid biosynthesis, degradation, and modification[S

    PubMed Central

    Pérez, Alexander J.; Bode, Helge B.

    2014-01-01

    FAs play a central role in the metabolism of almost all known cellular life forms. Although GC-MS is regarded as a standard method for FA analysis, other methods, such as HPLC/MS, are nowadays widespread but are rarely applied to FA analysis. Here we present azido-FAs as probes that can be used to study FA biosynthesis (elongation, desaturation) or degradation (β-oxidation) upon their uptake, activation, and metabolic conversion. These azido-FAs are readily accessible by chemical synthesis and their metabolic products can be easily detected after click-chemistry based derivatization with high sensitivity by HPLC/MS, contributing a powerful tool to FA analysis, and hence, lipid analysis in general. PMID:25013232

  19. Docosahexaenoic acid biosynthesis via fatty acyl elongase and Δ4-desaturase and its modulation by dietary lipid level and fatty acid composition in a marine vertebrate.

    PubMed

    Morais, Sofia; Mourente, Gabriel; Martínez, Almudena; Gras, Noélia; Tocher, Douglas R

    2015-05-01

    The present study presents the first "in vivo" evidence of enzymatic activity and nutritional regulation of a Δ4-desaturase-dependent DHA synthesis pathway in the teleost Solea senegalensis. Juvenile fish were fed diets containing 2 lipid levels (8 and 18%, LL and HL) with either 100% fish oil (FO) or 75% of the FO replaced by vegetable oils (VOs). Fatty acyl elongation (Elovl5) and desaturation (Δ4Fad) activities were measured in isolated enterocytes and hepatocytes incubated with radiolabeled α-linolenic acid (ALA; 18:3n-3) and eicosapentaenoic acid (EPA; 20:5n-3). Tissue distributions of elovl5 and Δ4fad transcripts were also determined, and the transcriptional regulation of these genes in liver and intestine was assessed at fasting and postprandially. DHA biosynthesis from EPA occurred in both cell types, although Elovl5 and Δ4Fad activities tended to be higher in hepatocytes. In contrast, no Δ6Fad activity was detected on (14)C-ALA, which was only elongated to 20:3n-3. Enzymatic activities and gene transcription were modulated by dietary lipid level (LL>HL) and fatty acid (FA) composition (VO>FO), more significantly in the liver than in the intestine, which was reflected in tissue FA compositions. Dietary VO induced a significant up-regulation of Δ4fad transcripts in the liver 6h after feeding, whereas in fasting conditions the effect of lipid level possibly prevailed over or interacted with FA composition in regulating the expression of elovl5 and Δ4fad, which were down-regulated in the liver of fish fed the HL diets. Results indicated functionality and biological relevance of the Δ4 LC-PUFA biosynthesis pathway in S. senegalensis.

  20. Biosynthetic mechanism for L-Gulose in main polar lipids of Thermoplasma acidophilum and possible resemblance to plant ascorbic acid biosynthesis.

    PubMed

    Yamauchi, Noriaki; Nakayama, Yusuke

    2013-01-01

    L-Gulose is a very rare sugar, but appears as a sugar component of the main polar lipids characteristic in such a thermophilic archaeon as Thermoplasma acidophilum that lives without cell walls in a highly acidic environment. The biosynthesis of L-gulose in this thermophilic organism was investigated with deuterium-labeling experiments. L-Gulose was found to be biosynthesized from D-glucose via stepwise stereochemical inversion at C-2 and C-5. The involvement of an epimerase related to GDP-mannose 3,5-epimerase, the key enzyme of plant ascorbate biosynthesis, was also suggested in this C-5 inversion. The resemblance of L-gulose biosynthesis in archaea and plants might be suggested from these results.

  1. 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...

  2. 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.

  3. Heteroconium chaetospira induces resistance to clubroot via upregulation of host genes involved in jasmonic acid, ethylene, and auxin biosynthesis.

    PubMed

    Lahlali, Rachid; McGregor, Linda; Song, Tao; Gossen, Bruce D; Narisawa, Kazuhiko; Peng, Gary

    2014-01-01

    An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc), suppressed clubroot (Plasmodiophora brassicae -Pb) on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001) with the severity of clubroot at 5 weeks after treatment at a low (2×10(5) spores pot(-1)) but not high (2×10(5) spores pot(-1)) dose of pathogen inoculum. Transcript levels of nine B. napus (Bn) genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL). These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL) involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2), ethylene (BnACO), auxin (BnAAO1), and PR-2 protein (BnPR-2) biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte.

  4. Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

    PubMed Central

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

    2014-01-01

    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. PMID:25287924

  5. Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.

    PubMed

    Shlaifer, Irina; Turnbull, Joanne L

    2016-07-01

    Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity. PMID:27290727

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

    PubMed

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

    2004-10-01

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

  7. 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.

  8. New approaches to target the mycolic acid biosynthesis pathway for the development of tuberculosis therapeutics.

    PubMed

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

    2014-01-01

    Mycolic acids are the major lipid components 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

  9. 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

  10. Stereospecific synthesis of threo- and erythro-beta-hydroxyglutamic acid during kutzneride biosynthesis.

    PubMed

    Strieker, Matthias; Nolan, Elizabeth M; Walsh, Christopher T; Marahiel, Mohamed A

    2009-09-23

    The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one alpha-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the beta-position of glutamic acid. KtzO generates threo-L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs nonhydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering approaches.

  11. Pseudomonas aeruginosa directly shunts β-oxidation degradation intermediates into de novo fatty acid biosynthesis.

    PubMed

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

    2012-10-01

    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 C(8)-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.

  12. FgIlv5 is required for branched-chain amino acid biosynthesis and full virulence in Fusarium graminearum.

    PubMed

    Liu, Xin; Wang, Jian; Xu, Jianhong; Shi, Jianrong

    2014-04-01

    In this study, we characterized FgIlv5, a homologue of the Saccharomyces cerevisiae keto-acid reductoisomerase (KARI) from the important wheat head scab fungus Fusarium graminearum. KARI is a key enzyme in the branched-chain amino acid (BCAA, including leucine, isoleucine and valine) biosynthetic pathway that exists in a variety of organisms from bacteria to fungi and higher plants, but not in mammals. The FgILV5 deletion mutant ΔFgIlv5-4 failed to grow when the culture medium was nutritionally limited for BCAAs. When grown on potato-dextrose agar plates, ΔFgIlv5-4 exhibited a significant decrease in aerial hyphae formation and red pigmentation. Conidia formation was also blocked in ΔFgIlv5-4. Exogenous addition of 1 mM isoleucine and valine was able to rescue the defects of mycelial growth and conidial morphogenesis. Cellular stress assays showed that ΔFgIlv5-4 was more sensitive to osmotic and oxidative stresses than the wild-type strain. In addition, virulence of ΔFgIlv5-4 was dramatically reduced on wheat heads, and a low level of deoxynivalenol production was detected in ΔFgIlv5-4 in wheat kernels. The results of this study indicate that FgIlv5 is involved in valine and isoleucine biosynthesis and is required for full virulence in F. graminearum.

  13. Indole-3-acetic acid biosynthesis in the biocontrol strain Pseudomonas fluorescens Psd and plant growth regulation by hormone overexpression.

    PubMed

    Kochar, Mandira; Upadhyay, Ashutosh; Srivastava, Sheela

    2011-05-01

    Pseudomonas fluorescens is an important biological component of agricultural soils that bestows a number of direct and indirect beneficial attributes to the plants. We analyzed the biocontrol strain P. fluorescens Psd for indole-3-acetic acid (IAA) biosynthesis and studied the effect of its consequent manipulation on its plant-growth-promoting (PGP) potential. While the indole pyruvic acid (IPyA) pathway commonly associated with PGP bacteria was lacking, the indole acetamide (IAM) pathway generally observed in phytopathogens was expressed in strain Psd. Overexpression of IAM pathway genes iaaM-iaaH, from Pseudomonas syringae subsp. savastanoi drastically increased IAA levels and showed a detrimental effect on sorghum root development. On the other hand, heterologous expression of the indole-3-pyruvate decarboxylase/phenylpyruvate decarboxylase gene (ipdC/ppdC) of the IPyA pathway from the PGP bacterium Azospirillum brasilense SM led to enhancement of the IAA level. A more favorable effect of this recombinant strain on sorghum root growth and development suggests that metabolic engineering could be used to generate strains with improved PGP function.

  14. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

    PubMed

    Gatto, Gregory J; Boyne, Michael T; Kelleher, Neil L; Walsh, Christopher T

    2006-03-22

    Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner. PMID:16536560

  15. 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

  16. Biosynthesis of mercapturic acids from allyl alcohol, allyl esters and acrolein

    PubMed Central

    Kaye, Clive M.

    1973-01-01

    1. 3-Hydroxypropylmercapturic acid, i.e. N-acetyl-S-(3-hydroxypropyl)-l-cysteine, was isolated, as its dicyclohexylammonium salt, from the urine of rats after the subcutaneous injection of each of the following compounds: allyl alcohol, allyl formate, allyl propionate, allyl nitrate, acrolein and S-(3-hydroxypropyl)-l-cysteine. 2. Allylmercapturic acid, i.e. N-acetyl-S-allyl-l-cysteine, was isolated from the urine of rats after the subcutaneous injection of each of the following compounds: triallyl phosphate, sodium allyl sulphate and allyl nitrate. The sulphoxide of allylmercapturic acid was detected in the urine excreted by these rats. 3. 3-Hydroxypropylmercapturic acid was identified by g.l.c. as a metabolite of allyl acetate, allyl stearate, allyl benzoate, diallyl phthalate, allyl nitrite, triallyl phosphate and sodium allyl sulphate. 4. S-(3-Hydroxypropyl)-l-cysteine was detected in the bile of a rat dosed with allyl acetate. PMID:4762754

  17. Biosynthesis of gamma-linolenic acid and beta-carotene by Zygomycetes fungi.

    PubMed

    Klempova, Tatiana; Basil, Eva; Kubatova, Alena; Certik, Milan

    2013-07-01

    Due to increasing demand for natural sources of both polyunsaturated fatty acids (PUFAs) and beta-carotene, 28 Zygomycetes fungal soil isolates were screened for their potential to synthesize these biologically active compounds. Although all fungi produced C18 PUFAs, only nine strains also formed beta-carotene. Although Actinomucor elegans CCF 3218 was the best producer of gamma-linolenic acid (GLA) (251 mg/L), Umbelopsis isabellina CCF 2412 was found to be the most valuable fungus because of the dual production of GLA (217 mg/L) and beta-carotene (40.7 mg/L). The calculated ratio of formed PUFAs provided new insight into activities of individual fatty acid desaturases involved in biosynthetic pathways for various types of PUFAs. The maximal activity of delta-9 desaturase was accompanied by high accumulation of storage lipids in fungal cells. On the other hand, maximal activity of delta-15 desaturase was found in strains synthesizing low amounts of oleic acid due to diminished delta-9 desaturase. Activities of delta-6 desaturase showed competition for fatty acids engaged in n3, n6, and n9 biosynthetic pathways. Such knowledge about fatty acid desaturase activities provides new challenges for the regulation of biotechnological production of PUFAs by Zygomycetes fungi. PMID:23625863

  18. 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 (...

  19. Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

    PubMed Central

    Darzins, A; Wang, S K; Vanags, R I; Chakrabarty, A M

    1985-01-01

    A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min. Images PMID:3932325

  20. Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression.

    PubMed

    Lee, Dong Wan; Ng, Bee Gek; Kim, Beom Seok

    2015-11-01

    Streptomyces sp. M10 is a valinomycin-producing bacterial strain that shows potent bioactivity against Botrytis blight of cucumber plants. During studies to increase the yield of valinomycin (a cyclododecadepsipeptide) in strain M10, additional antifungal metabolites, including bafilomycin derivatives (macrolide antibiotics), were identified. To examine the effect of bafilomycin biosynthesis on valinomycin production, the bafilomycin biosynthetic gene cluster was cloned from the genome of strain M10, as were two branched-chain α-keto acid dehydrogenase (BCDH) gene clusters related to precursor supply for bafilomycin biosynthesis. A null mutant (M10bafm) of one bafilomycin biosynthetic gene (bafV) failed to produce bafilomycin, but resulted in a 1.2- to 1.5-fold increase in the amount of valinomycin produced. In another null mutant (M10bkdFm) of a gene encoding a subunit of the BCDH complex (bkdF), bafilomycin production was completely abolished and valinomycin production increased fourfold relative to that in the wild-type M10 strain. The higher valinomycin yield was likely the result of redistribution of the metabolic flux from bafilomycin to valinomycin biosynthesis, because the two antibiotics share a common precursor, 2-ketoisovaleric acid, a deamination product of valine. The results show that directing precursor flux toward active ingredient biosynthesis could be used as a prospective tool to increase the competence of biofungicides.

  1. Biochemical and Genetic Engineering of Diatoms for Polyunsaturated Fatty Acid Biosynthesis

    PubMed Central

    Li, Hong-Ye; Lu, Yang; Zheng, Jian-Wei; Yang, Wei-Dong; Liu, Jie-Sheng

    2014-01-01

    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. PMID:24402175

  2. 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

  3. Biosynthesis of conjugated triene-containing fatty acids by a novel isomerase from the red marine alga Ptilota filicina.

    PubMed

    Wise, M L; Hamberg, M; Gerwick, W H

    1994-12-27

    The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was used to determine that the major protein in the purified enzyme is composed of similar or identical subunits of M(r) 58,119 Da. The native enzyme emerges with an apparent M(r) of 174,000 Da from a gel permeation chromatography column. While this enzyme catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)- eicosatetraenoate) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatrienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,14Z,17Z)- eicosapentaenoate], which is likely the native substrate. Through a series of experiments utilizing gamma-linolenates stereospecifically labeled with deuterium, we have determined that the enzyme intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position. Furthermore, the bis-allylic pro-R hydrogen at C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes protonated by a solvent-derived proton. There appears to be no requirement for molecular oxygen, and the transformation is catalyzed by this single enzyme. PMID:7803384

  4. 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.

  5. 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

    2016-05-01

    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. PMID:26945769

  6. 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

    2016-05-01

    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.

  7. 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

  8. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes. PMID:27370696

  9. Mutational analysis reveals that all tailoring region genes are required for production of polyketide antibiotic mupirocin by pseudomonas fluorescens: pseudomonic acid B biosynthesis precedes pseudomonic acid A.

    PubMed

    Hothersall, Joanne; Wu, Ji'en; Rahman, Ayesha S; Shields, Jennifer A; Haddock, James; Johnson, Nicola; Cooper, Sian M; Stephens, Elton R; Cox, Russell J; Crosby, John; Willis, Christine L; Simpson, Thomas J; Thomas, Christopher M

    2007-05-25

    The Pseudomonas fluorescens mupirocin biosynthetic cluster encodes six proteins involved in polyketide biosynthesis and 26 single polypeptides proposed to perform largely tailoring functions. In-frame deletions in the tailoring open reading frames demonstrated that all are required for mupirocin production. A bidirectional promoter region was identified between mupF, which runs counter to other open reading frames and its immediate neighbor macpC, implying the 74-kb cluster consists of two transcriptional units. mupD/E and mupJ/K must be cotranscribed as pairs for normal function implying co-assembly during translation. MupJ and K belong to a widely distributed enzyme pair implicated, with MupH, in methyl addition. Deletion of mupF, a putative ketoreductase, produced a mupirocin analogue with a C-7 ketone. Deletion of mupC, a putative dienoyl CoA reductase, generated an analogue whose structure indicated that MupC is also implicated in control of the oxidation state around the tetrahydropyran ring of monic acid. Double mutants with DeltamupC and DeltamupO, DeltamupU, DeltamupV, or DeltamacpE produced pseudomonic acid B but not pseudomonic acid A, as do the mupO, U, V, and macpE mutants, indicating that MupC must work after MupO, U, and V.

  10. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.

  11. Analysis of the pmsCEAB Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain Pseudomonas fluorescens WCS374

    PubMed Central

    Mercado-Blanco, Jesús; van der Drift, Koen M. G. M.; Olsson, Per E.; Thomas-Oates, Jane E.; van Loon, Leendert C.; Bakker, Peter A. H. M.

    2001-01-01

    Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production. PMID:11222588

  12. 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.

  13. 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...

  14. 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.

  15. The function of galactosyl phosphorylpolyprenol in biosynthesis of lipoteichoic acid in Bacillus coagulans.

    PubMed

    Yokoyama, K; Araki, Y; Ito, E

    1988-04-15

    Incubation of UDP-[14C]galactose with membranes of Bacillus coagulans led to the formation of a radioactive glycolipid, which was tentatively characterized as beta-galactosyl phosphorylpolyprenol (Gal-P-prenol) on the basis of its chromatographic behavior and data from structural analysis of its sugar 1-phosphate moiety. The sugar moiety of [14C]Gal-P-prenol was shown to be incorporated into a membrane-bound polymer, which coincided with the diacyl form of lipoteichoic acid in its chromatographic behavior on columns of Sephacryl S-300, DEAE-Sephacel and octyl-Sepharose. Hydrogen fluoride hydrolysis of the polymer afforded an alpha-galactoside identical with Gal(alpha 1----2)Gro obtained from lipoteichoic acids. The incorporation of galactose residues from [14C]Gal-P-prenol into the polymer was greatly enhanced by exogenous lipoteichoic acids, especially of the diacyl and monoacyl forms. The optimal pH and metal concentration for the Gal-P-prenol formation, respectively, were found to be 8.4 and 10 mM (MgCl2), whereas those for the transfer of galactose from this lipid intermediate to polymer were 4.5 and 16 mM (CaCl2). The above results lead to the conclusion that Gal-P-prenol serves as the direct galactosyl donor in the synthesis of lipoteichoic acids in B. coagulans.

  16. Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production.

    PubMed

    Haushalter, Robert W; Groff, Dan; Deutsch, Samuel; The, Lionadi; Chavkin, Ted A; Brunner, Simon F; Katz, Leonard; Keasling, Jay D

    2015-07-01

    Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.

  17. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines.

    PubMed

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome". Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression.

  18. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines

    PubMed Central

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the “kinome”. Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression. PMID:27362937

  19. Preparation of (228)Ra standard solution.

    PubMed

    Havelka, Miroslav

    2016-03-01

    For the preparation of a standard solution of (228)Ra, (228)Ra was isolated from (232)Th salt. Two simple methods were developed for Th-Ra separation. Both are based on a very good solubility of thorium nitrate in organic solvents. The first one used Ra co-precipitation with Pb in the form of Pb(NO3)2 from acetic acid solution. The second method was based on solvent extraction, remaining Th in the organic phase, while Ra was concentrated in the aqueous phase. The activity of (228)Ra (up to 20kBq) in the standard solution was related to the (232)Th standard by means of gamma ray spectrometry measurement. The obtained uncertainty was less than 0.7% (k=1). The standard solution was free of (232)Th and contained the carrier in the usual concentration (1gL(-1) BaCl2, 10gL(-1) HCl). PMID:26651171

  20. GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

    PubMed

    Kriel, Allison; Brinsmade, Shaun R; Tse, Jessica L; Tehranchi, Ashley K; Bittner, Alycia N; Sonenshein, Abraham L; Wang, Jue D

    2014-01-01

    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  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 the Escherichia coli K1 group 2 polysialic acid capsule occurs within a protected cytoplasmic compartment.

    PubMed

    Steenbergen, Susan M; Vimr, Eric R

    2008-06-01

    Capsular polysaccharides are important virulence determinants in a wide range of invasive infectious diseases. Although capsule synthesis has been extensively investigated, understanding polysaccharide export from the cytoplasm to the external environment has been more difficult. Here we present the results of a novel protection assay indicating that synthesis and export of the Escherichia coli K1 group 2 capsular polysialic acid (K1 antigen) occur within a protected subcellular compartment designated the sialisome. In addition to the polymerase encoded by neuS, localization and complementation analyses indicated that the sialisome includes the accessory membrane protein NeuE. The requirement for NeuE was suppressed by overproducing NeuS, suggesting that NeuE functions by stabilizing the polymerase or facilitating its assembly in the sialisome. Although an interaction between NeuE and NeuS could not be demonstrated with a bacterial two-hybrid system that reconstitutes an intracellular cell-signalling pathway, interactions between NeuS and KpsC as well as other sialisome components were detected. The combined results provide direct evidence for specific protein-protein interactions in the synthesis and export of group 2 capsular polysaccharides under in vivo conditions. The approaches developed here will facilitate further dissection of the sialisome, suggesting similar methodology for understanding the biosynthesis of other group 2 capsules.

  3. 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

  4. 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.

  5. 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.

  6. 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. PMID:22608992

  7. 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

  8. The Mechanism of MIO-Based Aminomutases in beta-Amino Acid Biosynthesis

    SciTech Connect

    Christianson,C.; Montavon, T.; Festin, G.; Cooke, H.; Shen, B.; Bruner, S.

    2007-01-01

    {beta}-Amino acids are widely used building blocks in both natural and synthetic compounds. Aromatic {beta}-amino acids can be biosynthesized directly from proteinogenic a-amino acids by the action of MIO (4-methylideneimidazole-5-one)-based aminomutase enzymes. The uncommon cofactor MIO plays a role in both ammonia lyases and 2, 3-aminomutases; however, the precise mechanism of the cofactor has not been resolved. Here we provide evidence that the electrophilic cofactor uses covalent catalysis through the substrate amine to direct the elimination and subsequent readdition of ammonia. A mechanism-based inhibitor was synthesized and the X-ray cocomplex structure was determined to 2.0 Angstroms resolution. The inhibitor halts the chemistry of the reverse reaction, providing a stable complex that establishes the mode of substrate binding and the importance of tyrosine 63 in the chemistry. The proposed mechanism is consistent with the biochemistry of aminomutases and ammonia lyases and provides strong support for an amine-adduct mechanism of catalysis for this enzyme class.

  9. Genetic engineering activates biosynthesis of aromatic fumaric acid amides in the human pathogen Aspergillus fumigatus.

    PubMed

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H; Dahse, Hans-Martin; Brakhage, Axel A; Hoffmeister, Dirk

    2015-03-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

  10. Genetic Engineering Activates Biosynthesis of Aromatic Fumaric Acid Amides in the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H.; Dahse, Hans-Martin; Brakhage, Axel A.

    2014-01-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds. PMID:25527545

  11. 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.

  12. 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

  13. Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects.

    PubMed

    Hamberger, Björn; Ohnishi, Toshiyuki; Hamberger, Britta; Séguin, Armand; Bohlmann, Jörg

    2011-12-01

    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

  14. Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects.

    PubMed

    Hamberger, Björn; Ohnishi, Toshiyuki; Hamberger, Britta; Séguin, Armand; Bohlmann, Jörg

    2011-12-01

    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.

  15. 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 ...

  16. Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.

    PubMed

    Bordewick, U; Heese, M; Börchers, T; Robenek, H; Spener, F

    1989-03-01

    Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.

  17. In silico analysis of amino acid biosynthesis and proteolysis in Lactobacillus delbrueckii subsp. bulgaricus 2038 and the implications for bovine milk fermentation.

    PubMed

    Zheng, Huajun; Liu, Enuo; Hao, Pei; Konno, Tomonobu; Oda, Munehiro; Ji, Zai-Si

    2012-08-01

    The amino acid biosynthesis pathway and proteolytic system of Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038), a mainstay of large-scale yogurt production, were modeled based on its genomic sequence. L. bulgaricus 2038 retains more potential for amino acid synthesis and a more powerful proteolytic system than other L. bulgaricus strains, but favors amino acid uptake over de novo synthesis. Free amino acids and peptides in bovine milk provide the main nitrogen sources; whey is more important than casein for L. bulgaricus during fermentation. Free amino acids are imported by amino acid permeases and by ABC-type transport systems whereas exogenous oligopeptides are imported by ABC-type proteins only. Histidine is neither synthesized nor imported singly, which might explain why L. bulgaricus cannot grow in synthetic media.

  18. Brassinosteroids Improve Quality of Summer Tea (Camellia sinensis L.) by Balancing Biosynthesis of Polyphenols and Amino Acids.

    PubMed

    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

  19. Brassinosteroids Improve Quality of Summer Tea (Camellia sinensis L.) by Balancing Biosynthesis of Polyphenols and Amino Acids.

    PubMed

    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

  20. 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

  1. 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

  2. Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis.

    PubMed

    Bhatt, Apoorva; Kremer, Laurent; Dai, Annie Z; Sacchettini, James C; Jacobs, William R

    2005-11-01

    Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development. PMID:16267284

  3. The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

    PubMed Central

    Tahlan, Kapil; Anders, Cecilia; Jensen, Susan E.

    2004-01-01

    Carboxyethylarginine synthase, encoded by the paralogous ceaS1 and ceaS2 genes, catalyzes the first reaction in the shared biosynthetic pathway leading to clavulanic acid and the other clavam metabolites in Streptomyces clavuligerus. The nutritional regulation of ceaS1 and ceaS2 expression was analyzed by reverse transcriptase PCR and by the use of the enhanced green fluorescent protein-encoding gene (egfp) as a reporter. ceaS1 was transcribed in complex soy medium only, whereas ceaS2 was transcribed in both soy and defined starch-asparagine (SA) media. The transcriptional start points of the two genes were also mapped to a C residue 98 bp upstream of ceaS1 and a G residue 51 bp upstream of the ceaS2 start codon by S1 nuclease protection and primer extension analyses. Furthermore, transcriptional mapping of the genes encoding the beta-lactam synthetase (bls1) and proclavaminate amidinohydrolase (pah1) isoenzymes from the paralogue gene cluster indicated that a single polycistronic transcript of ∼4.9 kb includes ceaS1, bls1, and pah1. The expression of ceaS1 and ceaS2 in a mutant strain defective in the regulatory protein CcaR was also examined. ceaS1 transcription was not affected in the ccaR mutant, whereas that of ceaS2 was greatly reduced compared to the wild-type strain. Overall, our results suggest that different mechanisms are involved in regulating the expression of ceaS1 and ceaS2, and presumably also of other paralogous genes that encode proteins involved in the early stages of clavulanic acid and clavam metabolite biosynthesis. PMID:15342599

  4. 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.

  5. 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

  6. Biosynthesis of fatty acids and cholesterol in squid giant nerve fiber

    SciTech Connect

    Tanaka, T.; Kishimoto, Y.; Gould, R.M.

    1987-05-01

    The giant nerve fiber of squid/sub 3/ (loligo pealel) was incubated with (1-/sup 14/C)acetate and (/sup 3/H)myristate. Fifty-five percent of the radioactivity incorporated from acetate was recovered in fatty acid methyl esters (FAME). The FAME was fractionated by AgNO/sub 3/-impregnated TLC plate; and 52%, 31%, 0.3%, 5%, and 11% of the radioactivity was recovered in spots associated with saturated, monoenes, dienes, trienes, and polyenes, respectively. The saturated FAME was further fractionated by reverse-phase HPLC; and 4, 47, 1, and 48% of the radioactivity was recovered in 14:0, 16:0, 17:0, and 18:0, respectively. Most radioactivity from polyenes migrated to the spot containing saturated FAME, after catalytical hydrogenation. Cholesterol was isolated and converted to dibromoderivative and the derivative was recrystallized. No radioactivity was found in purified dibromocholesterol. Nearly all radioactivity from myristate-labeled tissue was recovered in saturated FAME. Reverse-phase HPLC showed that 71%, 25%, and 3% of radioactivity was associated with 14:0, 16:0, and 18:0, respectively. These results indicate that the squid giant nerve fibers can synthesize fatty acids by de novo and also by chain elongation and desaturation, though at a slower rate.

  7. Ethanol Effects On Physiological Retinoic Acid Levels

    PubMed Central

    Napoli, Joseph L.

    2011-01-01

    Summary All-trans-retinoic acid (atRA) serves essential functions during embryogenesis and throughout post-natal vertebrate life. Insufficient or excess atRA causes teratogenic and/or toxic effects in the developing embryo: interference with atRA biosynthesis or signaling likely underlies some forms of cancer. Many symptoms of vitamin A (atRA precursor) deficiency and/or toxicity overlap with those of another pleiotropic agent—ethanol. These overlapping symptoms have prompted research to understand whether interference with atRA biosynthesis and/or action may explain (in part) pathology associated with excess ethanol consumption. Ethanol affects many aspects of retinoid metabolism and mechanisms of action site-specifically, but no robust data support inhibition of vitamin A metabolism, resulting in decreased atRA in vivo during normal vitamin A nutriture. Actually, ethanol either has no effect on or increases atRA at select sites. Despite this realization, insight into whether interactions between ethanol and retinoids represent cause vs. effect requires additional research. PMID:21766417

  8. Distribution of sulphuric acid aerosols in the clouds and upper haze of Venus using Venus Express VAST and VeRa temperature profiles

    NASA Astrophysics Data System (ADS)

    Parkinson, Christopher D.; Gao, Peter; Schulte, Rick; Bougher, Stephen W.; Yung, Yuk L.; Bardeen, Charles G.; Wilquet, Valérie; Vandaele, Ann Carine; Mahieux, Arnaud; Tellmann, Silvia; Pätzold, Martin

    2015-08-01

    Observations from Pioneer Venus and from SPICAV/SOIR aboard Venus Express (VEx) have shown the upper haze (UH) of Venus to be highly spatially and temporally variable, and populated by multiple particle size modes. Previous models of this system (e.g., Gao et al., 2014. Icarus 231, 83-98), using a typical temperature profile representative of the atmosphere (viz., equatorial VIRA profile), did not investigate the effect of temperature on the UH particle distributions. We show that the inclusion of latitude-dependent temperature profiles for both the morning and evening terminators of Venus helps to explain how the atmospheric aerosol distributions vary spatially. In this work we use temperature profiles obtained by two instruments onboard VEx, VeRa and SPICAV/SOIR, to represent the latitudinal temperature dependence. We find that there are no significant differences between results for the morning and evening terminators at any latitude and that the cloud base moves downwards as the latitude increases due to decreasing temperatures. The UH is not affected much by varying the temperature profiles; however, the haze does show some periodic differences, and is slightly thicker at the poles than at the equator. We also find that the sulphuric acid "rain" seen in previous models may be restricted to the equatorial regions of Venus, such that the particle size distribution is relatively stable at higher latitudes and at the poles.

  9. Lysosomal Acid Phosphatase Biosynthesis and Dysfunction: A Mini Review Focused on Lysosomal Enzyme Dysfunction in Brain.

    PubMed

    Ashtari, N; Jiao, X; Rahimi-Balaei, M; Amiri, S; Mehr, S E; Yeganeh, B; Marzban, H

    2016-01-01

    Lysosomes are membrane-bound organelles that are responsible for degrading and recycling macromolecules. Lysosomal dysfunction occurs in enzymatic and non-enzymatic deficiencies, which result in abnormal accumulation of materials. Although lysosomal storage disorders affect different organs, the central nervous system is the most vulnerable. Evidence shows the role of lysosomal dysfunction in different neurodegenerative diseases, such as Niemann-Pick Type C disease, juvenile neuronal ceroid lipofuscinosis, Alzheimer's disease and Parkinson's disease. Lysosomal enzymes such as lysosomal acid phosphatase 2 (Acp2) play a critical role in mannose-6-phosphate removal and Acp2 controls molecular and cellular functions in the brain during development and adulthood. Acp2 is essential in cerebellar development, and mutations in this gene cause severe cerebellar neurodevelopmental and neurodegenerative disorders. In this mini-review, we highlight lysosomal dysfunctions in the pathogenesis of neurodevelopmental and/or neurodegenerative diseases with special attention to Acp2 dysfunction. PMID:27132795

  10. Biosynthesis of isoxazolin-5-one and 3-nitropropanoic acid containing glucosides in juvenile Chrysomelina.

    PubMed

    Becker, Tobias; Ploss, Kerstin; Boland, Wilhelm

    2016-07-14

    Stable-isotope-labeled precursors were used to establish the biosynthetic pathway leading from β-alanine towards isoxazolin-5-one glucoside 1 and its 3-nitropropanoate (3-NPA) ester 2 in Chrysomelina larvae. Both structural elements originate from sequestered plant-derived β-alanine or from propanoyl-CoA that is derived from the degradation of some essential amino acids, e.g. valine. β-Alanine is converted into 3-NPA and isoxazolinone 5 by consecutive oxidations of the amino group of β-Ala. Substituting the diphospho group of α-UDP-glucose with 5 generates the isoxazolin-5-one glucoside 1, which serves in the circulating hemolymph of the larva as a platform for esterification with 3-nitropropanoyl-CoA. The pathway was validated with larvae of Phaedon cochleariae, Chrysomela populi as well as Gastrophysa viridula. PMID:27272952

  11. The dlt operon in the biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei.

    PubMed

    Neuhaus, F C; Heaton, M P; Debabov, D V; Zhang, Q

    1996-01-01

    The D-alanine incorporation system allows Lactobacillus casei to modulate the chemical properties of lipoteichoic acid (LTA) and hence control its proposed functions, i.e., regulation of autolysin action, metal ion binding, and the electromechanical properties of the cell wall. The system requires the D-alanine-D-alanyl carrier protein ligase (Dcl) and the D-alanyl carrier protein (Dcp). Our results indicate that the genes for these proteins are encoded in the dlt operon and that this operon contains at least 2 other genes, dltB and dltD. The aim of this paper is to describe the genetic organization of the operon, the role of the D-alanyl carrier protein, and the function of the putative protein encoded by dltB in the intramembranal translocation of the activated D-alanine. PMID:9158726

  12. 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.

  13. Occurrence and in Vivo Biosynthesis of Indole-3-Butyric Acid in Corn (Zea mays L.) 1

    PubMed Central

    Ludwig-Müller, Jutta; Epstein, Ephraim

    1991-01-01

    Indole-3-butyric acid (IBA) was identified as an endogenous compound in leaves and roots of maize (Zea mays L.) var Inrakorn by thin layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Its presence was also confirmed in the variety Hazera 224. Indole-3-acetic acid (IAA) was metabolized to IBA in vivo by seedlings of the two maize varieties. The reaction product was identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry after incubating the corn seedlings with [14C]IAA and [13C6]IAA. The in vivo conversion of IAA to IBA and the characteristics of IBA formation in two different maize varieties of Zea mays L. (Hazera 224 and Inrakorn) were investigated. IBA-forming activity was examined in the roots, leaves, and coleoptiles of both maize varieties. Whereas in the variety Hazera 224, IBA was formed mostly in the leaves, in the variety Inrakorn, IBA synthesis was detected in the roots as well as in the leaves. A time course study of IBA formation showed that maximum activity was reached in Inrakorn after 1 hour and in Hazera after 2 hours. The pH optimum for the uptake of IAA was 6.0, and that for IBA formation was 7.0. The Km value for IBA formation was 17 micromolar for Inrakorn and 25 micromolar for Hazera 224. The results are discussed with respect to the possible functions of IBA in the plant. ImagesFigure 5 PMID:16668464

  14. Characterization of the RNA Required for Biosynthesis of δ-Aminolevulinic Acid from Glutamate 1

    PubMed Central

    Schneegurt, Mark A.; Beale, Samuel I.

    1988-01-01

    The heme and chlorophyll precursor δ-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3′ terminus of yeast tRNAPhe(GAA) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNAGlu(UUC) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A260 unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in

  15. Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids

    PubMed Central

    Chang, Perng-Kuang; Hua, Sui Sheng T.; Sarreal, Siov Bouy L.; Li, Robert W.

    2015-01-01

    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 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 branched-chain amino acid degradation

  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

    2015-10-01

    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. Xanthophylls and abscisic acid biosynthesis in water-stressed bean leaves

    SciTech Connect

    Li, Y.; Walton, D.C.

    1987-12-01

    Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to /sup 14/CO/sub 2/ and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in /sup 12/CO/sub 2/ for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from /sup 14/CO/sub 2/ were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of /sup 14/C from /sup 14/CO/sub 2/ into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of /sup 18/O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with /sup 18/O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves.

  18. A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis.

    PubMed

    Guan, Xin; Chen, Hui; Abramson, Alex; Man, Huimin; Wu, Jinxia; Yu, Oliver; Nikolau, Basil J

    2015-11-01

    In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo-mtACP isoforms. The mitochondrial localization of the AT3G11470-encoded proteins was validated by the ability of their N-terminal 80-residue leader sequence to guide a chimeric GFP protein to this organelle. A T-DNA-tagged null mutant mtppt-1 allele shows an embryo-lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non-photorespiratory condition (i.e. 1% CO2 atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase. PMID:26402847

  19. 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.

  20. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

    PubMed Central

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

  1. Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid:coenzyme A ligase protein complex formation, regulation, and numerical modeling.

    PubMed

    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

    2014-03-01

    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.

  2. LCAA, a Novel Factor Required for Magnesium Protoporphyrin Monomethylester Cyclase Accumulation and Feedback Control of Aminolevulinic Acid Biosynthesis in Tobacco1[W][OA

    PubMed Central

    Albus, Christin Anne; Salinas, Annabel; Czarnecki, Olaf; Kahlau, Sabine; Rothbart, Maxi; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Grimm, Bernhard; Schöttler, Mark Aurel

    2012-01-01

    Low Chlorophyll Accumulation A (LCAA) antisense plants were obtained from a screen for genes whose partial down-regulation results in a strong chlorophyll deficiency in tobacco (Nicotiana tabacum). The LCAA mutants are affected in a plastid-localized protein of unknown function, which is conserved in cyanobacteria and all photosynthetic eukaryotes. They suffer from drastically reduced light-harvesting complex (LHC) contents, while the accumulation of all other photosynthetic complexes per leaf area is less affected. As the disturbed accumulation of LHC proteins could be either attributable to a defect in LHC biogenesis itself or to a bottleneck in chlorophyll biosynthesis, chlorophyll synthesis rates and chlorophyll synthesis intermediates were measured. LCAA antisense plants accumulate magnesium (Mg) protoporphyrin monomethylester and contain reduced protochlorophyllide levels and a reduced content of CHL27, a subunit of the Mg protoporphyrin monomethylester cyclase. Bimolecular fluorescence complementation assays confirm a direct interaction between LCAA and CHL27. 5-Aminolevulinic acid synthesis rates are increased and correlate with an increased content of glutamyl-transfer RNA reductase. We suggest that LCAA encodes an additional subunit of the Mg protoporphyrin monomethylester cyclase, is required for the stability of CHL27, and contributes to feedback-control of 5-aminolevulinic acid biosynthesis, the rate-limiting step of chlorophyll biosynthesis. PMID:23085838

  3. 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

  4. ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element.

    PubMed

    Chen, Hsing-Yu; Hsieh, En-Jung; Cheng, Mei-Chun; Chen, Chien-Yu; Hwang, Shih-Ying; Lin, Tsan-Piao

    2016-07-01

    ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress. PMID:26974851

  5. 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.

  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. PMID:20601479

  8. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

    PubMed Central

    Merski, Matthew

    2011-01-01

    The first committed biosynthetic step toward clavulanic acid, the clinically-important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N2-(2-carboxyethyl)arginine synthase (CEAS). This protein carries out a unique reaction among ThDP-dependent processes in which a C–N bond is formed, and an electrophilic acryloyl–thiazolium intermediate of ThDP is proposed to be involved, unlike the nucleophilic enamine species typically generated by this class of enzymes. Here we present evidence for the existence of the putative acryloyl adduct, and report the unexpected observation of a long-wavelength chromophore (λ = 433 nm), which we attribute to this enzyme bound species. Chemical models were synthesized that both confirm its expected absorption (λ = 310–320 nm), and exclude self-condensation and intramolecular imine formation with the cofactor as its cause. Circular dichroism experiments and others discount charge transfer as a likely explanation for the ~120 nm red shift of the chromophore (~25 kcal). Examples are well-known of charged molecules that exhibit significantly red-shifted UV-visible spectra compared to their neutral forms as, for example, polyene cations and dyes such as indigo and the cyanines. Rhodopsin is the classic biochemical example where the protein (opsin)-bound protonated Schiff base of retinal displays a remarkable range of red-shifted absorptions modulated by the protein environment. Similar tuning of the chromophoric behavior of the enzyme-bound CEAS acryloyl•ThDP species may be occurring. PMID:18052280

  9. Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra).

    PubMed

    Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2009-04-01

    The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.

  10. Global Effect of Indole-3-Acetic Acid Biosynthesis on Multiple Virulence Factors of Erwinia chrysanthemi 3937▿

    PubMed Central

    Yang, Shihui; Zhang, Qiu; Guo, Jianhua; Charkowski, Amy O.; Glick, Bernard R.; Ibekwe, A. Mark; Cooksey, Donald A.; Yang, Ching-Hong

    2007-01-01

    Production of the plant hormone indole-3-acetic acid (IAA) is widespread among plant-associated microorganisms. The non-gall-forming phytopathogen Erwinia chrysanthemi 3937 (strain Ech3937) possesses iaaM (ASAP16562) and iaaH (ASAP16563) gene homologues. In this work, the null knockout iaaM mutant strain Ech138 was constructed. The IAA production by Ech138 was reduced in M9 minimal medium supplemented with l-tryptophan. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected. The pectate lyase production of Ech138 was diminished. Compared with wild-type Ech3937, the expression levels of an oligogalacturonate lyase gene, ogl, and three endopectate lyase genes, pelD, pelI, and pelL, were reduced in Ech138 as determined by a green fluorescent protein-based fluorescence-activated cell sorting promoter activity assay. In addition, the transcription of type III secretion system (T3SS) genes, dspE (a putative T3SS effector) and hrpN (T3SS harpin), was found to be diminished in the iaaM mutant Ech138. Compared with Ech3937, reduced expression of hrpL (a T3SS alternative sigma factor) and gacA but increased expression of rsmA in Ech138 was also observed, suggesting that the regulation of T3SS and pectate lyase genes by IAA biosynthesis might be partially due to the posttranscriptional regulation of the Gac-Rsm regulatory pathway. PMID:17189441

  11. 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

  12. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

    PubMed

    Geisler, Katrin; Jensen, Niels Berg; Yuen, Macaire M S; Madilao, Lina; Bohlmann, Jörg

    2016-05-01

    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

  13. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

    PubMed

    Geisler, Katrin; Jensen, Niels Berg; Yuen, Macaire M S; Madilao, Lina; Bohlmann, Jörg

    2016-05-01

    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.

  14. Biosynthesis of a specifically deuteriated diunsaturated fatty acid (18:2/sub. delta. 6,9/) for /sup 2/H NMR membrane studies

    SciTech Connect

    Baenziger, J.E.; Smith, I.C.P.; Hill, R.J.

    1987-12-15

    A unique procedure for the biosynthesis and subsequent isolation of a series of specifically deuteriated cis,cis-octadeca-6,9-dienoic acids has been developed. An auxotroph of Tetrahymena, which lacks ..delta..9 and ..delta..12 desaturase activity, is supplemented with specifically deuteriated oleic acid and converts it to the corresponding deuteriated cis,cis-octadeca-6,9-dienoic acid, 18:2/sup ..delta..6,9/. The deuteriated fatty acid is subsequently isolated by argentation chromatography and HPLC. To demonstrate the utility of the procedure, we describe here the biosynthesis of cis,cis-octadeca-6,9-dienoic acid deuteriated at positions 9 and 10. Gas and thin-layer chromatography of the isolated fatty acid showed that it was greater than 99% pure while /sup 13/C NMR and mass spectrometry of the O-(trimethylsilyl) derivative confirmed that the 18-carbon fatty acid contains two double bonds located at positions 6 and 9. The yield, from an 11-L culture, was typically 100 mg of which 35% was found to be deuteriated at both the 9- and 10-positions. The deuteriated fatty acid was esterified to 1-hexadecanoyl-sn-glycero-3-phosphocholine, and aqueous, multilamellar dispersions of the lipid were studied by /sup 2/H NMR. Each spectrum consists of two overlapping powder patterns and therefore yields two quadrupolar splittings. Over a temperature range of 0 to 40/sup 0/C, one splitting decreases from 6.6 to 1.8 kHz while other increases from 4.5 to 5.3 kHz. The magnitudes of the two splittings are equivalent between 10 and 15/sup 0/ C. The values of the splittings, and their response to temperature, differ significantly from those of the corresponding deuteriated oleic acid in microbial membranes and in bilayers of 1-hexadecanoyl-2-cis-octadec-9-enoyl-sn-glycero-3-phosphocholine (POPC).

  15. 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

  16. Biosynthesis of C18 polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides.

    PubMed

    Jackson, F M; Fraser, T C; Smith, M A; Lazarus, C; Stobart, A K; Griffiths, G

    1998-03-15

    The biosynthesis of C18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained delta9, delta12 and delta6 desaturase activities. The delta9 desaturase exhibited characteristics similar to those of the animal and yeast delta9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the delta12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the delta12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine:acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. Delta6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the

  17. Deletion of the pyc Gene Blocks Clavulanic Acid Biosynthesis Except in Glycerol-Containing Medium: Evidence for Two Different Genes in Formation of the C3 Unit

    PubMed Central

    Pérez-Redondo, Rosario; Rodríguez-García, Antonio; Martín, Juan F.; Liras, Paloma

    1999-01-01

    The β-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the β-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing d-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin. PMID:10559157

  18. A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum.

    PubMed

    Nasution, Uly; van Gulik, Walter M; Ras, Cor; Proell, Angela; Heijnen, Joseph J

    2008-01-01

    The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium chrysogenum was studied by manipulating the steady-state flux in both pathways. A high producing industrial strain was cultivated at a growth rate mu=0.05 h(-1) in glucose-limited chemostat cultures, both under penicillin-G producing and non-producing conditions. Non-producing conditions were accomplished in two ways: (1) by cultivation without addition of the side chain precursor phenylacetic acid and (2) by cultivation of a mutant strain which lost all copies of the gene cluster coding for the penicillin biosynthesis pathway. Manipulation of the fluxes through central metabolism was obtained by cultivation on either glucose or ethanol as sole carbon source. A positive relation was observed between metabolite concentrations and carbon flux in central metabolism. Furthermore, in many cases a positive relation was found between the concentrations of free amino acids and their direct precursors in central metabolism. This corresponds with control of the biosynthesis of these amino acids via feed back inhibition by the end product. With respect to the penicillin production pathway, the flux seems not influenced by two of the three precursor amino acids, namely alphaAAA and valine but is only influenced by cysteine, which requires a large NADPH supply, and the ATP level. An interesting observation is that the absence of penicillin production seems to stimulate storage metabolism (trehalose metabolism). This leads to the final conclusion that the penicillin production flux appears to be mostly influenced by the availability of energy and redox cofactors, where ATP is supposed to exert its influence at ACV-synthetase and NADPH at the cysteine level.

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-01

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

  1. Evaluation of the impact of dietary petroselinic acid on the growth performance, fatty acid composition, and efficacy of long chain-polyunsaturated fatty acid biosynthesis of farmed Nile tilapia.

    PubMed

    Teoh, Chaiw-Yee; Ng, Wing-Keong

    2013-06-26

    The present study aimed to investigate the potential role of dietary petroselinic acid (PSA) in enhancing the n-3 long-chain polyunsaturated fatty acid (LC-PUFA) content in fish tissues. Three isolipidic casein-based diets were formulated to comprise graded levels of PSA (0, 10, or 20% of total fatty acid) with the incremented inclusion of coriander seed oil. Fish growth and nutrient digestibility were not significantly (P > 0.05) influenced by dietary PSA level. In general, dietary PSA affected the fatty acid composition of tilapia tissues and whole-body, which reflected dietary fatty acid ratios. Dietary PSA significantly (P < 0.05) increased β-oxidation, particularly on α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6). This study provided evidence that PSA, a pseudoproduct mimicking the structure of 18:3n-6, did reduce Δ-6 desaturation on 18:2n-6 but, contrary to popular speculation, did not stimulate more Δ-6 desaturase activity on 18:3n-3. The overall Δ-6 desaturase enzyme activity may be suppressed at high dietary levels of PSA. Nevertheless, the n-3 and n-6 LC-PUFA biosynthesis was not significantly inhibited by dietary PSA, indicating that the bioconversion efficiency is not modulated only by Δ-6 desaturase. The deposition of n-3 LC-PUFA in liver and fillet lipids was higher in fish fed PSA-supplemented diets.

  2. Martin RA-30 Baltimore

    NASA Technical Reports Server (NTRS)

    1943-01-01

    Martin RA-30 Baltimore: The Martin RA-30 Baltimore was a light bomber ordered by the Royal Air Force. Some examples were retained in the United States as part of a 'Reverse Lend-Lease.' This example was flown by the NACA from June 1943 until March 1944.

  3. 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.

  4. 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.

  5. Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa.

    PubMed Central

    Serino, L; Reimmann, C; Visca, P; Beyeler, M; Chiesa, V D; Haas, D

    1997-01-01

    The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron

  6. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    PubMed

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus.

  7. 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

  8. Qualitative and quantitative aspects of the biosynthesis of ribonucleic acid and of protein in the liver and the lung of the Syrian golden hamster

    PubMed Central

    Witschi, Hanspeter

    1973-01-01

    1. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total acid-soluble UMP were measured in both organs. An estimation of the rate of RNA biosynthesis showed that hamster lung synthesizes RNA at about one-half of the rate of that of hamster liver. 2. The apparent Km and Vmax. values of a few enzymes involved in pyrimidine biosynthesis were measured in the 100000g supernatants of liver and lung. The apparent Km values were very similar in both organs. From the estimated Vmax., it was concluded that hamster lung cells have less capacity to metabolize orotic acid than have liver cells. 3. A time–response and a dose–response study showed that actinomycin D inhibits pulmonary RNA synthesis as efficiently as hepatic RNA synthesis. 4. Protein synthesis, measured as the incorporation of leucine, was inhibited in both organs 30min after a dose of 2mg of cycloheximide/kg. The dose–response patterns were similar in both liver and lung 3h after cycloheximide. 5. It is concluded that RNA and protein synthesis in vivo in hamster lung are very similar to the corresponding reactions in liver. Alterations of RNA and protein synthesis by toxic agents can therefore be evaluated in lung with a similar approach to that used to study the pathological biochemistry of liver. PMID:4780700

  9. Transcription factors FabR and FadR regulate both aerobic and anaerobic pathways for unsaturated fatty acid biosynthesis in Shewanella oneidensis

    PubMed Central

    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. PMID:25566241

  10. 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.

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

    PubMed

    Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

    2015-11-01

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

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

    PubMed

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

    2016-09-01

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

  13. 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. PMID:24910972

  14. Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus

    PubMed Central

    Tahlan, Kapil; Park, Hyeon Ung; Wong, Annie; Beatty, Perrin H.; Jensen, Susan E.

    2004-01-01

    Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (pah1), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (ceaS) and β-lactam synthetase (bls) enzymes. These new paralogues are given the gene designations ceaS1 and bls1 and are located immediately upstream of pah1 on the chromosome. Furthermore, sequence analysis of the region downstream of pah1 revealed a second copy of a gene encoding ornithine acetyltransferase (oat1), thus indicating the presence of a cluster of paralogue genes. ceaS1, bls1, and oat1 display 73, 60, and 63% identities, respectively, at the nucleotide level to the original ceaS2, bls2, and oat2 genes from the clavulanic acid gene cluster. Single mutants defective in ceaS1, bls1, or oat1 were prepared and characterized and were found to be affected to variable degrees in their ability to produce clavulanic acid and clavam metabolites. Double mutants defective in both copies of the genes were also prepared and tested. The ceaS1/ceaS2 and the bls1/bls2 mutant strains were completely blocked in clavulanic acid and clavam metabolite biosynthesis. On the other hand, oat1/oat2 double mutants still produced some clavulanic acid and clavam metabolites. This may be attributed to the presence of the argJ gene in S. clavuligerus, which encodes yet another ornithine acetyltransferase enzyme that may be able to compensate for the lack of OAT1 and -2 in the double mutants. PMID:14982786

  15. Cellular and Subcellular Localization of S-Adenosyl-l-Methionine:Benzoic Acid Carboxyl Methyltransferase, the Enzyme Responsible for Biosynthesis of the Volatile Ester Methylbenzoate in Snapdragon Flowers1

    PubMed Central

    Kolosova, Natalia; Sherman, Debra; Karlson, Dale; Dudareva, Natalia

    2001-01-01

    The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators. PMID:11457946

  16. Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis.

    PubMed

    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

  17. 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

  18. Artemisinic Acid Serves as a Novel ORCA3 Inducer to Enhance Biosynthesis of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells.

    PubMed

    Wang, Mingxuan; Zi, Jiachen; Zhu, Jianhua; Chen, Shan; Wang, Pu; Song, Liyan; Yu, Rongmin

    2016-06-01

    To investigate the effect of artemisinic acid (AA) on improving the production of terpenoid indole alkaloids (TIAs) of Catharanthus roseus cambial meristematic cells (CMCs), feeding AA to C. roseus CMCs caused 2.35-fold and 2.51-fold increases in the production of vindoline and catharanthine, respectively, compared with those of the untreated CMCs. qRT-PCR experiments showed that AA resulted in a 1.36-8.52 fold increase in the transcript levels of several related genes, including octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3), tryptophan decarboxylase (TDC), strictosidine synthase (STR) and desacetoxyvindoline 4-hydroxylase (D4H). However, no effect was observed on the concentration of either jasmonic acid (JA), or the octadecanoid-pathway inhibitors block TIA accumulation caused by AA. The results indicated that AA might serve as a novel ORCA3 inducer to manipulate biosynthesis of TIAs in C. roseus CMCs via an unknown mechanism. PMID:27534099

  19. The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis.

    PubMed

    Keating, D H; Zhang, Y; Cronan, J E

    1996-05-01

    Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis. Jackowski and Rock (J. Biol. Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected. We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates.

  20. Upregulated mRNA expression of desaturase and elongase, two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

    PubMed Central

    Ishak, Sairatul D; Tan, Sze-Huey; Khong, Hou-Keat; Jaya-Ram, Annette; Enyu, Yee-Ling; Kuah, Meng-Kiat; Shu-Chien, Alexander Chong

    2008-01-01

    Background Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed. Methods mRNA levels of fadsd6 and elovl5 in different ovarian follicle stages were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. For analysis of the ovarian follicular fatty acid composition, gas chromatography was used. Results Our results have shown that desaturase displayed significant upregulation in expression during the oocyte maturation stage. Expression of elongase was significantly highest in pre-vitellogenic follicles, followed by maturation stage. Fatty acid composition analysis of different ovarian follicle stages also showed that ARA level was significantly highest in pre-vitellogenic and matured follicles. DHA level was highest in both late vitellogenic and maturation stage. Conclusion Collectively, our findings seem to suggest the existence of a HUFA synthesis system, which could be responsible for the synthesis of HUFA to promote oocyte maturation and possibly ovulation processes. The many advantages of zebrafish as model system to understand folliculogenesis will be useful platform to

  1. Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis.

    PubMed

    Mahesh, Venkataramaiah; Million-Rousseau, Rachel; Ullmann, Pascaline; Chabrillange, Nathalie; Bustamante, José; Mondolot, Laurence; Morant, Marc; Noirot, Michel; Hamon, Serge; de Kochko, Alexandre; Werck-Reichhart, Danièle; Campa, Claudine

    2007-05-01

    Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

  2. The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions.

    PubMed

    Veyron-Churlet, Romain; Bigot, Sarah; Guerrini, Olivier; Verdoux, Sébastien; Malaga, Wladimir; Daffé, Mamadou; Zerbib, Didier

    2005-11-01

    Tuberculosis kills about two million people every year and remains one of the leading causes of mortality worldwide. As a result of the increasing antibiotic resistance of Mycobacterium tuberculosis (Mtb) strains, there is an urgent need for new antitubercular drugs. Several efficient antibiotics, including isoniazid, specifically target the fatty acid synthase-II (FAS-II) complex of mycolic acid biosynthesis. We have previously shown that there are protein-protein interactions between the components of FAS-II that are essential for mycobacterial survival. We have now looked at the potential partners of FAS-II, mtFabD, the methyltransferases MmaAs, and Pks13. A combination of yeast two-hybrid and co-immunoprecipitation experiments showed that mtFabD interacts with each beta-ketoacyl-synthase (KasA, KasB and mtFabH) and with the core of FAS-II (InhA and MabA). The methyltransferases have a greater affinity for KasA and KasB than for mtFabH, suggesting that modifications on the meromycolic chains may occur during their elongation. Finally, Pks13, which catalyzes the final Claisen condensation of mycolic acids, interacts specifically with KasB. These data allowed us to determine the architecture of the multiple specialized FAS-II complexes, giving us insights into the organization of the complete mycolic acids biosynthesis. Our studies suggest a new and crucial interaction (KasB-Pks13) as a putative target for peptidomimetic antibiotics. PMID:16213523

  3. Measuring the radium quartet (228Ra, 226Ra, 224Ra, 223Ra) in seawater samples using gamma spectrometry.

    PubMed

    van Beek, P; Souhaut, M; Reyss, J-L

    2010-07-01

    Radium isotopes are widely used in marine studies (eg. to trace water masses, to quantify mixing processes or to study submarine groundwater discharge). While 228Ra and 226Ra are usually measured using gamma spectrometry, short-lived Ra isotopes (224Ra and 223Ra) are usually measured using a Radium Delayed Coincidence Counter (RaDeCC). Here we show that the four radium isotopes can be analyzed using gamma spectrometry. We report 226Ra, 228Ra, 224Ra, 223Ra activities measured using low-background gamma spectrometry in standard samples, in water samples collected in the vicinity of our laboratory (La Palme and Vaccarès lagoons, France) but also in seawater samples collected in the plume of the Amazon river, off French Guyana (AMANDES project). The 223Ra and 224Ra activities determined in these samples using gamma spectrometry were compared to the activities determined using RaDeCC. Activities determined using the two techniques are in good agreement. Uncertainties associated with the 224Ra activities are similar for the two techniques. RaDeCC is more sensitive for the detection of low 223Ra activities. Gamma spectrometry thus constitutes an alternate method for the determination of short-lived Ra isotopes.

  4. Evolution of conifer diterpene synthases: diterpene resin acid biosynthesis in lodgepole pine and jack pine involves monofunctional and bifunctional diterpene synthases.

    PubMed

    Hall, Dawn E; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L; Yuen, Macaire; Bohlmann, Jörg

    2013-02-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. PMID:23370714

  5. Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes.

    PubMed

    Argyris, Jason; Dahal, Peetambar; Hayashi, Eiji; Still, David W; Bradford, Kent J

    2008-10-01

    Lettuce (Lactuca sativa 'Salinas') seeds fail to germinate when imbibed at temperatures above 25 degrees C to 30 degrees C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37 degrees C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

  6. Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases1[W][OA

    PubMed Central

    Hall, Dawn E.; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L.; Yuen, Macaire; Bohlmann, Jörg

    2013-01-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. PMID:23370714

  7. Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

    PubMed Central

    de la Fuente, A.; Martín, J. F.; Rodríguez-García, A.; Liras, P.

    2004-01-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations. PMID:15375131

  8. Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways

    PubMed Central

    de la Fuente, Alvaro; Lorenzana, Luis M.; Martín, Juan F.; Liras, Paloma

    2002-01-01

    A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a β-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin. PMID:12426344

  9. 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. PMID:26732752

  10. 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 Central

    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. PMID:25133787

  11. 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.

  12. Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis.

    PubMed Central

    Wu, T K; Busby, R W; Houston, T A; McIlwaine, D B; Egan, L A; Townsend, C A

    1995-01-01

    Proclavaminate amidino hydrolase (PAH) catalyzes the reaction of guanidinoproclavaminic acid to proclavaminic acid and urea, a central step in the biosynthesis of the beta-lactamase inhibitor clavulanic acid. The gene encoding this enzyme (pah) was tentatively identified within the clavulanic acid biosynthetic cluster in Streptomyces clavuligerus by translation to a protein of the correct molecular mass (33 kDa) and appreciable sequence homology to agmatine ureohydrolase (M.B.W. Szumanski and S.M. Boyle, J. Bacteriol. 172:538-547, 1990) and several arginases, a correlation similarly recognized by Aidoo et al. (K. A. Aidoo, A. Wong, D. C. Alexander, R. A. R. Rittammer, and S. E. Jensen, Gene 147:41-46, 1994). Overexpression of the putative open reading frame as a 76-kDa fusion to the maltose-binding protein gave a protein having the catalytic activity sought. Cleavage of this protein with factor Xa gave PAH whose N terminus was slightly modified by the addition of four amino acids but exhibited unchanged substrate specificity and kinetic properties. Directly downstream of pah lies the gene encoding clavaminate synthase 2, an enzyme that carries out three distinct oxidative transformations in the in vivo formation of clavulanic acid. After the first of these oxidations, however, no further reaction was found to occur in vitro without the intervention of PAH. We have demonstrated that concurrent use of recombinant clavaminate synthase 2 and PAH results in the successful conversion of deoxyguanidinoproclavaminic acid to clavaminic acid, a four-step transformation. PAH has a divalent metal requirement, pH activity profile, and kinetic properties similar to those of other proteins of the broader arginase class. PMID:7601835

  13. 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

  14. 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

  15. Activation of the Ustilagic Acid Biosynthesis Gene Cluster in Ustilago maydis by the C2H2 Zinc Finger Transcription Factor Rua1▿

    PubMed Central

    Teichmann, Beate; Liu, Lidan; Schink, Kay Oliver; Bölker, Michael

    2010-01-01

    The phytopathogenic basidiomycetous fungus Ustilago maydis secretes, under conditions of nitrogen starvation, large amounts of the biosurfactant ustilagic acid (UA). This secreted cellobiose glycolipid is toxic for many microorganisms and confers biocontrol activity to U. maydis. Recently, a large gene cluster that is responsible for UA biosynthesis was identified. Here, we show that expression of all cluster genes depends on Rua1, a nuclear protein of the C2H2 zinc finger family, whose gene is located within the gene cluster. While deletion of rua1 results in complete loss of UA production, overexpression of rua1 promotes increased UA synthesis even in the presence of a good nitrogen source. Bioinformatic analysis allowed us to identify a conserved sequence element that is present in the promoters of all structural genes involved in UA biosynthesis. Deletion analysis of several promoters within the cluster revealed that this DNA element serves as an upstream activating sequence (UAS) and mediates Rua1-dependent expression. We used the yeast one-hybrid system to demonstrate specific recognition of this DNA element by Rua1. Introduction of nucleotide exchanges into the consensus sequence interfered with Rua1-dependent activation, suggesting that this sequence element acts as a direct binding site for Rua1. PMID:20173069

  16. 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. PMID:25250906

  17. Activation of the ustilagic acid biosynthesis gene cluster in Ustilago maydis by the C2H2 zinc finger transcription factor Rua1.

    PubMed

    Teichmann, Beate; Liu, Lidan; Schink, Kay Oliver; Bölker, Michael

    2010-04-01

    The phytopathogenic basidiomycetous fungus Ustilago maydis secretes, under conditions of nitrogen starvation, large amounts of the biosurfactant ustilagic acid (UA). This secreted cellobiose glycolipid is toxic for many microorganisms and confers biocontrol activity to U. maydis. Recently, a large gene cluster that is responsible for UA biosynthesis was identified. Here, we show that expression of all cluster genes depends on Rua1, a nuclear protein of the C(2)H(2) zinc finger family, whose gene is located within the gene cluster. While deletion of rua1 results in complete loss of UA production, overexpression of rua1 promotes increased UA synthesis even in the presence of a good nitrogen source. Bioinformatic analysis allowed us to identify a conserved sequence element that is present in the promoters of all structural genes involved in UA biosynthesis. Deletion analysis of several promoters within the cluster revealed that this DNA element serves as an upstream activating sequence (UAS) and mediates Rua1-dependent expression. We used the yeast one-hybrid system to demonstrate specific recognition of this DNA element by Rua1. Introduction of nucleotide exchanges into the consensus sequence interfered with Rua1-dependent activation, suggesting that this sequence element acts as a direct binding site for Rua1. PMID:20173069

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of mC7N units in ansamycin and mitomycin antibiotics: a review.

    PubMed

    Floss, Heinz G; Yu, Tin-Wein; Arakawa, Kenji

    2011-01-01

    The aminoshikimate pathway of formation of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of ansamycin and other antibiotics is reviewed. In this biosynthesis, genes for kanosamine formation have been recruited from other genomes, to provide a nitrogenous precursor. Kanosamine is then phosphorylated and converted by common cellular enzymes into 1-deoxy-1-imino-erythrose 4-phosphate, the substrate for the formation of aminoDAHP. This is converted via 5-deoxy-5-aminodehydroquinic acid and 5-deoxy-5-aminodehydroshikimic acid into AHBA. Remarkably, the pyridoxal phosphate enzyme AHBA synthase seems to have two catalytic functions: As a homodimer, it catalyzes the last reaction in the pathway, the aromatization of 5-deoxy-5-aminodehydroshikimic acid, and at the beginning of the pathway in a complex with the oxidoreductase RifL it catalyzes the transamination of UDP-3-keto-D-glucose. The AHBA synthase gene also serves as a useful tool in the genetic screening for new ansamycins and other AHBA-derived natural products.

  3. Kinetic, inhibition and structural studies on 3-oxoacyl-ACP reductase from Plasmodium falciparum, a key enzyme in fatty acid biosynthesis

    PubMed Central

    Wickramasinghe, Sasala R.; Inglis, Kirstine A.; Urch, Jonathan E.; Müller, Sylke; van Aalten, Daan M. F.; Fairlamb, Alan H.

    2005-01-01

    Type II fatty acid biosynthesis represents an attractive target for the discovery of new antimalarial drugs. Previous studies have identified malarial ENR (enoyl acyl-carrier-protein reductase, or FabI) as the target for the antiseptic triclosan. In the present paper, we report the biochemical properties and 1.5 Å (1 Å=0.1 nm) crystal structure of OAR (3-oxoacyl acyl-carrier-protein reductase, or FabG), the second reductive step in fatty acid biosynthesis and its inhibition by hexachlorophene. Under optimal conditions of pH and ionic strength, Plasmodium falciparum OAR displays kinetic properties similar to those of OAR from bacteria or plants. Activity with NADH is <3% of that with NADPH. Fluorescence enhancement studies indicate that NADPH can bind to the free enzyme, consistent with kinetic and product inhibition studies suggesting a steady-state ordered mechanism. The crystal structure reveals a tetramer with a sulphate ion bound in the cofactor-binding site such that the side chains of the catalytic triad of serine, tyrosine and lysine are aligned in an active conformation, as previously observed in the Escherichia coli OAR–NADP+ complex. A cluster of positively charged residues is positioned at the entrance to the active site, consistent with the proposed recognition site for the physiological substrate (3-oxoacyl-acyl-carrier protein) in E. coli OAR. The antibacterial and anthelminthic agent hexachlorophene is a potent inhibitor of OAR (IC50 2.05 μM) displaying non-linear competitive inhibition with respect to NADPH. Hexachlorophene (EC50 6.2 μM) and analogues such as bithionol also have antimalarial activity in vitro, suggesting they might be useful leads for further development. PMID:16225460

  4. Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway.

    PubMed

    Merrill, A H; Wang, E; Mullins, R E

    1988-01-12

    Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells. PMID:3126810

  5. An RA Big Think

    ERIC Educational Resources Information Center

    Wyatt, Neal

    2007-01-01

    Readers' advisory (RA) librarians use appeal to translate between readers and books, to make connections between readers and myriad other title possibilities based on the effects of the story. Appeal addresses the elements of books that readers respond to, the features that "draw a reader into a book" and help shape a particular reading…

  6. Transcriptome analysis and identification of genes associated with omega-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Perilla (Perilla frutescens (L.) var frutescens) produces high levels of a-linolenic acid (ALA), an omega-3 fatty acid important to health and development. To uncover key genes involved in fatty acid (FA) and triacylglycerol (TAG) synthesis in perilla, we conducted deep sequencing of cD...

  7. Determination of 226Ra, 224Ra, 223Ra and 228Ra in mineral water samples of the Slovak Republic

    NASA Astrophysics Data System (ADS)

    Durecová, A.; Durec, F.; Bursová, D.

    2006-01-01

    The Slovak Republic is very rich in mineral water sources. In recent years, it has been discovered that a number of mineral waters in the Slovak Republic contain high levels of 226Ra and 228Ra. Moreover, there is a lack of information on 224Ra and 223Ra concentrations in mineral waters as well. The currently approved techniques for alpha emitting radium isotopes are based on radon emanation methods. Due to the long ingrowth periods required by these techniques, any 224Ra and 223Ra in the sample decay away and go undetected. For this reason, we have used an alpha spectrometric method for the simultaneous determination of 226Ra, 223Ra and 224Ra. Radium was concentrated by a lead sulphate co-precipitation. The precipitate was dissolved in EDTA and the radium isotopes were separated from possible interfering radionuclides using barium sulphate micro precipitation. The radium-barium precipitate was filtered and counted by alpha spectrometry. 133Ba was used to quantify the yield by gamma spectrometry. In our laboratory, gamma spectrometry was also used for the determination of 228Ra in mineral water samples. Radium was concentrated by a lead-barium sulphate co-precipitation. 133Ba was used to quantify the yield, found to be 97% on the average, by gamma spectrometry. Furthermore, the committed effective doses for 226Ra, 224Ra, 223Ra, 228Ra intake via ingestion of mineral waters for the members of public were calculated.

  8. Inhibition of retinoic acid biosynthesis by the bisdichloroacetyldiamine WIN 18,446 markedly suppresses spermatogenesis and alters retinoid metabolism in mice.

    PubMed

    Paik, Jisun; Haenisch, Michael; Muller, Charles H; Goldstein, Alex S; Arnold, Samuel; Isoherranen, Nina; Brabb, Thea; Treuting, Piper M; Amory, John K

    2014-05-23

    Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.

  9. Inhibition of Retinoic Acid Biosynthesis by the Bisdichloroacetyldiamine WIN 18,446 Markedly Suppresses Spermatogenesis and Alters Retinoid Metabolism in Mice*

    PubMed Central

    Paik, Jisun; Haenisch, Michael; Muller, Charles H.; Goldstein, Alex S.; Arnold, Samuel; Isoherranen, Nina; Brabb, Thea; Treuting, Piper M.; Amory, John K.

    2014-01-01

    Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives. PMID:24711451

  10. Expression of genes controlling unsaturated fatty acids biosynthesis and oil deposition in developing seeds of Sacha inchi (Plukenetia volubilis L.).

    PubMed

    Wang, Xiaojuan; Liu, Aizhong

    2014-10-01

    Sacha inchi (Plukenetia volubilis L., Euphorbiaceae) seed oil is rich in α-linolenic acid, a kind of n-3 fatty acids with many health benefits. To discover the mechanism underlying α-linolenic acid accumulation in sacha inchi seeds, preliminary research on sacha inchi seed development was carried out from one week after fertilization until maturity, focusing on phenology, oil content, and lipid profiles. The results suggested that the development of sacha inchi seeds from pollination to mature seed could be divided into three periods. In addition, investigations on the effect of temperature on sacha inchi seeds showed that total oil content decreased in the cool season, while unsaturated fatty acid and linolenic acid concentrations increased. In parallel, expression profiles of 17 unsaturated fatty acid related genes were characterized during seed development and the relationships between gene expression and lipid/unsaturated fatty acid accumulation were discussed.

  11. Expression of genes controlling unsaturated fatty acids biosynthesis and oil deposition in developing seeds of Sacha inchi (Plukenetia volubilis L.).

    PubMed

    Wang, Xiaojuan; Liu, Aizhong

    2014-10-01

    Sacha inchi (Plukenetia volubilis L., Euphorbiaceae) seed oil is rich in α-linolenic acid, a kind of n-3 fatty acids with many health benefits. To discover the mechanism underlying α-linolenic acid accumulation in sacha inchi seeds, preliminary research on sacha inchi seed development was carried out from one week after fertilization until maturity, focusing on phenology, oil content, and lipid profiles. The results suggested that the development of sacha inchi seeds from pollination to mature seed could be divided into three periods. In addition, investigations on the effect of temperature on sacha inchi seeds showed that total oil content decreased in the cool season, while unsaturated fatty acid and linolenic acid concentrations increased. In parallel, expression profiles of 17 unsaturated fatty acid related genes were characterized during seed development and the relationships between gene expression and lipid/unsaturated fatty acid accumulation were discussed. PMID:25119487

  12. An ipdC gene knock-out of Azospirillum brasilense strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion.

    PubMed

    Malhotra, Mandira; Srivastava, Sheela

    2008-05-01

    The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (~50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by approximately 50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.

  13. 40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.6120 Phosphoric acid, 1,2-eth-a-ne... subject to reporting. (1) The chemical substance identified as phosphoric acid, 1,2-ethanediyl...

  14. 40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.6120 Phosphoric acid, 1,2-eth-a-ne... subject to reporting. (1) The chemical substance identified as phosphoric acid, 1,2-ethanediyl...

  15. 40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.6120 Phosphoric acid, 1,2-eth-a-ne... subject to reporting. (1) The chemical substance identified as phosphoric acid, 1,2-ethanediyl...

  16. The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

    PubMed Central

    Gao, Shouhong; Saechao, Saengking; Di, Peng; Chen, Junfeng; Chen, Wansheng

    2011-01-01

    Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors. PMID:22242141

  17. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza.

    PubMed

    Zhou, Yangyun; Sun, Wei; Chen, Junfeng; Tan, Hexin; Xiao, Ying; Li, Qing; Ji, Qian; Gao, Shouhong; Chen, Li; Chen, Shilin; Zhang, Lei; Chen, Wansheng

    2016-01-01

    Salvia miltiorrhiza Bunge, which contains tanshinones and phenolic acids as major classes of bioactive components, is one of the most widely used herbs in traditional Chinese medicine. Production of tanshinones and phenolic acids is enhanced by methyl jasmonate (MeJA). Transcription factor MYC2 is the switch of jasmontes signaling in plants. Here, we focused on two novel JA-inducible genes in S. miltiorrhiza, designated as SmMYC2a and SmMYC2b, which were localized in the nucleus. SmMYC2a and SmMYC2b were also discovered to interact with SmJAZ1 and SmJAZ2, implying that the two MYC2s might function as direct targets of JAZ proteins. Ectopic RNA interference (RNAi)-mediated knockdown experiments suggested that SmMYC2a/b affected multiple genes in tanshinone and phenolic acid biosynthetic pathway. Besides, the accumulation of tanshinones and phenolic acids was impaired by the loss of function in SmMYC2a/b. Meanwhile, SmMYC2a could bind with an E-box motif within SmHCT6 and SmCYP98A14 promoters, while SmMYC2b bound with an E-box motif within SmCYP98A14 promoter, through which the regulation of phenolic acid biosynthetic pathway might achieve. Together, these results suggest that SmMYC2a and SmMYC2b are JAZ-interacting transcription factors that positively regulate the biosynthesis