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Sample records for 6-methylsalicylic acid synthase

  1. Methyl 6-methylsalicylate: a female-produced pheromone component of the parasitoid wasp Spalangia endius.

    PubMed

    Nichols, William J; Cossé, Allard A; Bartelt, Robert J; King, Bethia H

    2010-10-01

    Sex-pheromone-related behavior and chemistry were studied in the wasp Spalangia endius Walker (Hymenoptera: Pteromalidae), a pupal parasitoid of the house fly, Musca domestica L. (Diptera: Muscidae). Males responded behaviorally to female extracts by arrestment, whereas females did not arrest to male extracts. In a comparison of male and female extracts by gas chromatography-mass spectrometry (GC-MS), two female-specific compounds were found. One was identified as methyl 6-methylsalicylate (gas chromatographic retention time and mass spectrum versus an authentic standard), but the chemical structure of the second compound is still unknown. Male antennae were sensitive to both compounds in electrophysiological tests (GC-EAD). Males responded behaviorally to methyl 6-methylsalicylate by arrestment, but did not arrest to the second compound. Methyl 6-methylsalicylate has been reported previously from some ant and beetle species, but never from the Pteromalidae. Chemical analysis of the extracts and the male behavioral results are consistent with the hypothesis that methyl 6-methylsalicylate functions as a female-emitted pheromone component at short range, but the exact role of both compounds in intersexual interactions in S. endius remains to be determined. PMID:20820889

  2. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    PubMed

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  3. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  4. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  5. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  6. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  7. An In Planta-Expressed Polyketide Synthase Produces (R)-Mellein in the Wheat Pathogen Parastagonospora nodorum

    PubMed Central

    Krill, Christian; Barrow, Russell A.; Chen, Shasha; Trengove, Robert; Oliver, Richard P.; Solomon, Peter S.

    2014-01-01

    Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of (R)-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of (R)-mellein. The P. nodorum ΔSN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, (R)-mellein at 200 μg/ml inhibited the germination of wheat (Triticum aestivum) and barrel medic (Medicago truncatula) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases. PMID:25326302

  8. Structural and functional organization of the animal fatty acid synthase.

    PubMed

    Smith, Stuart; Witkowski, Andrzej; Joshi, Anil K

    2003-07-01

    The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase. Each subunit contains three N-terminal domains, the beta-ketoacyl synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thiosterase. The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by beta-ketoacyl synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product. Only in the fatty acid synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for fatty acid synthesis at the two acyl carrier protein centers. Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric fatty acid synthases carrying different mutations on each subunit. Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein. The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and beta-ketoacyl synthase domains of either subunit. In contrast, the reactions involved in processing of the beta-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit. These findings suggest a revised model for the fatty acid synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between

  9. Producing a trimethylpentanoic acid using hybrid polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2014-10-07

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

  10. Structural organization of the multifunctional animal fatty-acid synthase.

    PubMed

    Witkowski, A; Rangan, V S; Randhawa, Z I; Amy, C M; Smith, S

    1991-06-15

    The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/acetyl transferase, residues 1630-1850 to the enoyl reductase, residues 1870-2100 to the oxyreductase, residues 2114-2190 to the acyl-carrier protein and residues 2200-2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.

  11. The type I fatty acid and polyketide synthases: a tale of two megasynthases

    PubMed Central

    Tsai, Shiou-Chuan

    2008-01-01

    This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

  12. The type I fatty acid and polyketide synthases: a tale of two megasynthases.

    PubMed

    Smith, Stuart; Tsai, Shiou-Chuan

    2007-10-01

    This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity.

  13. Molecular Genetic Characterization of Terreic Acid Pathway in Aspergillus terreus

    DOE PAGES

    Guo, Chun-Jun; Sun, Wei-wen; Bruno, Kenneth S.; Wang, Clay C.

    2014-09-29

    Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, in combined with bioinformatic analyses, allowed us to propose a biosynthetic pathway for terreic acid. Lastly, defining the pathway and the genes involved will facilitate the engineering of this molecule with interesting antimicrobial and antitumor bioactivities.

  14. Mechanism of the beta-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase.

    PubMed

    Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2002-09-01

    The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form

  15. Expression of fatty acid synthase in nonalcoholic fatty liver disease.

    PubMed

    Dorn, Christoph; Riener, Marc-Oliver; Kirovski, Georgi; Saugspier, Michael; Steib, Kathrin; Weiss, Thomas S; Gäbele, Erwin; Kristiansen, Glen; Hartmann, Arndt; Hellerbrand, Claus

    2010-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD. PMID:20606731

  16. Studies on tetrahydrocannabinolic acid synthase that produces the acidic precursor of tetrahydrocannabinol, the pharmacologically active cannabinoid in marijuana.

    PubMed

    Taura, F

    2009-06-01

    Tetrahydrocannabinol (THC), the psychoactive component of marijuana, is now regarded as a promising medicine because this cannabinoid has been shown to exert a variety of therapeutic activities. It has been demonstrated that THC is generated from the acidic precursor, tetrahydrocannabinolic acid (THCA) by nonenzymatic decarboxylation, and that THCA is biosynthesized by THCA synthase, which catalyzes a unique biosynthetic reaction, the stereospecific oxidative cyclization of the geranyl group of the substrate cannabigerolic acid. Molecular characterization of THCA synthase has revealed its structural characteristics and reaction mechanism. THCA synthase is the first cannabinoid synthase to be studied and is potentially attractive target for various biotechnological applications as it produces the direct precursor of THC. This review describes the research history of this enzyme, i.e., purification, molecular cloning, biochemical characterization, and possible biotechnological application of THCA synthase. PMID:22495534

  17. Effects and mechanism of acid rain on plant chloroplast ATP synthase.

    PubMed

    Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2016-09-01

    Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent. PMID:27278067

  18. Effects and mechanism of acid rain on plant chloroplast ATP synthase.

    PubMed

    Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2016-09-01

    Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

  19. The action of exogenous abscisic acid on malate-synthase synthesis in germinating castor-bean seeds.

    PubMed

    Dommes, J; Northcote, D H

    1985-12-01

    The presence of 30 μM abscisic acid inhibited development of malate-synthase activity in the endosperm of germinating castor-bean seeds. Malate synthase was purified from castor-bean endosperms and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of malate-synthase mRNA using an in-vitro translation system. The effect of abscisic acid appeared to be greater on malate-synthase mRNA than on the bulk of mRNA, indicating some specificity of abscisic-acid action. The extent of the inhibition of malate-synthase activity and of malate-synthase mRNA accumulation were similar. This indicates that abscisic acid inhibits malate-synthase activity by lowering levels of translatable malate-synthase mRNA rather than by affecting the translation rate of this mRNA.

  20. Acyl-carrier protein - Phosphopantetheinyltransferase partnerships in fungal fatty acid synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis of fatty acids is an essential primary metabolic process for energy storage and cellular structural integrity. Assembly of saturated fatty acids is achieved by fatty acid synthases (FASs) that combine acetyl- and malonyl-CoAs by repetitive decarboxylative Claisen condensations with su...

  1. Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples 1

    PubMed Central

    Bufler, Gebhard

    1984-01-01

    Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO2 production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO2; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene. Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO2, but not by 1.25% CO2. However, each of these CO2 concentrations reduced the rate of increase of ACC synthase activity. PMID:16663569

  2. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

    PubMed Central

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-01

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C22–C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C22–C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer. PMID:18199837

  3. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

    PubMed

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-22

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

  4. Isolation and partial characterization of the gene for goose fatty acid synthase.

    PubMed

    Kameda, K; Goodridge, A G

    1991-01-01

    Fatty acid synthase is regulated by diet and hormones, with regulation being primarily transcriptional. In chick embryo hepatocytes in culture, triiodothyronine stimulates accumulation of enzyme and transcription of the gene. Since the 5'-flanking region of this gene is likely involved in hormonal regulation of its expression, we have isolated and partially characterized an avian fatty acid synthase gene. A genomic DNA library was constructed in a cosmid vector and screened with cDNA clones that contained sequence complementary to the 3' end of goose fatty acid synthase mRNA. A genomic clone (approximately 35 kilobase pairs (kb] was isolated, and a 6.5-kb EcoRI fragment thereof contained DNA complementary to the 3' noncoding region of fatty acid synthase mRNA. Additional cosmid libraries were screened with 5' fragments of previously isolated genomic clones, resulting in the isolation of five overlapping cosmid DNAs. The entire region of cloned DNA spans approximately 105 kb. Exon-containing fragments were identified by hybridization with end-labeled poly(A)+ RNA and by hybridization of labeled exon-containing genomic DNA fragments to fatty acid synthase mRNA. A new set of cDNA clones spanning approximately 3.2 kb was isolated from a lambda-ZAP goose liver cDNA library using the 5'-most exon-containing fragment of the 5'-most genomic DNA clone. This region of mRNA contains a 5'-untranslated sequence and a continuous open reading frame which includes a region that codes for the essential cysteine of the beta-ketoacyl synthase domain. The entire fatty acid synthase gene spans about 50 kb. The 5' 15 kb of the gene contain 7 exons. S1 nuclease and primer extension analyses were used to identify a single site for initiation of transcription, 174 nucleotides upstream from the putative translation initiation codon. Putative "TATA" and "CCAAT" boxes are located 28 and 60 base pairs (bp), respectively, upstream of the site of initiation of transcription. The 5'-flanking 597

  5. Human fatty acid synthase: Structure and substrate selectivity of the thioesterase domain

    PubMed Central

    Chakravarty, Bornali; Gu, Ziwei; Chirala, Subrahmanyam S.; Wakil, Salih J.; Quiocho, Florante A.

    2004-01-01

    Human fatty acid synthase is a large homodimeric multifunctional enzyme that synthesizes palmitic acid. The unique carboxyl terminal thioesterase domain of fatty acid synthase hydrolyzes the growing fatty acid chain and plays a critical role in regulating the chain length of fatty acid released. Also, the up-regulation of human fatty acid synthase in a variety of cancer makes the thioesterase a candidate target for therapeutic treatment. The 2.6-Å resolution structure of human fatty acid synthase thioesterase domain reported here is comprised of two dissimilar subdomains, A and B. The smaller subdomain B is composed entirely of α-helices arranged in an atypical fold, whereas the A subdomain is a variation of the α/β hydrolase fold. The structure revealed the presence of a hydrophobic groove with a distal pocket at the interface of the two subdomains, which constitutes the candidate substrate binding site. The length and largely hydrophobic nature of the groove and pocket are consistent with the high selectivity of the thioesterase for palmitoyl acyl substrate. The structure also set the identity of the Asp residue of the catalytic triad of Ser, His, and Asp located in subdomain A at the proximal end of the groove. PMID:15507492

  6. Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro.

    PubMed

    Rangan, V S; Joshi, A K; Smith, S

    2001-09-11

    An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.

  7. Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.

    PubMed

    Gemperlein, Katja; Zipf, Gregor; Bernauer, Hubert S; Müller, Rolf; Wenzel, Silke C

    2016-01-01

    Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. PMID:26617065

  8. Evidence for a cyclic diguanylic acid-dependent cellulose synthase in plants.

    PubMed Central

    Amor, Y; Mayer, R; Benziman, M; Delmer, D

    1991-01-01

    Because numerous attempts to detect an activity for a cellulose synthase in plants have failed, we have taken a different approach toward detecting polypeptides involved in this process. The uniqueness of the structure and function of cyclic diguanylic acid (c-di-GMP) as an activator of the cellulose synthase of the bacterium Acetobacter xylinum makes it an attractive probe to use in a search for a c-di-GMP receptor that might be involved in the process in plants. Direct photolabeling with 32P-c-di-GMP has been used, therefore, to identify in plants two membrane polypeptides of 83 and 48 kD derived from cotton fibers that possess properties consistent with their being components of a c-di-GMP-dependent cellulose synthase. Based upon several criteria, the 48-kD species is proposed to be derived by proteolytic cleavage of the 83-kD polypeptide. Both polypeptides bind c-di-GMP with high affinity and specificity and show antigenic relatedness to the bacterial cellulose synthase, and the N-terminal sequence of the 48-kD polypeptide also indicates relatedness to the bacterial synthase. Ability to detect both cotton fiber polypeptides by photolabeling increases markedly in extracts derived from fibers entering the active phase of secondary wall cellulose synthesis. These results provide a basis for future work aimed at identifying and characterizing genes involved in cellulose synthesis in plants. PMID:1668373

  9. Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

    SciTech Connect

    Dotson, G.D.; Woodard, R.W.

    1994-12-01

    The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3{sup 2}H)PEP, (2-{sup 13}C)PEP, and (2-{sup 13}C,{sup 18}O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our {sup 1}H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3-{sup 2}H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3-{sup 2}H)PEP gave predominantly (3S)-(3{sup 2}H)KDO 8-P and (E)-(3-{sup 2}H)PEP gave predominantly (3R)-(3{sup 2}H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2-{sup 13}C, {sup 18}O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both {sup 13}C- and {sup 31}P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the {sup 18}O.

  10. Engineering of an active animal fatty acid synthase dimer with only one competent subunit.

    PubMed

    Joshi, Anil K; Rangan, Vangipuram S; Witkowski, Andrzej; Smith, Stuart

    2003-02-01

    Animal fatty acid synthases are large polypeptides containing seven functional domains that are active only in the dimeric form. Inactivity of the monomeric form has long been attributed to the obligatory participation of domains from both subunits in catalysis of substrate loading and condensation reactions. However, we have engineered a fatty acid synthase containing one wild-type subunit and one subunit compromised by mutations in all seven functional domains that is active in fatty acid synthesis. This finding indicates that a single subunit, in the context of a dimer, is able to catalyze the entire biosynthetic pathway and suggests that, in the natural complex, each of the two subunits forms a scaffold that optimizes the conformation of the companion subunit.

  11. Crystallization of Δ{sup 1}-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    SciTech Connect

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi; Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota; Shoyama, Yukihiro; Morimoto, Satoshi

    2005-08-01

    Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å{sup 3} Da{sup −1} assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

  12. Coexpressing Escherichia coli cyclopropane synthase with Sterculia foetida Lysophosphatidic acid acyltransferase enhances cyclopropane fatty acid accumulation.

    PubMed

    Yu, Xiao-Hong; Prakash, Richa Rawat; Sweet, Marie; Shanklin, John

    2014-01-01

    Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation. PMID:24204024

  13. Effect of abscisic and gibberellic acids on malate synthase transcripts in germinating castor bean seeds.

    PubMed

    Rodriguez, D; Dommes, J; Northcote, D H

    1987-05-01

    Several clones complementary to malate synthase mRNA have been identified in a complementary-DNA library to mRNA from castor bean endosperm. One of these clones has been used as a probe to measure levels of transcripts during seed germination and the effects of gibberellic acid and abscisic acid on these levels have been examined.Malate synthase transcripts increased during germination and GA3 advanced their appearance in the endosperm. Exogenously applied ABA inhibited the accumulation of transcripts over a time course of germination but the addition of GA3 counteracted its inhibitory effects. The data confirmed previous reports which indicated that the action of both growth regulators was on transcript accumulation and that there is a coordinated induction of the enzymes involved in the lipid metabolism in oil seeds.

  14. Deletion of a Chitin Synthase Gene in a Citric Acid Producing Strain of Aspergillus niger

    SciTech Connect

    Rinker, Torri E.; Baker, Scott E.

    2007-01-29

    Citric acid production by the filamentous fungus Aspergillus niger is carried out in a process that causes the organism to drastically alter its morphology. This altered morphology includes hyphal swelling and highly limited polar growth resulting in clumps of swollen cells that eventually aggregate into pellets of approximately 100 microns in diameter. In this pelleted form, A. niger has increased citric acid production as compared to growth in filamentous form. Chitin is a crucial component of the cell wall of filamentous fungi. Alterations in the deposition or production of chitin may have profound effects on the morphology of the organism. In order to study the role of chitin synthesis in pellet formation we have deleted a chitin synthase gene (csmA) in Aspergillus niger strain ATCC 11414 using a PCR based deletion construct. This class of chitin synthases is only found in filamentous fungi and is not present in yeasts. The csmA genes contain a myosin motor domain at the N-terminus and a chitin synthesis domain at the C-terminus. They are believed to contribute to the specialized polar growth observed in filamentous fungi that is lacking in yeasts. The csmA deletion strain (csmAΔ) was subjected to minimal media with and without osmotic stabilizers as well as tested in citric acid production media. Without osmotic stabilizers, the mutant germlings were abnormally swollen, primarily in the subapical regions, and contained large vacuoles. However, this swelling is ultimately not inhibitory to growth as the germlings are able to recover and undergo polar growth. Colony formation was largely unaffected in the absence of osmotic stabilizers. In citric acid production media csmAΔ was observed to have a 2.5 fold increase in citric acid production. The controlled expression of this class of chitin synthases may be useful for improving production of organic acids in filamentous fungi.

  15. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

    PubMed

    Salmon, Melissa; Thimmappa, Ramesha B; Minto, Robert E; Melton, Rachel E; Hughes, Richard K; O'Maille, Paul E; Hemmings, Andrew M; Osbourn, Anne

    2016-07-26

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  16. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  17. Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

    PubMed

    Walsh, Terence A; Bevan, Scott A; Gachotte, Daniel J; Larsen, Cory M; Moskal, William A; Merlo, P A Owens; Sidorenko, Lyudmila V; Hampton, Ronnie E; Stoltz, Virginia; Pareddy, Dayakar; Anthony, Geny I; Bhaskar, Pudota B; Marri, Pradeep R; Clark, Lauren M; Chen, Wei; Adu-Peasah, Patrick S; Wensing, Steven T; Zirkle, Ross; Metz, James G

    2016-08-01

    Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving. PMID:27398790

  18. Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

    PubMed

    Pasta, Saloni; Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2007-12-01

    Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.

  19. Carnosol and carnosic acids from Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1.

    PubMed

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-07-01

    Prostaglandin E(2) (PGE(2)), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE(2) synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE(2) in a cell-free assay by direct interference with microsomal PGE(2) synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC(50) values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC(50) values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE(2) generation upon stimulation with lipopolysaccharide (IC(50) = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF(1α), 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B(2)] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE(2) formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE(2) formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

  20. Carnosol and Carnosic Acids from Salvia officinalis Inhibit Microsomal Prostaglandin E2 Synthase-1

    PubMed Central

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M.; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-01-01

    Prostaglandin E2 (PGE2), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE2 synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE2 in a cell-free assay by direct interference with microsomal PGE2 synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC50 values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC50 values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE2 generation upon stimulation with lipopolysaccharide (IC50 = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF1α, 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B2] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE2 formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE2 formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis. PMID:22511203

  1. Natural fatty acid synthase inhibitors as potent therapeutic agents for cancers: A review.

    PubMed

    Zhang, Jia-Sui; Lei, Jie-Ping; Wei, Guo-Qing; Chen, Hui; Ma, Chao-Ying; Jiang, He-Zhong

    2016-09-01

    Context Fatty acid synthase (FAS) is the only mammalian enzyme to catalyse the synthesis of fatty acid. The expression level of FAS is related to cancer progression, aggressiveness and metastasis. In recent years, research on natural FAS inhibitors with significant bioactivities and low side effects has increasingly become a new trend. Herein, we present recent research progress on natural fatty acid synthase inhibitors as potent therapeutic agents. Objective This paper is a mini overview of the typical natural FAS inhibitors and their possible mechanism of action in the past 10 years (2004-2014). Method The information was collected and compiled through major databases including Web of Science, PubMed, and CNKI. Results Many natural products induce cancer cells apoptosis by inhibiting FAS expression, with fewer side effects than synthetic inhibitors. Conclusion Natural FAS inhibitors are widely distributed in plants (especially in herbs and foods). Some natural products (mainly phenolics) possessing potent biological activities and stable structures are available as lead compounds to synthesise promising FAS inhibitors.

  2. Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase.

    PubMed Central

    Gould, S J; Guo, J

    1994-01-01

    Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase), the first prokaryotic UDP-GT and a key enzyme in the biosynthesis of the antibiotic blasticidin S, was purified 870-fold. It has optimum activity at a pH of 8.4 to 8.6, Kms of 6.0 (UDP-glucuronic acid) and 243 (cytosine) microM, and a maximum rate of metabolism of 14.6 mumol/min/mg. The apparent M(r) is 43,000. Activity was slightly enhanced by Mg2+ or Ca2+ but was not inhibited by EDTA. Activity was strongly inhibited by UDP. Cytosylglucuronic acid differs from eukaryotic UDP-glucuronosyltransferases in being a soluble protein with no apparent phospholipid requirement. Images PMID:8113166

  3. Quinic acids from Aster caucasicus and from transgenic callus expressing a beta-amyrin synthase.

    PubMed

    Pecchia, Paola; Cammareri, Maria; Malafronte, Nicola; Consiglio, M Federica; Gualtieri, Maria Josefina; Conicella, Clara

    2011-11-01

    Several different classes of secondary metabolites, including flavonoids, triterpenoid saponins and quinic acid derivatives, are found in Aster spp. (Fam. Asteraceae). Several Aster compounds revealed biological as well as pharmacological activities. In this work, a phytochemical investigation of A. caucasicus evidenced the presence of quinic acid derivatives, as well as the absence of triterpene saponins. To combine in one species the production of different phytochemicals, including triterpenes, an Agrobacterium-mediated transformation of A. caucasicus was set up to introduce A. sedifolius beta-amyrin synthase (AsOXA1)-encoding gene under the control of the constitutive promoter CaMV35S. The quali-quantitative analysis of transgenic calli with ectopic expression of AsOXA1 showed, in one sample, a negligible amount of triterpene saponins combined with higher amount of quinic acid derivatives as compared with the wild type callus.

  4. 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.

  5. 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

  6. Fatty acid synthase is preferentially degraded by autophagy upon nitrogen starvation in yeast

    PubMed Central

    Shpilka, Tomer; Welter, Evelyn; Borovsky, Noam; Amar, Nira; Shimron, Frida; Peleg, Yoav; Elazar, Zvulun

    2015-01-01

    Autophagy, an evolutionarily conserved intracellular catabolic process, leads to the degradation of cytosolic proteins and organelles in the vacuole/lysosome. Different forms of selective autophagy have recently been described. Starvation-induced protein degradation, however, is considered to be nonselective. Here we describe a novel interaction between autophagy-related protein 8 (Atg8) and fatty acid synthase (FAS), a pivotal enzymatic complex responsible for the entire synthesis of C16- and C18-fatty acids in yeast. We show that although FAS possesses housekeeping functions, under starvation conditions it is delivered to the vacuole for degradation by autophagy in a Vac8- and Atg24-dependent manner. We also provide evidence that FAS degradation is essential for survival under nitrogen deprivation. Our results imply that during nitrogen starvation specific proteins are preferentially recruited into autophagosomes PMID:25605918

  7. Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

    PubMed Central

    Velours, J; Esparza, M; Hoppe, J; Sebald, W; Guerin, B

    1984-01-01

    The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans. Images Fig. 2. PMID:6323165

  8. Isoniazid affects multiple components of the type II fatty acid synthase system of Mycobacterium tuberculosis.

    PubMed

    Slayden, R A; Lee, R E; Barry, C E

    2000-11-01

    Genetic and biochemical evidence has implicated two different target enzymes for isoniazid (INH) within the unique type II fatty acid synthase (FAS) system involved in the production of mycolic acids. These two components are an enoyl acyl carrier protein (ACP) reductase, InhA, and a beta-ketoacyl-ACP synthase, KasA. We compared the consequences of INH treatment of Mycobacterium tuberculosis (MTB) with two inhibitors having well-defined targets: triclosan (TRC), which inhibits InhA; and thiolactomycin (TLM), which inhibits KasA. INH and TLM, but not TRC, upregulate the expression of an operon containing five FAS II components, including kasA and acpM. Although all three compounds inhibit mycolic acid synthesis, treatment with INH and TLM, but not with TRC, results in the accumulation of ACP-bound lipid precursors to mycolic acids that were 26 carbons long and fully saturated. TLM-resistant mutants of MTB were more cross-resistant to INH than TRC-resistant mutants. Overexpression of KasA conferred more resistance to TLM and INH than to TRC. Overexpression of InhA conferred more resistance to TRC than to INH and TLM. Co-overexpression of both InhA and KasA resulted in strongly enhanced levels of INH resistance, in addition to cross-resistance to both TLM and TRC. These results suggest that these components of the FAS II complex are not independently regulated and that alterations in the expression level of InhA affect expression levels of KasA. Nonetheless, INH appeared to resemble TLM more closely in overall mode of action, and KasA levels appeared to be tightly correlated with INH sensitivity. PMID:11069675

  9. Para-aminobenzoic acid (PABA) synthase enhances thermotolerance of mushroom Agaricus bisporus.

    PubMed

    Lu, Zhonglei; Kong, Xiangxiang; Lu, Zhaoming; Xiao, Meixiang; Chen, Meiyuan; Zhu, Liang; Shen, Yuemao; Hu, Xiangyang; Song, Siyang

    2014-01-01

    Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom's innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA) synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase) under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins. PMID:24614118

  10. Para-aminobenzoic acid (PABA) synthase enhances thermotolerance of mushroom Agaricus bisporus.

    PubMed

    Lu, Zhonglei; Kong, Xiangxiang; Lu, Zhaoming; Xiao, Meixiang; Chen, Meiyuan; Zhu, Liang; Shen, Yuemao; Hu, Xiangyang; Song, Siyang

    2014-01-01

    Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom's innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA) synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase) under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins.

  11. Cloning and characterisation of rosmarinic acid synthase from Melissa officinalis L.

    PubMed

    Weitzel, Corinna; Petersen, Maike

    2011-05-01

    Lemon balm (Melissa officinalis L.; Lamiaceae) is a well-known medicinal plant mainly due to two groups of compounds, the essential oil and the phenylpropanoid derivatives. The prominent phenolic compound is rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA shows a number of interesting biological activities. Rosmarinic acid synthase (RAS; 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) catalyses the ester formation. Cell cultures of M. officinalis have been established in order to characterise the formation of RA in an important diploid medicinal plant. RAS activity as well as the expression of the RAS gene are closely correlated with the accumulation of RA in suspension cultures of M. officinalis. The RAS cDNA and gene (MoRAS) were isolated. The RAS gene was shown to be intron-free. MoRAS belongs to the BAHD superfamily of acyltransferases. Southern-blot analysis suggests the presence of only one RAS gene copy in the M. officinalis genome. The enzyme was characterised with respect to enzyme properties, substrate preferences and kinetic data in crude plant extracts and as heterologously synthesised protein from Escherichia coli. PMID:21354582

  12. Cloning and characterisation of rosmarinic acid synthase from Melissa officinalis L.

    PubMed

    Weitzel, Corinna; Petersen, Maike

    2011-05-01

    Lemon balm (Melissa officinalis L.; Lamiaceae) is a well-known medicinal plant mainly due to two groups of compounds, the essential oil and the phenylpropanoid derivatives. The prominent phenolic compound is rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA shows a number of interesting biological activities. Rosmarinic acid synthase (RAS; 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) catalyses the ester formation. Cell cultures of M. officinalis have been established in order to characterise the formation of RA in an important diploid medicinal plant. RAS activity as well as the expression of the RAS gene are closely correlated with the accumulation of RA in suspension cultures of M. officinalis. The RAS cDNA and gene (MoRAS) were isolated. The RAS gene was shown to be intron-free. MoRAS belongs to the BAHD superfamily of acyltransferases. Southern-blot analysis suggests the presence of only one RAS gene copy in the M. officinalis genome. The enzyme was characterised with respect to enzyme properties, substrate preferences and kinetic data in crude plant extracts and as heterologously synthesised protein from Escherichia coli.

  13. Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

    PubMed

    Shoyama, Yoshinari; Tamada, Taro; Kurihara, Kazuo; Takeuchi, Ayako; Taura, Futoshi; Arai, Shigeki; Blaber, Michael; Shoyama, Yukihiro; Morimoto, Satoshi; Kuroki, Ryota

    2012-10-12

    ∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

  14. Isolation and Molecular Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase Genes in Hevea brasiliensis

    PubMed Central

    Zhu, Jia-Hong; Xu, Jing; Chang, Wen-Jun; Zhang, Zhi-Li

    2015-01-01

    Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1–7) of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment.These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production. PMID:25690030

  15. Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by orlistat

    SciTech Connect

    Pemble,C.; Johnson, L.; Kridel, S.; Lowther, W.

    2007-01-01

    Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-{angstrom}-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

  16. Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana.

    PubMed

    Jiang, He Zhong; Quan, Xiao Fang; Tian, Wei Xi; Hu, Jiang Miao; Wang, Peng Cheng; Huang, Sheng Zhuo; Cheng, Zhong Quan; Liang, Wen Juan; Zhou, Jun; Ma, Xiao Feng; Zhao, You Xing

    2010-10-15

    Natural inhibitors of fatty acid synthase (FAS) are emerging as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Garcinia mangostana led to the isolation of 13 phenolic compounds (1-13) mainly including xanthone and benzophenone, in which compounds 7, 8, 9, 10, and 11 were isolated from this plant for the first time and compound 9 was a new natural product. These isolates possess strong inhibitory activity of FAS with the IC(50) values ranging from 1.24 to 91.07 μM. The study indicates that two types of natural products, xanthones and benzophenones, could be considered as promising FAS inhibitors.

  17. 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

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

    PubMed Central

    Burkart, Michael D.

    2015-01-01

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

  19. Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

    PubMed Central

    Yasumoto, Yuki; Miyazaki, Hirofumi; Vaidyan, Linda Koshy; Kagawa, Yoshiteru; Ebrahimi, Majid; Yamamoto, Yui; Ogata, Masaki; Katsuyama, Yu; Sadahiro, Hirokazu; Suzuki, Michiyasu; Owada, Yuji

    2016-01-01

    Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma. PMID:26808816

  20. Structure of Quinolinate Synthase from Pyrococcus horikoshii in the Presence of Its Product, Quinolinic Acid.

    PubMed

    Esakova, Olga A; Silakov, Alexey; Grove, Tyler L; Saunders, Allison H; McLaughlin, Martin I; Yennawar, Neela H; Booker, Squire J

    2016-06-15

    Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and aspartate-enamine by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which is proposed to bind the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. However, direct evidence for this role in catalysis has yet to be provided. Herein, we present the structure of NadA in the presence of the product of its reaction, QA. We find that N1 and the C7 carboxylate group of QA ligate to Fea in a bidentate fashion, which is confirmed by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding mode would place the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it. The structure shows that three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity. PMID:27224840

  1. Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

    PubMed Central

    Grogan, D W; Cronan, J E

    1984-01-01

    Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation. PMID:6325391

  2. Characterization of the beta-carbon processing reactions of the mammalian cytosolic fatty acid synthase: role of the central core.

    PubMed

    Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2004-08-17

    The properties of the beta-ketoacyl reductase, dehydrase, and enoyl reductase components of the animal fatty acid synthase responsible for the reduction of the beta-ketoacyl moiety formed at each round of chain elongation have been studied by engineering and characterizing mutants defective in each of these three catalytic domains. These "beta-carbon processing" mutants leak the stalled four-carbon intermediates by direct transfer to CoA. However, enoyl reductase mutants leak beta-ketobutyryl, beta-hydroxybutyryl, and crotonyl moieties, a finding explained, at least in part, by the observation that the equilibrium and rate constant for the dehydrase reaction favor the formation of beta-hydroxy rather than enoyl moieties. In this regard, the type I animal fatty acid synthase resembles its type II counterpart in Escherichia coli in that both systems rely on the enoyl reductase to pull the beta-carbon processing reactions to completion. Kinetic and nucleotide binding measurements on fatty acid synthases mutated in either of the two nucleotide binding domains revealed that the NADPH binding sites are nonidentical, the enoyl reductase exhibiting higher affinity. Surprisingly, NADPH binding is also completely compromised by certain deletions and mutations in the central core region distant from the nucleotide binding sites. Comparable central core sequences are present in the structurally related modular polyketide synthases, except in those modules that lack all three beta-carbon processing enzymes. These findings suggest that the central core region of fatty acid and polyketide synthases plays an important role in facilitating the beta-carbon processing reactions.

  3. Fatty acid synthase-positive hepatocytes and subsequent steatosis in rat livers by irinotecan

    PubMed Central

    SAWANO, TAKEYUKI; SHIMIZU, TAKESHI; YAMADA, TOSHIYUKI; NANASHIMA, NAOKI; MIURA, TAKUYA; MOROHASHI, SATOKO; KUDO, DAISUKE; HUI, FENG MAO; KIJIMA, HIROSHI; HAKAMADA, KENICHI; TSUCHIDA, SHIGEKI

    2015-01-01

    Using a rat model, we investigated factors contributing to the pathogenesis of irinotecan-associated fatty liver disease. Male Sprague-Dawley rats were administered 200 mg/kg irinotecan by intraperitoneal injection on days 1–4, but not on days 5–7. This schedule was repeated 3 times. Rats were sacrificed 4, 18 and 25 days after the last injection, and liver steatosis was evaluated by hematoxylin and eosin (H&E) staining, microarray analysis and immunohistochemistry. Panacinar intrahepatocyte vacuoles were absent on days 4 and 25, but present on day 18, and this alteration was more prominent around the bile ducts than the central veins. Microarray analysis showed that the expression of genes involved in the synthesis of cholesterol and fatty acids was upregulated on day 4. Immunohistochemistry detected fatty acid synthase (Fasn)-strongly positive hepatocytes as well as the activation of liver progenitor cells on day 4, whereas intracellular vacuoles were evident in carbonic anhydrase 3 (CA3)-positive hepatocytes on day 18. Thus, irinotecan-induced liver steatosis was preceded by Fasn-strongly-positive hepatocytes and liver progenitor cell activation. The magnitude of the decrease in the number of Fasn-strongly positive hepatocytes between days 4 and 18 was similar to that of the increase in the number of CA3-positive hepatocytes accompanying vacuoles. PMID:25708528

  4. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth.

    PubMed

    Gang, Xiaokun; Yang, Yinhui; Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-03-22

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment. PMID:26934656

  5. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth.

    PubMed

    Gang, Xiaokun; Yang, Yinhui; Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-03-22

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment.

  6. Evolutionary origins of the multienzyme architecture of giant fungal fatty acid synthase.

    PubMed

    Bukhari, Habib S T; Jakob, Roman P; Maier, Timm

    2014-12-01

    Fungal fatty acid synthase (fFAS) is a key paradigm for the evolution of complex multienzymes. Its 48 functional domains are embedded in a matrix of scaffolding elements, which comprises almost 50% of the total sequence and determines the emergent multienzymes properties of fFAS. Catalytic domains of fFAS are derived from monofunctional bacterial enzymes, but the evolutionary origin of the scaffolding elements remains enigmatic. Here, we identify two bacterial protein families of noncanonical fatty acid biosynthesis starter enzymes and trans-acting polyketide enoyl reductases (ERs) as potential ancestors of scaffolding regions in fFAS. The architectures of both protein families are revealed by representative crystal structures of the starter enzyme FabY and DfnA-ER. In both families, a striking structural conservation of insertions to scaffolding elements in fFAS is observed, despite marginal sequence identity. The combined phylogenetic and structural data provide insights into the evolutionary origins of the complex multienzyme architecture of fFAS.

  7. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

    PubMed

    Hagen, Andrew; Poust, Sean; Rond, Tristan de; Fortman, Jeffrey L; Katz, Leonard; Petzold, Christopher J; Keasling, Jay D

    2016-01-15

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

  8. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

    PubMed

    Hagen, Andrew; Poust, Sean; Rond, Tristan de; Fortman, Jeffrey L; Katz, Leonard; Petzold, Christopher J; Keasling, Jay D

    2016-01-15

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry. PMID:26501439

  9. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth

    PubMed Central

    Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R. Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-01-01

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment. PMID:26934656

  10. Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines.

    PubMed

    Niranjan, S K; Goyal, S; Dubey, P K; Kumari, N; Mishra, S K; Mukesh, M; Kataria, R S

    2016-01-10

    Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines

  11. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

    PubMed Central

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M.; Baerga-Ortiz, Abel

    2014-01-01

    Increasing the production of fatty acids by microbial fermentation remains an important step towards the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations towards accessible biodiesel precursors. PMID:24411456

  12. Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis

    SciTech Connect

    Guerinot, M.L.; Chelm, B.K.

    1986-03-01

    Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. The authors now show that a hemA mutant of Bradyrhizobium japonicum that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolated the wild-type hemA gene encoding delta-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the Rhizobium meliloti hemA gene as a hybridization probe. A deletion of the hemA gene region, generated in vitro, then was used to construct the analogous chromosomal mutation by gene-directed mutagenesis. The mutant strain had no delta-aminolevulinic acid synthase activity and was unable to grow in minimal medium unless delta-aminolevulinic acid was added. Despite its auxotrophy, the mutant strain incited nodules that appeared normal, contained heme, and were capable of high levels of acetylene reduction. These results rule out bacterial delta-aminolevulinic acid synthase activity as the exclusive source of delta-aminolevulinic acid for heme formation in soybean nodules.

  13. Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis

    SciTech Connect

    Yu X. H.; Shanklin J.; Rawat, R.

    2011-05-01

    Cyclopropane fatty acids (CPA) have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE) are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants from herbivory. Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA) distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed. In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model plant systems, it represents a strong candidate gene for

  14. In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.

    PubMed

    Yu, Xingye; Liu, Tiangang; Zhu, Fayin; Khosla, Chaitan

    2011-11-15

    Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.

  15. In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

    PubMed Central

    Yu, Xingye; Liu, Tiangang; Zhu, Fayin; Khosla, Chaitan

    2011-01-01

    Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels. PMID:22042840

  16. {alpha}-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

    SciTech Connect

    Lee, Young; Naseem, R. Haris; Park, Byung-Hyun; Garry, Daniel J.; Richardson, James A.; Schaffer, Jean E.; Unger, Roger H. . E-mail: roger.unger@utsouthwestern.edu

    2006-05-26

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

  17. Intron-exon organization of the gene for the multifunctional animal fatty acid synthase.

    PubMed Central

    Amy, C M; Williams-Ahlf, B; Naggert, J; Smith, S

    1992-01-01

    The complete intron-exon organization of the gene encoding a multifunctional mammalian fatty acid synthase has been elucidated, and specific exons have been assigned to coding sequences for the component domains of the protein. The rat gene is interrupted by 42 introns and the sequences bordering the splice-site junctions universally follow the GT/AG rule. However, of the 41 introns that interrupt the coding region of the gene, 23 split the reading frame in phase I, 14 split the reading frame in phase 0, and only 4 split the reading frame in phase II. Remarkably, 46% of the introns interrupt codons for glycine. With only one exception, boundaries between the constituent enzymes of the multifunctional polypeptide coincide with the location of introns in the gene. The significance of the predominance of phase I introns, the almost uniformly short length of the 42 introns and the overall small size of the gene, is discussed in relation to the evolution of multifunctional proteins. Images PMID:1736293

  18. Circulating Fatty Acid Synthase in pregnant women: Relationship to blood pressure, maternal metabolism and newborn parameters

    PubMed Central

    Carreras-Badosa, Gemma; Prats-Puig, Anna; Puig, Teresa; Vázquez-Ruíz, Montserrat; Bruel, Monserrat; Mendoza, Ericka; de Zegher, Francis; Ibáñez, Lourdes; López-Bermejo, Abel; Bassols, Judit

    2016-01-01

    The enzyme FASN (fatty acid synthase) is potentially related with hypertension and metabolic dysfunction. FASN is highly expressed in the human placenta. We aimed to investigate the relationship circulating FASN has with blood pressure, maternal metabolism and newborn parameters in healthy pregnant women. Circulating FASN was assessed in 115 asymptomatic pregnant women in the second trimester of gestation along with C-peptide, fasting glucose and insulin, post-load glucose lipids, HMW-adiponectin and blood pressure (the latter was assessed in each trimester of gestation). At birth, newborns and placentas were weighed. FASN expression was also able to be assessed in 80 placentas. Higher circulating FASN was associated with lower systolic blood pressure (SBP), with a more favourable metabolic phenotype (lower fasting glucose and insulin, post load glucose, HbAc1, HOMA-IR and C-peptide), and with lower placental and birth weight (all p < 0.05 to p < 0.001). Placental FASN expression related positively to circulating FASN (p < 0.005) and negatively to placental weight (p < 0.05). Our observations suggest a physiological role of placental FASN in human pregnancy. Future studies will clarify whether circulating FASN of placental origin does actually regulate placental and fetal growth, and (thereby) has a favourable influence on the pregnant mother’s insulin sensitivity and blood pressure. PMID:27090298

  19. Influence of Different Levels of Lipoic Acid Synthase Gene Expression on Diabetic Nephropathy

    PubMed Central

    Xu, Longquan; Hiller, Sylvia; Simington, Stephen; Nickeleit, Volker; Maeda, Nobuyo; James, Leighton R.; Yi, Xianwen

    2016-01-01

    Oxidative stress is implicated in the pathogenesis of diabetic nephropathy (DN) but outcomes of many clinical trials are controversial. To define the role of antioxidants in kidney protection during the development of diabetic nephropathy, we have generated a novel genetic antioxidant mouse model with over- or under-expression of lipoic acid synthase gene (Lias). These models have been mated with Ins2Akita/+ mice, a type I diabetic mouse model. We compare the major pathologic changes and oxidative stress status in two new strains of the mice with controls. Our results show that Ins2Akita/+ mice with under-expressed Lias gene, exhibit higher oxidative stress and more severe DN features (albuminuria, glomerular basement membrane thickening and mesangial matrix expansion). In contrast, Ins2Akita/+ mice with highly-expressed Lias gene display lower oxidative stress and less DN pathologic changes. Our study demonstrates that strengthening endogenous antioxidant capacity could be an effective strategy for prevention and treatment of DN. PMID:27706190

  20. Potent Inhibitory Effect of Chinese Dietary Spices on Fatty Acid Synthase.

    PubMed

    Jiang, Bing; Liang, Yan; Sun, Xuebing; Liu, Xiaoxin; Tian, Weixi; Ma, Xiaofeng

    2015-09-01

    Dietary spices have been adopted in cooking since ancient times to enhance flavor and also as food preservatives and disease remedies. In China, the use of spices and other aromatic plants as food flavoring is an integral part of dietary behavior, but relatively little is known about their functions. Fatty acid synthase (FAS) has been recognized as a remedy target, and its inhibitors might be applied in disease treatment. The present work was designed to assess the inhibitory activities on FAS of spices extracts in Chinese menu. The in vitro inhibitory activities on FAS of 22 extracts of spices were assessed by spectrophotometrically monitoring oxidation of NADPH at 340 nm. Results showed that 20 spices extracts (90.9 %) exhibited inhibitory activities on FAS, with half inhibition concentration (IC(50)) values ranging from 1.72 to 810.7 μg/ml. Among them, seven spices showed strong inhibitory effect with IC(50) values lower than 10 μg/ml. These findings suggest that a large proportion of the dietary spices studied possess promising inhibitory activities on FAS, and subsequently might be applied in the treatment of obesity and obesity-related human diseases.

  1. Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

    PubMed

    Valera, Maria José; Torija, Maria Jesús; Mas, Albert; Mateo, Estibaliz

    2015-02-01

    The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

  2. Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication.

    PubMed

    Wilsky, Steffi; Sobotta, Katharina; Wiesener, Nadine; Pilas, Johanna; Althof, Nadine; Munder, Thomas; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Coxsackievirus B3 (CVB3) is a human pathogen that causes acute and chronic infections, but an antiviral drug to treat these diseases has not yet been developed for clinical use. Several intracellular pathways are altered to assist viral transcription, RNA replication, and progeny release. Among these, fatty acid synthase (FAS) expression is increased. In order to test the potential of FAS inhibition as an anti-CVB3 strategy, several experiments were performed, including studies on the correlation of CVB3 replication and FAS expression in human Raji cells and an analysis of the time and dose dependence of the antiviral effect of FAS inhibition due to treatment with amentoflavone. The results demonstrate that CVB3 infection induces an up-regulation of FAS expression already at 1 h postinfection (p.i.). Incubation with increasing concentrations of amentoflavone inhibited CVB3 replication significantly up to 8 h p.i. In addition, suppression of p38 MAP kinase activity by treatment with SB239063 decreased FAS expression as well as viral replication. These data provide evidence that FAS inhibition via amentoflavone administration might present a target for anti-CVB3 therapy. PMID:22075919

  3. Differential regulation of host genes including hepatic fatty acid synthase in HBV-transgenic mice.

    PubMed

    Zhang, Hongmin; Li, Hong; Yang, Yixuan; Li, Sanglin; Ren, Hong; Zhang, Dazhi; Hu, Huaidong

    2013-06-01

    Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans, and it is considered to be a major global health problem. To gain a better understanding of HBV pathogenesis, and identify novel putative targets for anti-HBV therapy, this study was designed to elucidate the differential expression of host proteins in liver tissue from HBV-transgenic mice. Liver samples from two groups, (1) HBV-transgenic (Tg) mice, (2) corresponding background normal mice, wild-type (WT) mice, were collected and subjected to iTRAQ and mass spectrometry analysis. In total, 1950 unique proteins were identified, and 68 proteins were found to be differentially expressed in HBV-Tg mice as compared with that in WT mice. Several differentially expressed proteins were further validated by real-time quantitative RT-PCR, Western blot and immunohistochemical analysis. Furthermore, the association of HBV replication with fatty acid synthase (FASN), one of the highly expressed proteins in HBV-Tg mice, was verified. Silencing of FASN expression in HepG2.2.15 cells suppressed viral replication through the IFN signaling pathway, and some downstream antiviral effectors. The implicated role of FASN in HBV replication provides an opportunity to test existing compounds against FASN for adjuvant therapy and/or treatment of HBV replication. PMID:23675653

  4. Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase.

    PubMed

    Joshi, Anil K; Witkowski, Andrzej; Berman, Harvey A; Zhang, Lei; Smith, Stuart

    2005-03-15

    A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long

  5. The smoking-associated oxidant hypothiocyanous acid induces endothelial nitric oxide synthase dysfunction.

    PubMed

    Talib, Jihan; Kwan, Jair; Suryo Rahmanto, Aldwin; Witting, Paul K; Davies, Michael J

    2014-01-01

    Smokers have an elevated risk of cardiovascular disease but the origin(s) of this increased risk are incompletely defined. Considerable evidence supports an accumulation of the oxidant-generating enzyme MPO (myeloperoxidase) in the inflamed artery wall, and smokers have high levels of SCN(-), a preferred MPO substrate, with this resulting in HOSCN (hypothiocyanous acid) formation. We hypothesized that this thiol-specific oxidant may target the Zn(2+)-thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signalling molecule NO•. Decreased NO• bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smoking-associated disease. In the present study it is shown that exposure of isolated eNOS to HOSCN or MPO/H2O2/SCN(-) decreased active dimeric eNOS levels, and increased inactive monomer and Zn(2+) release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl(-)-treated samples. eNOS activity was increasingly compromised by MPO/H2O2/Cl(-) with increasing SCN(-) concentrations. Exposure of HCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl(-) with increasing SCN(-), increased eNOS monomerization and Zn(2+) release, and decreased activity. Intact HCAECs exposed to HOCl and HOSCN had decreased eNOS activity and NO2(-)/NO3(-) formation (products of NO• decomposition), and increased free Zn(2+). Exposure of isolated rat aortic rings to HOSCN resulted in thiol loss, and decreased eNOS activity and cGMP levels. Overall these data indicate that high SCN(-) levels, as seen in smokers, can increase HOSCN formation and enhance eNOS dysfunction in human endothelial cells, with this potentially contributing to increased atherogenesis in smokers. PMID:24112082

  6. Inhibition of Mycobacterium tuberculosis dihydrodipicolinate synthase by alpha-ketopimelic acid and its other structural analogues

    PubMed Central

    Shrivastava, Priyanka; Navratna, Vikas; Silla, Yumnam; Dewangan, Rikeshwer P.; Pramanik, Atreyi; Chaudhary, Sarika; Rayasam, GeethaVani; Kumar, Anuradha; Gopal, Balasubramanian; Ramachandran, Srinivasan

    2016-01-01

    The Mycobacterium tuberculosis dihydrodipicolinate synthase (Mtb-dapA) is an essential gene. Mtb-DapA catalyzes the aldol condensation between pyruvate and L-aspartate-beta-semialdehyde (ASA) to yield dihydrodipicolinate. In this work we tested the inhibitory effects of structural analogues of pyruvate on recombinant Mtb-DapA (Mtb-rDapA) using a coupled assay with recombinant dihydrodipicolinate reductase (Mtb-rDapB). Alpha-ketopimelic acid (α-KPA) showed maximum inhibition of 88% and IC50 of 21 μM in the presence of pyruvate (500 μM) and ASA (400 μM). Competition experiments with pyruvate and ASA revealed competition of α-KPA with pyruvate. Liquid chromatography-mass spectrometry (LC-MS) data with multiple reaction monitoring (MRM) showed that the relative abundance peak of final product, 2,3,4,5-tetrahydrodipicolinate, was decreased by 50%. Thermal shift assays showed 1 °C Tm shift of Mtb-rDapA upon binding α-KPA. The 2.4 Å crystal structure of Mtb-rDapA-α-KPA complex showed the interaction of critical residues at the active site with α-KPA. Molecular dynamics simulations over 500 ns of pyruvate docked to Mtb-DapA and of α-KPA-bound Mtb-rDapA revealed formation of hydrogen bonds with pyruvate throughout in contrast to α-KPA. Molecular descriptors analysis showed that ligands with polar surface area of 91.7 Å2 are likely inhibitors. In summary, α-hydroxypimelic acid and other analogues could be explored further as inhibitors of Mtb-DapA. PMID:27501775

  7. Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation1[W][OPEN

    PubMed Central

    Yu, Xiao-Hong; Prakash, Richa Rawat; Sweet, Marie; Shanklin, John

    2014-01-01

    Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation. PMID:24204024

  8. 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

  9. 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

  10. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

    PubMed Central

    Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-01-01

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure–function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids. PMID:26842837

  11. Head-to-head coiled arrangement of the subunits of the animal fatty acid synthase.

    PubMed

    Witkowski, Andrzej; Ghosal, Alokesh; Joshi, Anil K; Witkowska, H Ewa; Asturias, Francisco J; Smith, Stuart

    2004-12-01

    The role of the beta-ketoacyl synthase domains in dimerization of the 2505 residue subunits of the multifunctional animal FAS has been evaluated by a combination of crosslinking and characterization of several truncated forms of the protein. Polypeptides containing only the N-terminal 971 residues can form dimers, but polypeptides lacking only the N-terminal 422 residue beta-ketoacyl synthase domain cannot. FAS subunits can be crosslinked with spacer lengths as short as 6 A, via cysteine residues engineered near the N terminus of the full-length polypeptides. The proximity of the N-terminal beta-ketoacyl synthase domains and their essential role in dimerization is consistent with a revised model for the FAS in which a head-to-head arrangement of two coiled subunits facilitates functional interactions between the dimeric beta-ketoacyl synthase and the acyl carrier protein domains of either subunit.

  12. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    PubMed

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

  13. Gene identification and functional analysis of methylcitrate synthase in citric acid-producing Aspergillus niger WU-2223L.

    PubMed

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2013-01-01

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

  14. Gene identification and functional analysis of methylcitrate synthase in citric acid-producing Aspergillus niger WU-2223L.

    PubMed

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2013-01-01

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity. PMID:23832368

  15. Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits 1

    PubMed Central

    Hubbard, Natalie L.; Huber, Steven C.; Pharr, D. Mason

    1989-01-01

    Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 ± 0.3 micromole per hour per gram fresh weight and 2.2 ± 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality. PMID:16667212

  16. MicroRNA-24 can control triacylglycerol synthesis in goat mammary epithelial cells by targeting the fatty acid synthase gene.

    PubMed

    Wang, H; Luo, J; Chen, Z; Cao, W T; Xu, H F; Gou, D M; Zhu, J J

    2015-12-01

    In nonruminants it has been demonstrated that microRNA-24 (miR-24) is involved in preadipocyte differentiation, hepatic lipid, and plasma triacylglycerol synthesis. However, its role in ruminant mammary gland remains unclear. In this study we measured miR-24 expression in goat mammary gland tissue at 4 different stages of lactation and observed that it had highest expression at peak lactation when compared with the dry period. Overexpression or downregulation of miR-24 in goat mammary epithelial cells (GMEC) strongly affected fatty acid profiles; in particular, miR-24 enhanced unsaturated fatty acid concentration. Additional effects of miR-24 included changes in triacylglycerol content and the expression of fatty acid synthase, sterol regulatory element binding transcription protein 1, stearoyl-CoA desaturase, glycerol-3-phosphate acyltransferase mitochondrial, and acetyl-CoA carboxylase. Luciferase reporter assay confirmed that fatty acid synthase is a target of miR-24. Taken together, these results not only highlight the physiological importance of miR-24 in fatty acid metabolism in GMEC, but also laid the foundation for further research on regulatory mechanisms among miR-24 and other microRNA expressed in GMEC. PMID:26476938

  17. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    SciTech Connect

    Chang, Soo-Ik ); Hammes, G.G. )

    1989-11-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.

  18. Fatty Acid Synthase Cooperates with Glyoxalase 1 to Protect against Sugar Toxicity

    PubMed Central

    Garrido, Damien; Rubin, Thomas; Poidevin, Mickael; Maroni, Brigitte; Le Rouzic, Arnaud; Parvy, Jean-Philippe; Montagne, Jacques

    2015-01-01

    Fatty acid (FA) metabolism is deregulated in several human diseases including metabolic syndrome, type 2 diabetes and cancers. Therefore, FA-metabolic enzymes are potential targets for drug therapy, although the consequence of these treatments must be precisely evaluated at the organismal and cellular levels. In healthy organism, synthesis of triacylglycerols (TAGs)—composed of three FA units esterified to a glycerol backbone—is increased in response to dietary sugar. Saturation in the storage and synthesis capacity of TAGs is associated with type 2 diabetes progression. Sugar toxicity likely depends on advanced-glycation-end-products (AGEs) that form through covalent bounding between amine groups and carbonyl groups of sugar or their derivatives α-oxoaldehydes. Methylglyoxal (MG) is a highly reactive α-oxoaldehyde that is derived from glycolysis through a non-enzymatic reaction. Glyoxalase 1 (Glo1) works to neutralize MG, reducing its deleterious effects. Here, we have used the power of Drosophila genetics to generate Fatty acid synthase (FASN) mutants, allowing us to investigate the consequence of this deficiency upon sugar-supplemented diets. We found that FASN mutants are lethal but can be rescued by an appropriate lipid diet. Rescued animals do not exhibit insulin resistance, are dramatically sensitive to dietary sugar and accumulate AGEs. We show that FASN and Glo1 cooperate at systemic and cell-autonomous levels to protect against sugar toxicity. We observed that the size of FASN mutant cells decreases as dietary sucrose increases. Genetic interactions at the cell-autonomous level, where glycolytic enzymes or Glo1 were manipulated in FASN mutant cells, revealed that this sugar-dependent size reduction is a direct consequence of MG-derived-AGE accumulation. In summary, our findings indicate that FASN is dispensable for cell growth if extracellular lipids are available. In contrast, FA-synthesis appears to be required to limit a cell

  19. Identification of an insulin response element in the fatty acid synthase promoter.

    PubMed

    Moustaïd, N; Beyer, R S; Sul, H S

    1994-02-25

    We have previously reported that insulin increases fatty acid synthase (FAS) gene transcription, and that sequences responsible for positive regulation are located within the first 332 base pairs of the FAS promoter. To define minimal sequences required for insulin regulation within this region, chimeric constructs containing serial 5' deletions starting at -318 and extending through position +67 of the rat FAS gene ligated to the luciferase reporter gene were transfected into 3T3-L1 adipocytes. Insulin treatment at 10 nM increased luciferase activity 2-3-fold in 3T3-L1 adipocytes transfected with constructs containing progressive deletions from -318 to -67. This stimulation of the FAS promoter activity by insulin was dose-dependent. However, no effect of insulin was observed when fusion constructs containing FAS promoter sequences spanning from -25 or from -19 to +67 were transfected into adipocytes. These results suggest that the insulin response sequences of the FAS gene may be located in the region from -67 to -25. DNase I footprinting using liver nuclear extracts revealed a protected region spanning -71 and -50 in addition to a region near the putative TATA box. Gel mobility shift assays using the sequence from -71 to -50 as a probe revealed nuclear factor(s) from mouse liver and 3T3-L1 adipocytes that specifically complexed with this sequence. Mutational analysis of this region showed that sequences between -68 and -60 are essential for recognition and interaction with a trans-acting factor(s). Moreover, when three tandem repeats of the sequences spanning -68 to -52 were linked to the SV40 promoter and used for transfection, luciferase activity increased 3.6-fold in response to insulin treatment. Thus, we have identified novel cis-acting DNA sequences responsible for insulin regulation of the FAS gene, which interact with nuclear protein(s) from liver and adipocytes and which are found to share limited homology to insulin response sequences present in other

  20. Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

    PubMed Central

    Boini, Krishna M.; Xia, Min; Abais, Justine M.; Xu, Ming; Li, Cai-xia; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs+/− and Asm+/− mice. Given that the homozygotes of Cbs−/−/Asm−/− mice could not survive for 3 weeks. Cbs+/−/Asm+/+, Cbs+/−/Asm+/− and Cbs+/−/Asm−/− as well as their Cbs wild type littermates were used to study the role of Asm−/− under a background of Cbs+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs+/−) mice with different copies of Asm gene compared to Cbs+/+ mice with different Asm gene copies. Cbs+/−/Asm+/+ mice had significantly increased renal Asm activity, ceramide production and O2.− level compared to Cbs+/+/Asm+/+, while Cbs+/−/Asm−/− mice showed significantly reduced renal Asm activity, ceramide production and O2.− level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs+/−/Asm−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O2.− production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or

  1. 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

  2. The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation.

    PubMed

    Molle, Virginie; Brown, Alistair K; Besra, Gurdyal S; Cozzone, Alain J; Kremer, Laurent

    2006-10-01

    Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages. PMID:16873379

  3. Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.

    PubMed

    Kremer, Laurent; Dover, Lynn G; Carrère, Séverine; Nampoothiri, K Madhavan; Lesjean, Sarah; Brown, Alistair K; Brennan, Patrick J; Minnikin, David E; Locht, Camille; Besra, Gurdyal S

    2002-06-01

    Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA. PMID:12023885

  4. An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction.

    PubMed

    E, Guangqi; Drujon, Thierry; Correia, Isabelle; Ploux, Olivier; Guianvarc'h, Dominique

    2013-12-01

    We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.

  5. Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I

    SciTech Connect

    Enderle, Mathias; McCarthy, Andrew; Paithankar, Karthik Shivaji; Grininger, Martin

    2015-10-23

    Bacterial and fungal type I fatty-acid synthases (FAS I) are evolutionarily connected, as bacterial FAS I is considered to be the ancestor of fungal FAS I. In this work, the production, crystallization and X-ray diffraction data analysis of a bacterial FAS I are reported. While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.

  6. Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants

    SciTech Connect

    Mayer, R.; Ross, P.; Weinhouse, H.; Amikam, D.; Volman, G.; Ohana, P.; Benziman, M. ); Calhoon, R.D.; Wong, Hing C.; Emerick, A.W. )

    1991-06-15

    To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

  7. Analogs of the antituberculous agent pyrazinamide are competitive inhibitors of NADPH binding to M. tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Pugliese, Kaitlin M; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2012-11-01

    Analogs of pyrazinamide (=pyrazine-2-carboxamide; PZA), an essential component of short-course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl-POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding.

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

    SciTech Connect

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

    2009-08-14

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

  9. 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

  10. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    SciTech Connect

    Hopperton, Kathryn E.; Duncan, Robin E.; Bazinet, Richard P.; Archer, Michael C.

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from {sup 14}C-labeled acetate to those supplied exogenously as {sup 14}C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare

  11. An Intronless β-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea.

    PubMed

    Liu, Yanling; Zhao, Zhongjuan; Xue, Zheyong; Wang, Long; Cai, Yunfei; Wang, Peng; Wei, Tiandi; Gong, Jing; Liu, Zhenhua; Li, Juan; Li, Shuo; Xiang, Fengning

    2016-01-01

    Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a β-amyrin synthase (βAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known β-amyrin synthases (βASs). Heterologously expressed GsAS2 generates more β-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in β-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less β-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more β-amyrin than native GsAS1. PMID:27624821

  12. An Intronless β-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea

    PubMed Central

    Liu, Yanling; Zhao, Zhongjuan; Xue, Zheyong; Wang, Long; Cai, Yunfei; Wang, Peng; Wei, Tiandi; Gong, Jing; Liu, Zhenhua; Li, Juan; Li, Shuo; Xiang, Fengning

    2016-01-01

    Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a β-amyrin synthase (βAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known β-amyrin synthases (βASs). Heterologously expressed GsAS2 generates more β-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in β-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less β-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more β-amyrin than native GsAS1. PMID:27624821

  13. Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation

    PubMed Central

    Du, Xueliang; Edelstein, Diane; Obici, Silvana; Higham, Ninon; Zou, Ming-Hui; Brownlee, Michael

    2006-01-01

    Insulin resistance markedly increases cardiovascular disease risk in people with normal glucose tolerance, even after adjustment for known risk factors such as LDL, triglycerides, HDL, and systolic blood pressure. In this report, we show that increased oxidation of FFAs in aortic endothelial cells without added insulin causes increased production of superoxide by the mitochondrial electron transport chain. FFA-induced overproduction of superoxide activated a variety of proinflammatory signals previously implicated in hyperglycemia-induced vascular damage and inactivated 2 important antiatherogenic enzymes, prostacyclin synthase and eNOS. In 2 nondiabetic rodent models — insulin-resistant, obese Zucker (fa/fa) rats and high-fat diet–induced insulin-resistant mice — inactivation of prostacyclin synthase and eNOS was prevented by inhibition of FFA release from adipose tissue; by inhibition of the rate-limiting enzyme for fatty acid oxidation in mitochondria, carnitine palmitoyltransferase I; and by reduction of superoxide levels. These studies identify what we believe to be a novel mechanism contributing to the accelerated atherogenesis and increased cardiovascular disease risk occurring in people with insulin resistance. PMID:16528409

  14. Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin.

    PubMed

    Klatt, P; Pfeiffer, S; List, B M; Lehner, D; Glatter, O; Bächinger, H P; Werner, E R; Schmidt, K; Mayer, B

    1996-03-29

    Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

  15. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  16. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

    PubMed Central

    Paradkar, A S; Jensen, S E

    1995-01-01

    A Streptomyces clavuligerus mutant disrupted in cas2, encoding the clavaminate synthase (CAS2) isoenzyme, was constructed by a gene replacement procedure. The resulting cas2 mutant showed no clavulanic acid production when grown in starch-asparagine medium. However, in soy medium, the cas2 mutant did produce clavulanic acid, although in amounts less than those produced by wild-type cultures. This medium-dependent leaky phenotype correlated well with the presence of the cas1 transcript, encoding the CAS1 isoenzyme, in cultures grown in soy medium and with its absence from those grown in starch-asparagine medium. This suggested that CAS1 and CAS2 both contribute to clavulanic acid production but that their production is regulated differently. Under nutritional conditions in which cas1 expression is blocked, cas2 becomes essential for clavulanic acid production. Northern (RNA) analysis revealed that while cas1 is transcribed as a 1.4-kb monocistronic transcript only, cas2 is transcribed both as a 1.2-kb monocistronic transcript and as part of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease analysis located the transcription start point of the monocistronic cas2 transcript at a C residue 103 nucleotides upstream from the cas2 start codon. PMID:7868606

  17. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  18. Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I

    PubMed Central

    Enderle, Mathias; McCarthy, Andrew; Paithankar, Karthik Shivaji; Grininger, Martin

    2015-01-01

    While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution. PMID:26527268

  19. Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I.

    PubMed

    Enderle, Mathias; McCarthy, Andrew; Paithankar, Karthik Shivaji; Grininger, Martin

    2015-11-01

    While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.

  20. In vitro evidence that D-serine disturbs the citric acid cycle through inhibition of citrate synthase activity in rat cerebral cortex.

    PubMed

    Zanatta, Angela; Schuck, Patrícia Fernanda; Viegas, Carolina Maso; Knebel, Lisiane Aurélio; Busanello, Estela Natacha Brandt; Moura, Alana Pimentel; Wajner, Moacir

    2009-11-17

    The present work investigated the in vitro effects of D-serine (D-Ser) on important parameters of energy metabolism in cerebral cortex of young rats. The parameters analyzed were CO(2) generation from glucose and acetate, glucose uptake and the activities of the respiratory chain complexes I-IV, of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase and of creatine kinase and Na(+),K(+)-ATPase. Our results show that D-Ser significantly reduced CO(2) production from acetate, but not from glucose, reflecting an impairment of the citric acid cycle function. Furthermore, D-Ser did not affect glucose uptake. We also observed that the activity of the mitochondrial enzyme citrate synthase from mitochondrial preparations and purified citrate synthase was significantly inhibited by D-Ser, whereas the other activities of the citric acid cycle as well as the activities of complexes I-III, II-III, II and IV of the respiratory chain, creatine kinase and Na(+),K(+)-ATPase were not affected by this D-amino acid. We also found that L-serine did not affect citrate synthase activity from mitochondrial preparations and purified enzyme. The data indicate that D-Ser impairs the citric acid cycle activity via citrate synthase inhibition, therefore compromising energy metabolism production in cerebral cortex of young rats. Therefore, it is presumed that this mechanism may be involved at least in part in the neurological damage found in patients affected by disorders in which D-Ser metabolism is impaired, with altered cerebral concentrations of this D-amino acid.

  1. Retinoic acid activates human inducible nitric oxide synthase gene through binding of RAR{alpha}/RXR{alpha} heterodimer to a novel retinoic acid response element in the promoter

    SciTech Connect

    Zou Fang; Liu Yan; Liu Li; Wu Kailang; Wei Wei; Zhu Ying . E-mail: yingzhu@whu.edu.cn; Wu Jianguo . E-mail: wu9988@vip.sina.com

    2007-04-06

    Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RAR{alpha}/RXR{alpha} heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5 bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RAR{alpha}/RXR{alpha} directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RAR{alpha}/RXR{alpha}) in the induction of hiNOS by RA.

  2. Fatty Acid Synthase as a Factor Required for Exercise-Induced Cognitive Enhancement and Dentate Gyrus Cellular Proliferation

    PubMed Central

    Chorna, Nataliya E.; Santos-Soto, Iván J.; Carballeira, Nestor M.; Morales, Joan L.; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P.; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis. PMID:24223732

  3. Fatty acid synthase as a factor required for exercise-induced cognitive enhancement and dentate gyrus cellular proliferation.

    PubMed

    Chorna, Nataliya E; Santos-Soto, Iván J; Carballeira, Nestor M; Morales, Joan L; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis.

  4. 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

  5. Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

    PubMed

    Zaccardi, Margot J; Yezdimer, Eric M; Boehr, David D

    2013-09-13

    The tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase is a proposed target for new antimicrobials and is a favored starting framework in enzyme engineering studies. Forty years ago, Parry proposed that the enzyme mechanism proceeds through two intermediates in a series of condensation, decarboxylation, and dehydration steps. X-ray crystal structures have suggested that Lys-110 (numbering according to the Sulfolobus solfataricus enzyme) behaves as a general acid both in the condensation and dehydration steps, but did not reveal an efficient pathway for the reprotonation of this critical residue. Our mutagenesis and kinetic experiments suggest an alternative mechanism whereby Lys-110 acts as a general acid in the condensation step, but another invariant residue, Lys-53, acts as the general acid in the dehydration step. These studies also indicate that the conserved residue Glu-51 acts as the general base in the dehydration step. The revised mechanism effectively divides the active site into discrete regions where the catalytic surfaces containing Lys-110 and Lys-53/Glu-51 catalyze the ring closure (i.e. condensation and decarboxylation) and dehydration steps, respectively. These results can be leveraged toward the development of novel inhibitors against this validated antimicrobial target and toward the rational engineering of the enzyme to produce indole derivatives that are highly prized by the pharmaceutical and agricultural industries.

  6. Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

    PubMed

    Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

    2015-08-01

    Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

  7. Δ9-Tetrahydrocannabinolic acid synthase: The application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis.

    PubMed

    Lange, Kerstin; Schmid, Andreas; Julsing, Mattijs K

    2016-09-10

    Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize Δ(9)-tetrahydrocannabinolic acid (THCA), which is the direct precursor of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL(-1)h(-1) were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids. PMID:27369551

  8. Citrate enhances in vitro metastatic behaviours of PC-3M human prostate cancer cells: status of endogenous citrate and dependence on aconitase and fatty acid synthase.

    PubMed

    Mycielska, Maria E; Broke-Smith, Timothy P; Palmer, Christopher P; Beckerman, Rachel; Nastos, Theodoros; Erguler, Kamil; Djamgoz, Mustafa B A

    2006-01-01

    Prostate is a unique organ that produces and releases large amounts of citrate. This is reduced significantly in cancer and it is possible that citrate is (re)taken up and used as a metabolite to enhance cellular activity. The main purpose of this study was to determine how cytosolic citrate might affect in vitro metastatic cell behaviours (lateral motility, endocytosis and adhesion). Normal (PNT2-C2) and metastatic (PC-3M) human prostate cancer cells were used in a comparative approach. As regards intermediary metabolic enzymes, aconitase and fatty acid synthase, already implicated in prostate cancer, were evaluated. The level of intracellular citrate was significantly higher in PNT2-C2 cells under both control conditions and following preincubation in extracellular citrate. Supply of exogenous citrate enhanced endocytosis, lateral motility, decreased cell adhesion of PC-3M cells but failed to produce any effect on normal cells. Real-time PCR measurements showed that the mRNA levels of mitochondrial and cytosolic aconitases and fatty acid synthase were significantly higher in PC-3M cells. Correspondingly, aconitase activity was also higher in PC-3M cells. Using cerulenin (an inhibitor of fatty acid synthase), oxalomalate and fluorocitrate (inhibiting aconitases), we investigated the dependence of citrate-induced down-regulation of cellular adhesion on aconitase and fatty acid synthase activities. It was concluded: (1) that strongly metastatic PC-3M cells stored less/utilised more cytosolic citrate than the normal PNT2-C2 cells and (2) that cancer cells could metabolise cytoplasmic citrate via aconitase and fatty acid synthase to enhance their metastatic behaviour. PMID:16798056

  9. Associations of Uric Acid with Polymorphisms in the δ-Aminolevulinic Acid Dehydratase, Vitamin D Receptor, and Nitric Oxide Synthase Genes in Korean Lead Workers

    PubMed Central

    Weaver, Virginia M.; Schwartz, Brian S.; Jaar, Bernard G.; Ahn, Kyu-Dong; Todd, Andrew C.; Lee, Sung-Soo; Kelsey, Karl T.; Silbergeld, Ellen K.; Lustberg, Mark E.; Parsons, Patrick J.; Wen, Jiayu; Lee, Byung-Kook

    2005-01-01

    Recent research suggests that uric acid may be nephrotoxic at lower levels than previously recognized and that it may be one mechanism for lead-related nephrotoxicity. Therefore, in understanding mechanisms for lead-related nephrotoxicity, it would be of value to determine whether genetic polymorphisms that are associated with renal outcomes in lead workers and/or modify associations between lead dose and renal function are also associated with uric acid and/or modify associations between lead dose and uric acid. We analyzed data on three such genetic polymorphisms: δ-aminolevulinic acid dehydratase (ALAD), endothelial nitric oxide synthase (eNOS), and the vitamin D receptor (VDR). Mean (± SD) tibia, blood, and dimercaptosuccinic acid–chelatable lead levels were 37.2 ± 40.4 μg/g bone mineral, 32.0± 15.0 g/dL, and 0.77± 0.86 μg/mg creatinine, respectively, in 798 current and former lead workers. Participants with the eNOS Asp allele had lower mean serum uric acid compared with those with the Glu/Glu genotype. Among older workers (age ≥ median of 40.6 years), ALAD genotype modified associations between lead dose and uric acid levels. Higher lead dose was significantly associated with higher uric acid in workers with the ALAD1-1 genotype; associations were in the opposite direction in participants with the variant ALAD1-2 genotype. In contrast, higher tibia lead was associated with higher uric acid in those with the variant VDR B allele; however, modification was dependent on participants with the bb genotype and high tibia lead levels. We conclude that genetic polymorphisms may modify uric acid mediation of lead-related adverse renal effects. PMID:16263504

  10. (+)-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

  11. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  12. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  13. Expression and regulation of pear 1-aminocyclopropane-1-carboxylic acid synthase gene (PpACS1a) during fruit ripening, under salicylic acid and indole-3-acetic acid treatment, and in diseased fruit.

    PubMed

    Shi, Hai-Yan; Zhang, Yu-Xing

    2014-06-01

    In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.

  14. Delayed circulatory failure due to the induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in anaesthetized rats.

    PubMed Central

    De Kimpe, S J; Hunter, M L; Bryant, C E; Thiemermann, C; Vane, J R

    1995-01-01

    1. This study investigates the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus, a micro-organism without endotoxin, on haemodynamics and induction of nitric oxide synthase (iNOS) in the anaesthetized rat. 2. Intravenous injection of LTA (10 mg kg-1) resulted in a decrease in blood pressure from 123 +/- 1 mmHg to 83 +/- 7 mmHg after 270 min (P < 0.001) and a reduction of the pressor response to noradrenaline (1 microgram kg-1) from 33 +/- 1 mmHg.min to 23 +/- 3 mmHg.min after 270 min (P < 0.05). 3. The delayed circulatory failure (hypotension and vascular hyporeactivity) caused by LTA was prevented by pretreatment of rats with dexamethasone (10 mg kg-1, 60 min prior to LTA) or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg kg-1 h-1, i.v. infusion starting 30 min prior to LTA). 4. In contrast, treatment of rats with polymyxin B (0.05 mg kg-1), an agent which binds endotoxin (lipopolysaccharides, LPS), did not affect the delayed circulatory failure caused by LTA. Polymyxin B, however, attenuated the hypotension and vascular hyporeactivity to noradrenaline afforded by endotoxaemia (2 mg kg-1 LPS, i.v.) for 270 min. 5. The delayed circulatory failure caused by LTA was associated with a time-dependent increase in (i) the expression of iNOS protein in the lung (Western blot analysis), and (ii) iNOS activity. This increase in iNOS protein and activity was prevented by pretreatment of LTA-rats with dexamethasone (10 mg kg-1).(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 6 PMID:7542534

  15. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    PubMed

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro. PMID:27559295

  16. Function of heterologous Mycobacterium tuberculosis InhA, a type 2 fatty acid synthase enzyme involved in extending C20 fatty acids to C60-to-C90 mycolic acids, during de novo lipoic acid synthesis in Saccharomyces cerevisiae.

    PubMed

    Gurvitz, Aner; Hiltunen, J Kalervo; Kastaniotis, Alexander J

    2008-08-01

    We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C(20) fatty acids to form C(60)-to-C(90) mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Delta cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Delta cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C(4) to C(8)) than was previously thought (>C(12)). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis. PMID:18552191

  17. Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.

    PubMed

    Dye, J F; Vause, S; Johnston, T; Clark, P; Firth, J A; D'Souza, S W; Sibley, C P; Glazier, J D

    2004-01-01

    We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low. PMID:14597568

  18. Inhibitory effects of onion (Allium cepa L.) extract on proliferation of cancer cells and adipocytes via inhibiting fatty acid synthase.

    PubMed

    Wang, Yi; Tian, Wei-Xi; Ma, Xiao-Feng

    2012-01-01

    Onions (Allium cepa L.) are widely used in the food industry for its nutritional and aromatic properties. Our studies showed that ethyl acetate extract of onion (EEO) had potent inhibitory effects on animal fatty acid synthase (FAS), and could induce apoptosis in FAS over-expressing human breast cancer MDA-MB-231 cells. Furthermore, this apoptosis was accompanied by reduction of intracellular FAS activity and could be rescued by 25 mM or 50 mM exogenous palmitic acids, the final product of FAS catalyzed synthesis. These results suggest that the apoptosis induced by EEO occurs via inhibition of FAS. We also found that EEO could suppress lipid accumulation during the differentiation of 3T3-L1 adipocytes, which was also related to its inhibition of intracellular FAS activity. Since obesity is closely related to breast cancer and obese patients are at elevated risk of developing various cancers, these findings suggested that onion might be useful for preventing obesity-related malignancy.

  19. Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor α (RORα)

    PubMed Central

    Crumbley, Christine; Wang, Yongjun; Banerjee, Subhashis; Burris, Thomas P.

    2012-01-01

    The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression. PMID:22485150

  20. ACTIVATION OF VASCULAR ENDOTHELIAL NITRIC OXIDE SYNTHASE AND HEME OXYGENASE-1 EXPRESSION BY ELECTROPHILIC NITRO-FATTY ACIDS

    PubMed Central

    Khoo, Nicholas K.H.; Rudolph, Volker; Cole, Marsha P.; Golin-Bisello, Franca; Schopfer, Francisco J.; Woodcock, Steven R.; Batthyany, Carlos; Freeman, Bruce A.

    2010-01-01

    Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated byproducts of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yield electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO2) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO2 via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO2-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO2-FAs stimulated the phosphorylation of eNOS at Ser1179. These post-translational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO2-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders. PMID:19857569

  1. Low concentrations of salicylic acid delay methyl jasmonate-induced leaf senescence by up-regulating nitric oxide synthase activity.

    PubMed

    Ji, Yingbin; Liu, Jian; Xing, Da

    2016-09-01

    In plants, extensive efforts have been devoted to understanding the crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling in pathogen defenses, but this crosstalk has scarcely been addressed during senescence. In this study, the effect of SA application on methyl jasmonate (MeJA)-induced leaf senescence was assessed. We found that low concentrations of SA (1-50 μM) played a delayed role against the senescence promoted by MeJA. Furthermore, low concentrations of SA enhanced plant antioxidant defenses and restricted reactive oxygen species (ROS) accumulation in MeJA-treated leaves. When applied simultaneously with MeJA, low concentrations of SA triggered a nitric oxide (NO) burst, and the elevated NO levels were linked to the nitric oxide associated 1 (NOA1)-dependent pathway via nitric oxide synthase (NOS) activity. The ability of SA to up-regulate plant antioxidant defenses, reduce ROS accumulation, and suppress leaf senescence was lost in NO-deficient Atnoa1 plants. In a converse manner, exogenous addition of NO donors increased the plant antioxidant capacity and lowered the ROS levels in MeJA-treated leaves. Taken together, the results indicate that SA at low concentrations counteracts MeJA-induced leaf senescence through NOA1-dependent NO signaling and strengthening of the antioxidant defense. PMID:27440938

  2. Association between single-nucleotide polymorphisms of fatty acid synthase gene and meat quality traits in Datong Yak (Bos grunniens).

    PubMed

    Chu, M; Wu, X Y; Guo, X; Pei, J; Jiao, F; Fang, H T; Liang, C N; Ding, X Z; Bao, P J; Yan, P

    2015-03-30

    Fatty acid synthase (FASN) is a key enzyme in fatty acid anabolism that plays an important role in the fat deposit of eukaryotic cells. Therefore, in this study, we detected 2 novel single-nucleotide polymorphisms (SNPs) in the FASN gene in 313 adult individuals of Datong yak using polymerase chain reaction-single strand conformation polymorphism and DNA sequencing techniques. SNP g.5477C>T is located in intron 3 of FASN, and 3 genotypes, HH, HG, and GG, were detected in this mutation site. SNP g.16930T>A is located in exon 37 of FASN, and 2 genotypes, EE and EF, were detected in this site. Association analysis of these 2 SNPs with meat quality traits showed that in SNP g.5477C>T, yaks with the HH genotype and HG genotype had significantly higher intramuscular fat content than individuals with the GG genotype (P < 0.01). In SNP g.16930T>A, yaks with the EE genotype also had significantly higher IMF content than individuals with the EF genotype (P < 0.01). The results indicate that FASN may be used as a candidate gene affecting intramuscular fat content in Datong yaks.

  3. Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase.

    PubMed

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-12-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

  4. Enhancement of the inducible NO synthase activation by retinoic acid is mimicked by RARalpha agonist in vivo.

    PubMed

    Seguin-Devaux, Carole; Devaux, Yvan; Latger-Cannard, Véronique; Grosjean, Sandrine; Rochette-Egly, Cécile; Zannad, Faiez; Meistelman, Claude; Mertes, Paul-Michel; Longrois, Dan

    2002-09-01

    We have previously shown that all-trans retinoic acid (atRA), the active metabolite of vitamin A, enhances the activation of the inducible nitric oxide synthase (NOS II) pathway, a component of innate immunity, in rats in vivo. We investigated the relative contribution of retinoic acid receptor-alpha (RARalpha) and retinoid X receptors (RXRs) to NOS II activation triggered by LPS. Five-day supplementation with 10 mg/kg of either atRA or the RARalpha selective agonist Ro-40-6055, but not with 10 mg/kg of the pan-RXR agonist Ro-25-7386, enhanced the LPS-induced NOS II mRNA, protein expression in liver, and plasma nitrite/nitrate concentration. Both atRA and the RARalpha agonist (but not the RXR agonist) increased the number of peripheral T helper lymphocytes and plasma interferon-gamma concentration. Synergism between retinoids and LPS on NOS II activation within an organ coincided with synergism on interferon regulatory factor-1 mRNA expression but not with the level of expression of the RARalpha protein. These results suggest that, in vivo, atRA activates NOS II through RARalpha and contributes to characterizing the complex effect of retinoids on the host inflammatory/immune response.

  5. Effect of increased free fatty acid supply on glucose metabolism and skeletal muscle glycogen synthase activity in normal man.

    PubMed

    Johnson, A B; Argyraki, M; Thow, J C; Cooper, B G; Fulcher, G; Taylor, R

    1992-02-01

    1. Experimental elevation of plasma non-esterified fatty acid concentrations has been postulated to decrease insulin-stimulated glucose oxidation and storage rates. Possible mechanisms were examined by measuring skeletal muscle glycogen synthase activity and muscle glycogen content before and during hyperinsulinaemia while fasting plasma non-esterified fatty acid levels were maintained. 2. Fasting plasma non-esterified fatty acid levels were maintained in seven healthy male subjects by infusion of 20% (w/v) Intralipid (1 ml/min) for 120 min before and during a 240 min hyperinsulinaemic euglycaemic clamp (100 m-units h-1 kg-1) combined with indirect calorimetry. On the control day, 0.154 mol/l NaCl was infused. Vastus lateralis muscle biopsy was performed before and at the end of the insulin infusion. 3. On the Intralipid study day serum triacylglycerol (2.24 +/- 0.20 versus 0.67 +/- 0.10 mmol/l), plasma nonesterified fatty acid (395 +/- 13 versus 51 +/- 1 mumol/l), blood glycerol (152 +/- 2 versus 11 +/- 1 mumol/l) and blood 3-hydroxybutyrate clamp levels [mean (95% confidence interval)] [81 (64-104) versus 4 (3-5) mumol/l] were all significantly higher (all P less than 0.001) than on the control study day. Lipid oxidation rates were also elevated (1.07 +/- 0.07 versus 0.27 +/- 0.08 mg min-1 kg-1, P less than 0.001). During the clamp with Intralipid infusion, insulin-stimulated whole-body glucose disposal decreased by 28% (from 8.53 +/- 0.77 to 6.17 +/- 0.71 mg min-1 kg-1, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Increased expression of fatty acid synthase and acetyl-CoA carboxylase in the prefrontal cortex and cerebellum in the valproic acid model of autism

    PubMed Central

    Chen, Jianling; Wu, Wei; Fu, Yingmei; Yu, Shunying; Cui, Donghong; Zhao, Min; Du, Yasong; Li, Jijun; Li, Xiaohong

    2016-01-01

    The primary aim of the present study was to investigate alterations in enzymes associated with fatty acid synthesis, namely fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), in the prefrontal cortex and cerebellum of the valproic acid (VPA)-induced animal model of autism. In this model, pregnant rats were given a single intraperitoneal injection of VPA, and prefrontal cortex and cerebellum samples from their pups were analyzed. The results of western blotting and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that the protein and mRNA expression levels of FASN, ACC and phospho-ACC (pACC) were increased in the prefrontal cortex and cerebellum of the VPA model of autism. Furthermore, in the prefrontal cortex and cerebellum of the VPA model of autism, AMPK expression is increased, whereas PI3K and Akt expression are unchanged. This suggests that disorder of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/FASN and/or adenosine 5′-monophosphate-activated protein kinase (AMPK)/ACC pathway may be involved in the pathogenesis of autism. It is hypothesized that fatty acid synthesis participates in autism through PI3K/Akt/FASN and AMPK/ACC pathways. PMID:27602061

  7. Increased expression of fatty acid synthase and acetyl-CoA carboxylase in the prefrontal cortex and cerebellum in the valproic acid model of autism

    PubMed Central

    Chen, Jianling; Wu, Wei; Fu, Yingmei; Yu, Shunying; Cui, Donghong; Zhao, Min; Du, Yasong; Li, Jijun; Li, Xiaohong

    2016-01-01

    The primary aim of the present study was to investigate alterations in enzymes associated with fatty acid synthesis, namely fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), in the prefrontal cortex and cerebellum of the valproic acid (VPA)-induced animal model of autism. In this model, pregnant rats were given a single intraperitoneal injection of VPA, and prefrontal cortex and cerebellum samples from their pups were analyzed. The results of western blotting and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that the protein and mRNA expression levels of FASN, ACC and phospho-ACC (pACC) were increased in the prefrontal cortex and cerebellum of the VPA model of autism. Furthermore, in the prefrontal cortex and cerebellum of the VPA model of autism, AMPK expression is increased, whereas PI3K and Akt expression are unchanged. This suggests that disorder of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/FASN and/or adenosine 5′-monophosphate-activated protein kinase (AMPK)/ACC pathway may be involved in the pathogenesis of autism. It is hypothesized that fatty acid synthesis participates in autism through PI3K/Akt/FASN and AMPK/ACC pathways.

  8. Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses.

    PubMed

    Ohol, Yamini M; Wang, Zhaoti; Kemble, George; Duke, Gregory

    2015-01-01

    Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.

  9. Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses

    PubMed Central

    Ohol, Yamini M.; Wang, Zhaoti; Kemble, George; Duke, Gregory

    2015-01-01

    Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity. PMID:26659560

  10. Examining the Relationship Between Cu-ATSM Hypoxia Selectivity and Fatty Acid Synthase Expression in Human Prostate Cancer Cell Lines

    PubMed Central

    Vāvere, Amy L.; Lewis, Jason S.

    2013-01-01

    Introduction PET imaging with Cu-ATSM for delineating hypoxia has provided valuable clinical information, but investigations in animal models of prostate cancer have shown some inconsistencies. As a defense mechanism in prostate cancer cells, the fatty acid synthesis pathway harnesses its oxidizing power for improving the redox balance despite conditions of extreme hypoxia, potentially altering Cu-ATSM hypoxia-selectivity. Methods Human prostate tumor cultured cell lines (PC-3, 22Rv1, LNCaP, and LAPC-4), were treated with an FAS inhibitor (C75, 100 μM) under anoxia. 64Cu-ATSM uptake into these treated cells, and non-treated anoxic cells, was then examined. Fatty acid synthase (FAS) expression level in each cell line was subsequently quantified by ELISA. An additional study was performed in PC-3 cells to examine the relationship between the restoration of 64Cu-ATSM hypoxia-selectivity and the concentration of C75 (100, 20, 4, or 0.8 μM) administered to the cells. Results Inhibition of fatty acid synthesis with C75 resulted in a significant increase in 64Cu-ATSM retention into prostate tumor cells in vitro under anoxia over 60 mins. Inhibition studies demonstrated higher uptake values of 20.9 ± 3.27, 103.0 ± 32.6, 144.2 ± 32.3, and 200.1 ± 79.3% at 15 mins over control values for LAPC-4, PC-3, LNCaP, and 22Rv1 cells, respectively. A correlation was seen (R2 = 0.911) with FAS expression plotted against % change in 64Cu-ATSM uptake with C75 treatment. Conclusions Although Cu-ATSM has clinical relevance in the PET imaging of hypoxia in many tumor types, its translation to the imaging of prostate cancer may be limited by the over-expression of FAS associated with prostatic malignancies. PMID:18355682

  11. S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid: a model for potential bioreductively activated prodrugs for inhibitors of nitric oxide synthase (NOS) activity.

    PubMed

    Ulhaq, S; Naylor, M A; Chinje, E C; Threadgill, M D; Stratford, I J

    1997-01-01

    Treatment of 1,1-dimethylethyl S-(2-1,1-dimethylethoxycarbonylamino)-5-bromopentanoate with 1-potassio-2-nitroimidazole, followed by deprotection, afforded S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid, which was reduced to S-2-amino-5-(2-aminoimidazol-1-yl)pentanoic acid. This aminoimadazole inhibited rat brain nitric oxide synthase (NOS) activity 3.2 times more potently than did the nitro analogue. Thus S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid is a potent prodrug which may be bioreductively activated to a NOS inhibitor in hypoxic solid tumours.

  12. Divergence of cuticular hydrocarbons in two sympatric grasshopper species and the evolution of fatty acid synthases and elongases across insects

    PubMed Central

    Finck, Jonas; Berdan, Emma L.; Mayer, Frieder; Ronacher, Bernhard; Geiselhardt, Sven

    2016-01-01

    Cuticular hydrocarbons (CHCs) play a major role in the evolution of reproductive isolation between insect species. The CHC profiles of two closely related sympatric grasshopper species, Chorthippus biguttulus and C. mollis, differ mainly in the position of the first methyl group in major methyl-branched CHCs. The position of methyl branches is determined either by a fatty acid synthase (FAS) or by elongases. Both protein families showed an expansion in insects. Interestingly, the FAS family showed several lineage-specific expansions, especially in insect orders with highly diverse methyl-branched CHC profiles. We found five putative FASs and 12 putative elongases in the reference transcriptomes for both species. A dN/dS test showed no evidence for positive selection acting on FASs and elongases in these grasshoppers. However, one candidate FAS showed species-specific transcriptional differences and may contribute to the shift of the methyl-branch position between the species. In addition, transcript levels of four elongases were expressed differentially between the sexes. Our study indicates that complex methyl-branched CHC profiles are linked to an expansion of FASs genes, but that species differences can also mediated at the transcriptional level. PMID:27677406

  13. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    PubMed

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

  14. Curcumin inhibits intracellular fatty acid synthase and induces apoptosis in human breast cancer MDA-MB-231 cells.

    PubMed

    Fan, Huijin; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; Sun, Jia; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-05-01

    High levels of fatty acid synthase (FAS) expression have been found in many tumors, including prostate, breast, and ovarian cancers, and inhibition of FAS has been reported to obstruct tumor growth in vitro and in vivo. Curcumin is one of the major active ingredients of Curcuma longa, which has been proven to inhibit the growth of cancer cells. In the present study, we investigated the potential activity of curcumin as a FAS inhibitor for chemoprevention of breast cancer. As a result, curcumin induced human breast cancer MDA-MB-231 cell apoptosis with the half-inhibitory concentration value of 3.63 ± 0.26 µg/ml, and blocked FAS activity, expression and mRNA level in a dose-dependent manner. Curcumin also regulated B-cell lymphoma 2 (Bcl-2), Bax and p-Akt protein expression in MDA-MB-231 cells. Moreover, FAS knockdown showed similar effect as curcumin. All these results suggested that curcumin may induce cell apoptosis via inhibiting FAS. PMID:26985864

  15. Expression of Fatty Acid Synthase Depends on NAC1 and Is Associated with Recurrent Ovarian Serous Carcinomas.

    PubMed

    Ueda, Stefanie M; Yap, Kai Lee; Davidson, Ben; Tian, Yuan; Murthy, Vivek; Wang, Tian-Li; Visvanathan, Kala; Kuhajda, Francis P; Bristow, Robert E; Zhang, Hui; Shih, Ie-Ming

    2010-01-01

    Our previous reports demonstrated that NAC1, a BTB/POZ domain-containing nuclear protein, upregulates in recurrent ovarian serous carcinoma and participates in developing drug resistance in cancer cells. The current study applies quantitative proteomics to identify the proteins controlled by NAC1 by comparing the proteomes of SKOV3 cells with and without expression of a dominant negative NAC1 construct, N130. From the proteins that are downregulated by N130 (upregulated by NAC1), we chose to further characterize fatty acid synthase (FASN). Similar to change in protein level, the FASN transcript level in SKOV3 cells was significantly reduced by N130 induction or by NAC1 knockdown. Immunohistochemistry showed that NAC1 and FASN immunointensities in ovarian serous carcinoma tissues had a highly significant correlation (P < .0001). Moreover, we found that recurrent serous carcinomas exhibited higher FASN immunointensities than their matched primary tumors (P < .001). Multivariate analysis showed that an FASN staining score of >1 in serous carcinomas was associated with a worse overall survival time (P < .01). Finally, C93, a new FASN inhibitor, induced massive apoptosis in carboplatin/paclitaxel resistant ovarian cancer cells. In conclusion, we show that NAC1 is essential for FASN expression in ovarian serous carcinomas and the expression of FASN significantly correlates with tumor recurrence and disease aggressiveness. The dependence of drug resistant tumor cells on FASN suggests a potential application of FASN-based therapeutics for recurrent ovarian cancer patients.

  16. Pharmacophore Modeling and Virtual Screening for Novel Acidic Inhibitors of Microsomal Prostaglandin E2 Synthase-1 (mPGES-1)

    PubMed Central

    2011-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes prostaglandin E2 formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC50 values between 0.4 and 7.9 μM, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC50 values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis. PMID:21466167

  17. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    PubMed

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition. PMID:20127879

  18. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    PubMed

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

  19. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    SciTech Connect

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  20. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

    PubMed

    Choi, Yong-Geun; Park, Chin-Ju; Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa

    2015-02-01

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  1. Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability.

    PubMed

    Veyron-Churlet, Romain; Guerrini, Olivier; Mourey, Lionel; Daffé, Mamadou; Zerbib, Didier

    2004-12-01

    Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions. PMID:15554959

  2. Structure-function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD.

    PubMed

    Lee, C H; Um, P Y; Park, M H

    2001-05-01

    Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305-->Ala showed partial binding and activity. His-288-->Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137-->Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342-->Ala, Asp-313-->Ala and Asp-238-->Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction. PMID:11311149

  3. Pleiotropic Roles of Polyglycerolphosphate Synthase of Lipoteichoic Acid in Growth of Staphylococcus aureus Cells ▿ †

    PubMed Central

    Oku, Yusuke; Kurokawa, Kenji; Matsuo, Miki; Yamada, Sakuo; Lee, Bok-Luel; Sekimizu, Kazuhisa

    2009-01-01

    Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus ΔltaS mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, ΔltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of ΔltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the ΔltaS mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the ΔltaS mutation was found to be synthetically lethal with the ΔtagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function. PMID:18952789

  4. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  5. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  6. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    PubMed

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

    2008-10-24

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  7. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    PubMed

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

    2008-10-24

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

  8. Comparison of γδ T cell responses and farnesyl diphosphate synthase inhibition in tumor cells pretreated with zoledronic acid

    PubMed Central

    Idrees, Atif S. M.; Sugie, Tomoharu; Inoue, Chiyomi; Murata-Hirai, Kaoru; Okamura, Haruki; Morita, Craig T.; Minato, Nagahiro; Toi, Masakazu; Tanaka, Yoshimasa

    2013-01-01

    Summary Exposing human tumor cells to nitrogen-containing bisphosphonates (N-BPs), such as zoledronic acid (Zol), greatly increases their susceptibility to killing by γδ T cells. Based on this finding and other studies, cancer immunotherapy using γδ T cells and N-BPs has been studied in pilot clinical trials and has shown benefits. Although Zol treatment can render a wide variety of human tumor cells susceptible to γδ T cell killing, there has not been a systematic investigation to determine which types of tumor cells are the most susceptible to γδ T cell-mediated cytotoxicity. In this study, we determined the Zol concentrations required to stimulate half maximal tumor necrosis factor-α production by γδ T cells cultured with various tumor cell lines pretreated with Zol and compared these concentrations with those required for half maximal inhibition of farnesyl diphosphate synthase (FPPS) in the same tumor cell lines. The inhibition of tumor cell growth by Zol was also assessed. We found that FPPS inhibition strongly correlated with γδ T cell activation, confirming that the mechanism underlying γδ T cell activation by Zol is isopentenyl diphosphate (IPP) accumulation due to FPPS blockade. In addition, we showed that γδ TCR-mediated signaling correlated with γδ T cell tumor necrosis factor-α production and cytotoxicity. Some lymphoma, myeloid leukemia, and mammary carcinoma cell lines were relatively resistant to Zol treatment suggesting that assessing tumor sensitivity to Zol may help select those patients most likely to benefit from immunotherapy with γδ T cells. PMID:23387443

  9. The Nutrient-Dependent O-GlcNAc Modification Controls the Expression of Liver Fatty Acid Synthase.

    PubMed

    Baldini, Steffi F; Wavelet, Cindy; Hainault, Isabelle; Guinez, Céline; Lefebvre, Tony

    2016-08-14

    Liver Fatty Acid Synthase (FAS) is pivotal for de novo lipogenesis. Loss of control of this metabolic pathway contributes to the development of liver pathologies ranging from steatosis to nonalcoholic steatohepatitis (NASH) which can lead to cirrhosis and, less frequently, to hepatocellular carcinoma. Therefore, deciphering the molecular mechanisms governing the expression and function of key enzymes such as FAS is crucial. Herein, we link the availability of this lipogenic enzyme to the nutrient-dependent post-translational modification O-GlcNAc that is thought to be deregulated in metabolic diseases (diabetes, obesity, and metabolic syndrome). We demonstrate that expression and activity of liver FAS correlate with O-GlcNAcylation contents in ob/ob mice and in mice fed with a high-carbohydrate diet both in a transcription-dependent and -independent manner. More importantly, inhibiting the removal of O-GlcNAc residues in mice intraperitoneally injected with the selective and potent O-GlcNAcase (OGA) inhibitor Thiamet-G increases FAS expression. FAS and O-GlcNAc transferase (OGT) physically interact, and FAS is O-GlcNAc modified. Treatment of a liver cell line with drugs or nutrients that elevate the O-GlcNAcylation interferes with FAS expression. Inhibition of OGA increases the interaction between FAS and the deubiquitinase Ubiquitin-specific protease-2a (USP2A) in vivo and ex vivo, providing mechanistic insights into the control of FAS expression through O-GlcNAcylation. Together, these results reveal a new type of regulation of FAS, linked to O-GlcNAcylation status, and advance our knowledge on deregulation of lipogenesis in diverse forms of liver diseases.

  10. The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys

    PubMed Central

    Nawrocki, Andrea R.; Zhou, Dan; Wu, Margaret; Previs, Stephen; Miller, Corey; Liu, Haiying; Hines, Catherine D. G.; Madeira, Maria; Cao, Jin; Herath, Kithsiri; Wang, Liangsu; Kelley, David E.; Li, Cai

    2016-01-01

    Objectives Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. Methods We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. Results The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. Conclusions These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic

  11. The Nutrient-Dependent O-GlcNAc Modification Controls the Expression of Liver Fatty Acid Synthase.

    PubMed

    Baldini, Steffi F; Wavelet, Cindy; Hainault, Isabelle; Guinez, Céline; Lefebvre, Tony

    2016-08-14

    Liver Fatty Acid Synthase (FAS) is pivotal for de novo lipogenesis. Loss of control of this metabolic pathway contributes to the development of liver pathologies ranging from steatosis to nonalcoholic steatohepatitis (NASH) which can lead to cirrhosis and, less frequently, to hepatocellular carcinoma. Therefore, deciphering the molecular mechanisms governing the expression and function of key enzymes such as FAS is crucial. Herein, we link the availability of this lipogenic enzyme to the nutrient-dependent post-translational modification O-GlcNAc that is thought to be deregulated in metabolic diseases (diabetes, obesity, and metabolic syndrome). We demonstrate that expression and activity of liver FAS correlate with O-GlcNAcylation contents in ob/ob mice and in mice fed with a high-carbohydrate diet both in a transcription-dependent and -independent manner. More importantly, inhibiting the removal of O-GlcNAc residues in mice intraperitoneally injected with the selective and potent O-GlcNAcase (OGA) inhibitor Thiamet-G increases FAS expression. FAS and O-GlcNAc transferase (OGT) physically interact, and FAS is O-GlcNAc modified. Treatment of a liver cell line with drugs or nutrients that elevate the O-GlcNAcylation interferes with FAS expression. Inhibition of OGA increases the interaction between FAS and the deubiquitinase Ubiquitin-specific protease-2a (USP2A) in vivo and ex vivo, providing mechanistic insights into the control of FAS expression through O-GlcNAcylation. Together, these results reveal a new type of regulation of FAS, linked to O-GlcNAcylation status, and advance our knowledge on deregulation of lipogenesis in diverse forms of liver diseases. PMID:27185461

  12. Fatty acid synthase is a metabolic marker of cell proliferation rather than malignancy in ovarian cancer and its precursor cells.

    PubMed

    Veigel, Daniel; Wagner, Renate; Stübiger, Gerald; Wuczkowski, Michael; Filipits, Martin; Horvat, Reinhard; Benhamú, Bellinda; López-Rodríguez, María Luz; Leisser, Asha; Valent, Peter; Grusch, Michael; Hegardt, Fausto G; García, Jordi; Serra, Dolors; Auersperg, Nelly; Colomer, Ramón; Grunt, Thomas W

    2015-05-01

    Ovarian cancer (OC) is caused by genetic aberrations in networks that control growth and survival. Importantly, aberrant cancer metabolism interacts with oncogenic signaling providing additional drug targets. Tumors overexpress the lipogenic enzyme fatty acid synthase (FASN) and are inhibited by FASN blockers, whereas normal cells are FASN-negative and FASN-inhibitor-resistant. Here, we demonstrate that this holds true when ovarian/oviductal cells reside in their autochthonous tissues, whereas in culture they express FASN and are FASN-inhibitor-sensitive. Upon subculture, nonmalignant cells cease growth, express senescence-associated β-galactosidase, lose FASN and become FASN-inhibitor-resistant. Immortalized ovarian/oviductal epithelial cell lines—although resisting senescence—reveal distinct growth activities, which correlate with FASN levels and FASN drug sensitivities. Accordingly, ectopic FASN stimulates growth in these cells. Moreover, FASN levels and lipogenic activities affect cellular lipid composition as demonstrated by thin-layer chromatography. Correlation between proliferation and FASN levels was finally evaluated in cancer cells such as HOC-7, which contain subclones with variable differentiation/senescence and corresponding FASN expression/FASN drug sensitivity. Interestingly, senescent phenotypes can be induced in parental HOC-7 by differentiating agents. In OC cells, FASN drugs induce cell cycle blockade in S and/or G2/M and stimulate apoptosis, whereas in normal cells they only cause cell cycle deceleration without apoptosis. Thus, normal cells, although growth-inhibited, may survive and recover from FASN blockade, whereas malignant cells get extinguished. FASN expression and FASN drug sensitivity are directly linked to cell growth and correlate with transformation/differentiation/senescence only indirectly. FASN is therefore a metabolic marker of cell proliferation rather than a marker of malignancy and is a useful target for future drug

  13. Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

    PubMed Central

    Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

    2002-01-01

    α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

  14. Gibberellic acid, synthetic auxins, and ethylene differentially modulate alpha-L-Arabinofuranosidase activities in antisense 1-aminocyclopropane-1-carboxylic acid synthase tomato pericarp discs.

    PubMed

    Sozzi, Gabriel O; Greve, L Carl; Prody, Gerry A; Labavitch, John M

    2002-07-01

    Alpha-L-Arabinofuranosidases (alpha-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different alpha-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. alpha-Af I and II are active throughout fruit ontogeny. alpha-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. alpha-Af II activity accounts for over 80% of the total alpha-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, alpha-Af III is ethylene dependent and specifically active during ripening. alpha-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas alpha-Af II and III acted on Na(2)CO(3)-soluble pectins. Different alpha-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. alpha-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only alpha-Af III activity. Results suggest that tomato alpha-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.

  15. Gene-gene interactions of fatty acid synthase (FASN) using multifactor-dimensionality reduction method in Korean cattle.

    PubMed

    Lee, Jeayoung; Jin, Mehyun; Lee, Yoonseok; Ha, Jaejung; Yeo, Jungsou; Oh, Dongyep

    2014-01-01

    We examined the gene-gene interactions of five exonic single nucleotide polymorphisms (SNPs) in the gene encoding fatty acid synthase using 513 Korean cattle and using the model free and the non-parametrical multifactor dimensionality reduction method for the analysis. The five SNPs of g.12870 T>C, g.13126 T>C, g.15532 C>A, g.16907 T>C and g.17924 G>A associated with a variety of fatty acid compositions and marbling score were used in this study. The two-factor interaction between g.13126 T>C and g.15532 C>A had the highest training-balanced among the five-factor models and a testing-balanced accuracy at 70.18 % on C18:1 with a cross-validation consistency of 10 out of 10. Also, the two-factor interaction between g.13126 T>C and g.15532 C>A had the highest testing-balanced accuracy at 68.59 % with a 10 out of 10 cross-validation consistency, than any other models on MUFA. In MS, a single SNP g.15532 C>A had the best accuracy at 58.85 % and the two-factor interaction model g.12870 T>C and g.15532 C>A had the highest testing-balanced accuracy at 64.00 %. The three-factor interaction model g.12870 T>C, g.13126 T>C and g.15532 C>A was recorded as having a high testing-balanced accuracy of 63.24 %, but it was lower than the two-factor interaction model. We used likelihood ratio tests for interaction, and Chi square tests to validate our results, with all tests showing statistical significance. We also compared this with mean scores between the high-risk trait group and low-risk trait group. The genotypes of TTCA, TTAA and TCAA at g.15532 and g.13126 on C18:1, genotypes TTCC, TTCA, TTAA, TCAA CCAA at g.15532 and g.13126 on MUFA and genotypes CCCC, TCCA, CCCA, TTAA, TCAA and CCAA at g.15532 and g.12870 on MS were recommended for the genetic improvement of beef quality.

  16. The 10t,12c isomer of conjugated linoleic acid inhibits fatty acid synthase expression and enzyme activity in human breast, colon, and prostate cancer cells.

    PubMed

    Lau, Dominic S Y; Archer, Michael C

    2010-01-01

    The objective of this study was to determine whether downregulation of fatty acid synthase (FAS) expression and/or inhibition of its activity by the two major CLA isomers, 10t,12c and 9c,11t CLA, could contribute to their inhibitory effect on the growth of human breast (MCF-7), colon (HT-29) and prostate (LNCaP) cancer cell lines. We first confirmed and extended the results of others showing that the inhibitory action of CLA on proliferation is dependent on the cell type as well as the structure of the isomer, the 10,12 isomer being a more potent inhibitor than the 9,11 isomer in the concentration range 25-100 microM. By Western analysis, we showed that 10,12 CLA downregulated FAS expression in all of the cell lines in a concentration-dependent manner, but the 9,11 isomer had no effect. Both isomers inhibited FAS enzyme activity, but 10,12 CLA was again more potent than the 9,11 isomer. Our results suggest that downregulation of FAS by 10,12 CLA, but not by the 9,11 isomer, as well as inhibition of FAS enzyme activity by both isomers, may contribute to growth inhibition of cancer cells but only at relatively high concentrations.

  17. Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH).

    PubMed

    Jiang, Wen; Jiang, Yanfang; Bentley, Gayle J; Liu, Di; Xiao, Yi; Zhang, Fuzhong

    2015-08-01

    Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids.

  18. Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH).

    PubMed

    Jiang, Wen; Jiang, Yanfang; Bentley, Gayle J; Liu, Di; Xiao, Yi; Zhang, Fuzhong

    2015-08-01

    Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids. PMID:25788017

  19. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  20. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.

    PubMed

    Myers, Jennifer S; von Lersner, Ariana K; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  1. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.

    PubMed

    Myers, Jennifer S; von Lersner, Ariana K; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  2. Cystathionine-β-Synthase Inhibition for Colon Cancer: Enhancement of the Efficacy of Aminooxyacetic Acid via the Prodrug Approach

    PubMed Central

    Chao, Celia; Zatarain, John R; Ding, Ye; Coletta, Ciro; Mrazek, Amy A; Druzhyna, Nadiya; Johnson, Paul; Chen, Haiying; Hellmich, Judy L; Asimakopoulou, Antonia; Yanagi, Kazunori; Olah, Gabor; Szoleczky, Petra; Törö, Gabor; Bohanon, Fredrick J; Cheema, Minal; Lewis, Rachel; Eckelbarger, David; Ahmad, Akbar; Módis, Katalin; Untereiner, Ashley; Szczesny, Bartosz; Papapetropoulos, Andreas; Zhou, Jia; Hellmich, Mark R; Szabo, Csaba

    2016-01-01

    Colon cancer cells contain high levels of cystathionine-β-synthase (CBS). Its product, hydrogen sulfide (H2S), promotes the growth and proliferation of colorectal tumor cells. To improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle or AOAA (0.1, 0.5 or 1 mg/kg/d) for 3 wks. Tumor growth was significantly reduced by 9 mg/kg/d AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/d. Thus, the in vivo efficacy of YD0171 is nine times higher than that of AOAA. YD0171 (1 mg/kg/d) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/d) also reduced tumor growth in patient-derived tumor xenograft bearing athymic mice. YD0171 (3 mg/kg/d) induced the regression of established HCT116 tumors in vivo. A 5-d safety study in mice demonstrated that YD0171 at 20 mg/kg/d (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory

  3. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3β modulators in 3-nitropropionic acid-induced neurotoxicity in rats.

    PubMed

    Khan, A; Jamwal, S; Bijjem, K R V; Prakash, A; Kumar, P

    2015-02-26

    The present study has been designed to explore the possible interaction between hemeoxygenase-1 (HO-1) and glycogen synthase kinase-3β (GSK-3β) pathway in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats. 3-NP produces neurotoxicity by inhibition of the mitochondrial complex II (enzyme succinate dehydrogenase) and by sensitizing the N-methyl-D-aspartate receptor. Recent studies have reported the therapeutic potential of HO-1/GSK-3β modulators in different neurodegenerative disorders. However, their exact role is yet to be explored. The present study is an attempt to investigate the effect of pharmacological modulation of HO-1/GSK-3β pathway against 3-NP-induced behavioral, biochemical and molecular alterations in rat. Behavioral observation, oxidative stress, pro-inflammatory [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], HO-1 and GSK-3β activity were evaluated post 3-NP treatment. Findings of the present study demonstrate a significant alteration in the locomotor activity, motor coordination, oxidative burden (increased lipid peroxidation, nitrite concentration and decreased endogenous antioxidants), pro-inflammatory mediators [TNF-α, IL-1β], HO-1 and GSK-3β activity in 3-NP-treated animals. Further, administration of hemin (10- and 30-mg/kg; i.p.) and lithium chloride (LiCl) (25- and 50-mg/kg; i.p.) prevented the alteration in body weight, motor impairments, oxidative stress and cellular markers. In addition, combined administration of hemin (10-mg/kg) and LiCl (25-mg/kg) showed synergistic effect on 3-NP-treated rats. Pretreatment with Tin (IV) protoporphyrin (40 μM/kg), HO-1 inhibitor reversed the beneficial effect of LiCl and hemin. Outcomes of the present study suggest that HO-1 and GSK-3β enzymes are involved in the pathophysiology of HD. The modulators of both the pathways might be used as adjuvants or prophylactic therapy for the treatment of HD-like symptoms.

  4. 1-(Fluoroalkylidene)-1,1-bisphosphonic Acids are Potent and Selective Inhibitors of the Enzymatic Activity of Toxoplasma gondii Farnesyl Pyrophosphate Synthase

    PubMed Central

    Szajnman, Sergio H.; Rosso, Valeria S.; Malayil, Leena; Smith, Alyssa; Moreno, Silvia N. J.; Docampo, Roberto

    2012-01-01

    α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease and against Toxoplasma gondii, the responsible agent of toxoplasmosis and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) has proven to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range exhibiting an IC50 of 30 nM. This compound was two-fold more potent than risedronate (IC50 = 74 nM) taken as a positive control. This enzymatic activity was associated to a strong cell growth inhibition against tachyzoites of T. gondii having an IC50 value of 2.7 μM. PMID:22215028

  5. Disrupted short chain specific β-oxidation and improved synthase expression increase synthesis of short chain fatty acids in Saccharomyces cerevisiae.

    PubMed

    Leber, Christopher; Choi, Jin Wook; Polson, Brian; Da Silva, Nancy A

    2016-04-01

    Biologically derived fatty acids have gained tremendous interest as an alternative to petroleum-derived fuels and chemical precursors. We previously demonstrated the synthesis of short chain fatty acids in Saccharomyces cerevisiae by introduction of the Homo sapiens fatty acid synthase (hFAS) with heterologous phosphopantetheine transferases and heterologous thioesterases. In this study, short chain fatty acid production was improved by combining a variety of novel enzyme and metabolic engineering strategies. The use of a H. sapiens-derived thioesterase and phosphopantetheine transferase were evaluated. In addition, strains were engineered to disrupt either the full β-oxidation (by deleting FAA2, PXA1, and POX1) or short chain-specific β-oxidation (by deleting FAA2, ANT1, and PEX11) pathways. Prohibiting full β-oxidation increased hexanoic and octanoic acid levels by 8- and 79-fold relative to the parent strain expressing hFAS. However, by targeting only short chain β-oxidation, hexanoic and octanoic acid levels increased further to 31- and 140-fold over the parent. In addition, an optimized hFAS gene increased hexanoic, octanoic, decanoic and total short chain fatty acid levels by 2.9-, 2.0-, 2.3-, and 2.2-fold, respectively, relative to the non-optimized counterpart. By combining these unique enzyme and metabolic engineering strategies, octanoic acid was increased more than 181-fold over the parent strain expressing hFAS.

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

    PubMed

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

    1996-02-01

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

  7. Pseudouridine synthases.

    PubMed

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  8. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    PubMed

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

    2012-10-01

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

  9. Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype.

    PubMed

    Onofri, Chiara; de Meijer, Etienne P M; Mandolino, Giuseppe

    2015-08-01

    Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases.

  10. Application of chromatography technology in the separation of active alkaloids from Hypecoum leptocarpum and their inhibitory effect on fatty acid synthase.

    PubMed

    Zhang, Qiulong; Luan, Guangxiang; Ma, Tao; Hu, Na; Suo, Yourui; Wang, Xiaoyan; Ma, Xiaofeng; Ding, Chenxu

    2015-12-01

    A method that involved the combination of pH-zone-refining counter-current chromatography and semipreparative reversed-phase liquid chromatography has been established for the preparative separation of alkaloids from Hypecoum leptocarpum. From 1.2 g of crude sample, 31 mg N-feruloyltyramine, 27 mg oxohydrastinine, 47 mg hydroprotopine, 25 mg leptopidine, and 18 mg hypecocarpine have been obtained. The structure of the new compound, hypecocarpine, is confirmed based on the analysis of spectroscopic data, including NMR, UV, and IR spectroscopy and positive electrospray ionization mass spectrometry. The known chemical structures were characterized on the basis of (1) H and (13) C NMR spectroscopy. The purities of the five alkaloids are all over 92.7% as determined by high-performance liquid chromatography. The alkaloids' cytotoxicity in breast cancer cells is assessed by using a Cell Counting Kit assay and their inhibitory effect on fatty acid synthase expression is assessed by a Western blot assay. These results suggest that leptopidine could suppress growth and induce cytotoxicity in breast cancer cells and that the cytotoxicity of leptopidine may be related to its inhibitory effect on fatty acid synthase expression.

  11. Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12.

    PubMed

    Jackson, J H; Davis, E J; Madu, A C; Braxter, S E

    1981-01-01

    The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.

  12. Hybrid polyketide synthases

    DOEpatents

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  15. 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

  16. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    PubMed

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  17. Cloning, expression, and characterization of para-aminobenzoic acid (PABA) synthase from Agaricus bisporus 02, a thermotolerant mushroom strain.

    PubMed

    Deng, Li-Xin; Shen, Yue-Mao; Song, Si-Yang

    2015-01-01

    The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST-AbADCS or His-AbADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. AbADCS has maximum activity at a temperature of approximately 25°C and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H2O2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.

  18. Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

    DOE PAGES

    Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; Sisson, Lyle A.; Schneider, Philip E.; Posewitz, Matthew C.

    2016-05-26

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

  19. Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase

    PubMed Central

    Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; Sisson, Lyle A.; Schneider, Philip E.; Posewitz, Matthew C.

    2016-01-01

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions. PMID:27303412

  20. Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase.

    PubMed

    Gu, Huiya; Jinkerson, Robert E; Davies, Fiona K; Sisson, Lyle A; Schneider, Philip E; Posewitz, Matthew C

    2016-01-01

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions. PMID:27303412

  1. Increased thymidylate synthase in L1210 cells possessing acquired resistance to N10-propargyl-5,8-dideazafolic acid (CB3717): development, characterization, and cross-resistance studies

    SciTech Connect

    Jackman, A.L.; Alison, D.L.; Calvert, A.H.; Harrap, K.R.

    1986-06-01

    The properties are described of a mutant L1210 cell line (L1210:C15) with acquired resistance (greater than 200-fold) to the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid. TS was overproduced 45-fold and was accompanied by a small increase in the activity of dihydrofolate reductase (2.6-fold). Both the level of resistance and enzyme activities were maintained in drug-free medium (greater than 300 generations). Failure of N10-propargyl-5,8-dideazafolic acid to suppress the (/sup 3/H)-2'-deoxyuridine incorporation into the acid-precipitable material of the resistant line supported the evidence that TS overproduction was the mechanism of resistance; consequently the L1210:C15 cells were largely cross-resistant to another (but weaker) TS inhibitor, 5,8-dideazafolic acid. Minimal cross-resistance was observed to the dihydrofolate reductase inhibitors methotrexate and 5-methyl-5,8-dideazaaminopterin (5- and 2-fold, respectively). L1210 and L1210:C15 cells were, however, equally sensitive to 5-fluorodeoxyuridine (FdUrd), an unexpected finding since a metabolite, 5-fluorodeoxyuridine monophosphate, is a potent TS inhibitor; however, this cytotoxicity against the L1210:C15 cells was antagonized by coincubation with 5 microM folinic acid although folinic acid potentiated the cytotoxicity of FdUrd to the N10-propargyl-5,8-dideazafolic acid-sensitive L1210 line. Thymidine was much less effective as a FdUrd protecting agent in the L1210:C15 when compared with the L1210 cells; however, a combination of thymidine plus hypoxanthine was without any additional effect (compared with thymidine alone) against the sensitive line but effectively protected L1210:C15 cells.

  2. Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

    PubMed Central

    Zhou, Tao; Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Sato, Seizo; Igarashi, Yasuhiro

    2015-01-01

    The incorporation pattern of biosynthetic precursors into two structurally unique polyketides, akaeolide and lorneic acid A, was elucidated by feeding experiments with 13C-labeled precursors. In addition, the draft genome sequence of the producer, Streptomyces sp. NPS554, was performed and the biosynthetic gene clusters for these polyketides were identified. The putative gene clusters contain all the polyketide synthase (PKS) domains necessary for assembly of the carbon skeletons. Combined with the 13C-labeling results, gene function prediction enabled us to propose biosynthetic pathways involving unusual carbon-carbon bond formation reactions. Genome analysis also indicated the presence of at least ten orphan type I PKS gene clusters that might be responsible for the production of new polyketides. PMID:25603349

  3. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    PubMed

    Dung Pham, Van; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-01-01

    Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways.

  4. Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

    PubMed

    Pham, Van Dung; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2015-08-10

    Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

  5. Pu-erh tea, green tea, and black tea suppresses hyperlipidemia, hyperleptinemia and fatty acid synthase through activating AMPK in rats fed a high-fructose diet.

    PubMed

    Huang, Hsiu-Chen; Lin, Jen-Kun

    2012-02-01

    Although green tea extract has been reported to suppress hyperlipidemia, it is unclear how tea extracts prepared from green, oolong, black and pu-erh teas modulate fatty acid synthase expression in rats fed on a high-fructose diet. In this animal study, we evaluated the hypolipidemic and hypoleptinemia effect of these four different tea leaves fed to male Wistar rats for 12 weeks. The results showed that a fructose-rich diet significantly elevated serum triacylglycerols, cholesterol, insulin, and leptin concentrations, as compared with those in the control group. Interestingly, consuming tea leaves for 12 weeks almost normalized the serum triacylglycerols concentrations. Again, rats fed with fructose/green tea and fructose/pu-erh tea showed the greatest reduction in serum TG, cholesterol, insulin and leptin levels. In contrast, serum cholesterol and insulin concentrations of the fructose/oolong tea-fed rats did not normalize. The relative epididymal adipose tissue weight was lower in all rats supplemented with tea leaves than those fed with fructose alone. There was molecular evidence of improved lipid homeostasis according to fatty acid synthase (FAS) protein expression. Furthermore, supplementation of green, black, and pu-erh tea leaves significantly decreased hepatic FAS mRNA and protein levels, and increased AMPK phosphorylation, compared with those of rats fed with fructose only. These findings suggest that the intake of green, black, and pu-erh tea leaves ameliorated the fructose-induced hyperlipidemia and hyperleptinemia state in part through the suppression of FAS protein levels and increased AMPK phosphorylation.

  6. Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase.

    PubMed

    Howe, G A; Lee, G I; Itoh, A; Li, L; DeRocher, A E

    2000-06-01

    Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C(18) fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.

  7. Vanillic acid prevents the deregulation of lipid metabolism, endothelin 1 and up regulation of endothelial nitric oxide synthase in nitric oxide deficient hypertensive rats.

    PubMed

    Kumar, Subramanian; Prahalathan, Pichavaram; Saravanakumar, Murugesan; Raja, Boobalan

    2014-11-15

    Hypertension is one of the main factors causing cardiovascular diseases. The present study was designed to evaluate the protective effect of vanillic acid against nitric oxide deficient rats. Hypertension was induced in adult male albino rats of Wistar strain, weighing 180-220g, by oral administration of N(ω)-nitro-l arginine methyl ester (l-NAME) 40mg/kg in drinking water for 4 weeks. Vanillic acid was administered orally at a dose of 50mg/kg b.w. Nitric oxide deficient rats showed increased levels of mean arterial pressure (MAP), heart rate (HR) and decreased heart nitric oxide metabolites (NOx). A significant increase in the levels of plasma cholesterol, low density lipoprotein-cholesterol (LDL-C), very low density lipoprotein-cholesterol (VLDL-C), triglycerides (TG), free fatty acids (FFA), phospholipids (PL), 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase in the plasma, liver and kidney and decreased level of high density lipoprotein-cholesterol (HDL-C) are observed, whereas there is a decrease in the activities of plasma lipoprotein lipase (LPL) and lecithin cholesterol acyl transferase (LCAT) in nitric oxide deficient rats. l-NAME rats also showed an increase in TC, TG, FFA and PL levels in the liver and kidney tissues. Vanillic acid treatment brought the above parameters towards near normal level. Moreover the down regulated endothelial nitric oxide synthase (eNOS) and up regulated expression of endothelin 1 (ET1) components was also attenuated by vanillic acid treatment. All the above outcomes were confirmed by the histopathological examination. These results suggest that vanillic acid has enough potential to attenuate hypertension, dyslipidemia and hepatic and renal damage in nitric oxide deficient rats. PMID:25239071

  8. A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence

    PubMed Central

    Jiao, Yuntong; Xu, Weirong; Duan, Dong; Wang, Yuejin; Nick, Peter

    2016-01-01

    Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. ‘Carigane’ (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis. PMID:27702992

  9. Increase in nervonic acid content in transformed yeast and transgenic plants by introduction of a Lunaria annua L. 3-ketoacyl-CoA synthase (KCS) gene.

    PubMed

    Guo, Yiming; Mietkiewska, Elzbieta; Francis, Tammy; Katavic, Vesna; Brost, Jennifer M; Giblin, Michael; Barton, Dennis L; Taylor, David C

    2009-03-01

    Nervonic acid is a Very Long-Chain Monounsaturated Fatty Acid (VLCMFA), 24:1 Delta15 (cis-tetracos-15-enoic acid) found in the seed oils of Lunaria annua, borage, hemp, Acer (Purpleblow maple) and Tropaeolum speciosum (Flame flower). However, of these, only the "money plant" (Lunaria annua L.) has been studied and grown sparingly for future development as a niche crop and the outlook has been disappointing. Therefore, our goal was to isolate and characterize strategic new genes for high nervonic acid production in Brassica oilseed crops. To this end, we have isolated a VLCMFA-utilizing 3-Keto-Acyl-CoA Synthase (KCS; fatty acid elongase; EC 2.3.1.86) gene from Lunaria annua and functionally expressed it in yeast, with the recombinant KCS protein able to catalyze the synthesis of several VLCMFAs, including nervonic acid. Seed-specific expression of the Lunaria KCS in Arabidopsis resulted in a 30-fold increase in nervonic acid proportions in seed oils, compared to the very low quantities found in the wild-type. Similar transgenic experiments using B. carinata as the host resulted in a 7-10 fold increase in seed oil nervonic acid proportions. KCS enzyme activity assays indicated that upon using (14)C-22:1-CoA as substrate, the KCS activity from developing seeds of transgenic B. carinata was 20-30-fold higher than the low erucoyl-elongation activity exhibited by wild type control plants. There was a very good correlation between the Lun KCS transcript intensity and the resultant 22:1-CoA KCS activity in developing seed. The highest nervonic acid level in transgenic B. carinata expressing the Lunaria KCS reached 30%, compared to 2.8% in wild type plant. In addition, the erucic acid proportions in these transgenic lines were considerably lower than that found in native Lunaria oil. These results show the functional utility of the Lunaria KCS in engineering new sources of high nervonate/reduced erucic oils in the Brassicaceae. PMID:19082744

  10. Acetohydroxyacid synthases: evolution, structure, and function.

    PubMed

    Liu, Yadi; Li, Yanyan; Wang, Xiaoyuan

    2016-10-01

    Acetohydroxyacid synthase, a thiamine diphosphate-dependent enzyme, can condense either two pyruvate molecules to form acetolactate for synthesizing L-valine and L-leucine or pyruvate with 2-ketobutyrate to form acetohydroxybutyrate for synthesizing L-isoleucine. Because the key reaction catalyzed by acetohydroxyacid synthase in the biosynthetic pathways of branched-chain amino acids exists in plants, fungi, archaea, and bacteria, but not in animals, acetohydroxyacid synthase becomes a potential target for developing novel herbicides and antimicrobial compounds. In this article, the evolution, structure, and catalytic mechanism of acetohydroxyacid synthase are summarized. PMID:27576495

  11. Producing biofuels using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  12. Partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (indoleacetic acid-inositol synthase)

    NASA Technical Reports Server (NTRS)

    Kesy, J. M.; Bandurski, R. S.

    1990-01-01

    A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-beta-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-beta-d-glucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.

  13. Metformin-Induced Killing of Triple Negative Breast Cancer Cells is Mediated by Reduction in Fatty Acid Synthase via miRNA-193b

    PubMed Central

    Wahdan-Alaswad, Reema S.; Cochrane, Dawn R.; Spoelstra, Nicole S.; Howe, Erin N.; Edgerton, Susan M.; Anderson, Steven M.; Thor, Ann D.; Richer, Jennifer K.

    2015-01-01

    The anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) reduces both the incidence and mortality of several types of cancer. Metformin has been shown to selectively kill cancer stem cells and triple negative breast cancer (TNBC) cell lines are more sensitive to the effects of metformin. However, the mechanism underlying the enhanced susceptibility of TNBC to metformin had not been elucidated. Expression profiling of metformin-treated TNBC lines revealed fatty acid synthase (FASN) as one of the genes most significantly downregulated following 24 hours of treatment and a decrease in FASN protein was also observed. Since FASN is critical for de novo fatty acid synthesis, and is important for survival of TNBC, we hypothesized that FASN downregulation facilitates metformin-induced apoptosis. Profiling studies also exposed a rapid metformin-induced increase in miR-193 family members, and miR-193b was found to directly target the FASN 3′UTR. Addition of exogenous miR-193b mimic to untreated TNBC cells resulted in decreased FASN protein expression and increased apoptosis of TNBC cells, while spontaneously immortalized, non-transformed breast epithelial cells remained unaffected. Conversely, antagonizing miR-193 activity impaired the ability of metformin to decrease FASN and cause cell death. Further, the metformin-stimulated increase in miR-193 resulted in reduced mammosphere formation by TNBC lines. These studies provide mechanistic insight into the metformin-induced killing of TNBC. PMID:25213330

  14. Elevated salicylic acid levels conferred by increased expression of ISOCHORISMATE SYNTHASE 1 contribute to hyperaccumulation of SUMO1 conjugates in the Arabidopsis mutant early in short days 4.

    PubMed

    Villajuana-Bonequi, Mitzi; Elrouby, Nabil; Nordström, Karl; Griebel, Thomas; Bachmair, Andreas; Coupland, George

    2014-07-01

    Post-translational modification of proteins by attachment of small ubiquitin-like modifier (SUMO) is essential for plant growth and development. Mutations in the SUMO protease early in short days 4 (ESD4) cause hyperaccumulation of conjugates formed between SUMO and its substrates, and phenotypically are associated with extreme early flowering and impaired growth. We performed a suppressor mutagenesis screen of esd4 and identified a series of mutants called suppressor of esd4 (sed), which delay flowering, enhance growth and reduce hyperaccumulation of SUMO conjugates. Genetic mapping and genome sequencing indicated that one of these mutations (sed111) is in the gene salicylic acid induction-deficient 2 (SID2), which encodes ISOCHORISMATE SYNTHASE I, an enzyme required for biosynthesis of salicylic acid (SA). Analyses showed that compared with wild-type plants, esd4 contains higher levels of SID2 mRNA and about threefold more SA, whereas sed111 contains lower SA levels. Other sed mutants also contain lower SA levels but are not mutant for SID2, although most reduce SID2 mRNA levels. Therefore, higher SA levels contribute to the small size, early flowering and elevated SUMO conjugate levels of esd4. Our results support previous data indicating that SUMO homeostasis influences SA biosynthesis in wild-type plants, and also demonstrate that elevated levels of SA strongly increase the abundance of SUMO conjugates.

  15. Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid1

    PubMed Central

    Wang, Ning Ning; Yang, Shang Fa; Charng, Yee-yung

    2001-01-01

    Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower. PMID:11351088

  16. Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: a mechanism for uric acid-induced cardiovascular disease development.

    PubMed

    Park, Jung-Hyun; Jin, Yoon Mi; Hwang, Soojin; Cho, Du-Hyong; Kang, Duk-Hee; Jo, Inho

    2013-08-01

    The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.

  17. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGES

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  18. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    SciTech Connect

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  19. Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit.

    PubMed

    Tran, Lee; Hanavan, Paul D; Campbell, Latoya E; De Filippis, Elena; Lake, Douglas F; Coletta, Dawn K; Roust, Lori R; Mandarino, Lawrence J; Carroll, Chad C; Katsanos, Christos S

    2016-01-01

    Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = - 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish

  20. Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression

    PubMed Central

    Ventura, Richard; Mordec, Kasia; Waszczuk, Joanna; Wang, Zhaoti; Lai, Julie; Fridlib, Marina; Buckley, Douglas; Kemble, George; Heuer, Timothy S.

    2015-01-01

    Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20–200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K–AKT–mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics. Research in context Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for

  1. Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

    PubMed Central

    Tran, Lee; Hanavan, Paul D.; Campbell, Latoya E.; De Filippis, Elena; Lake, Douglas F.; Coletta, Dawn K.; Roust, Lori R.; Mandarino, Lawrence J.; Carroll, Chad C.; Katsanos, Christos S.

    2016-01-01

    Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish

  2. Characterization of FabG and FabI of the Streptomyces coelicolor dissociated fatty acid synthase.

    PubMed

    Singh, Renu; Reynolds, Kevin A

    2015-03-01

    Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched-chain β-ketoacyl-NAC and enoyl-NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis. PMID:25662938

  3. Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase.

    PubMed

    Roughan, P G

    1997-10-01

    Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10-20 microM, assuming a stromal volume of 66 microl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 microM. Malonyl-CoA concentrations were also very low (<0.1-3.0 microM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 microM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA-acyl-carrier protein transacylase reactions at the concentrations of metabolites measured here are < 0.1-4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

  4. Effects of bovine fatty acid synthase, stearoyl-coenzyme A desaturase, sterol regulatory element-binding protein 1, and growth hormone gene polymorphisms on fatty acid composition and carcass traits in Japanese Black cattle.

    PubMed

    Matsuhashi, T; Maruyama, S; Uemoto, Y; Kobayashi, N; Mannen, H; Abe, T; Sakaguchi, S; Kobayashi, E

    2011-01-01

    The quality of fat is an important factor in defining the quality of meat. Fat quality is determined by the composition of fatty acids. Among lipid metabolism-related genes, including fatty acid synthesis genes, several genetic variations have been reported in the bovine fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), sterol regulatory element-binding protein 1 (SREBP1), and GH genes. In the present study, we evaluated the single and epistatic effects of 5 genetic variations (4 SNP and 1 insertion/deletion) in 4 genes (FASN, SCD, SREBP1, and GH) on the fatty acid composition of the longissimus thoracis muscle and carcass and meat quality traits in 480 commercial Japanese Black cattle. Significant single effects of FASN, SCD, and GH(L127V) polymorphisms on the fatty acid composition of the longissimus thoracis muscle were detected. The A293V polymorphism of SCD had the largest effect on myristic acid (C14:0, P < 0.001), myristoleic acid (C14:1, P < 0.001), stearic acid (C18:0, P < 0.001), oleic acid (C18:1, P < 0.001), and MUFA (P < 0.001). Polymorphisms in the FASN, SCD, and SREBP1 genes showed no effect on any meat yield trait. There were no significant epistatic effects on fatty acid composition among pairs of the 3 genes (FASN, SCD, and SREBP1) involved in fatty acid synthesis. No epistatic interactions (P > 0.1) were detected between FASN and SCD for any carcass trait. When the genotypes of 3 markers (FASN, SCD, and GH(L127V)) were substituted from the lesser effect allele to the greater effect allele, the proportion of C18:1 increased by 4.46%. More than 20% of the genetic variance in the C18:1 level could be accounted for by these 3 genetic markers. The present results revealed that polymorphisms in 2 fatty acid synthesis genes (FASN and SCD) independently influenced fatty acid composition in the longissimus thoracis muscle. These results suggest that SNP in the FASN and SCD genes are useful markers for the improvement of fatty acid composition in

  5. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    SciTech Connect

    Kultti, Anne; Pasonen-Seppaenen, Sanna; Jauhiainen, Marjo; Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I.

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  6. Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission.

    PubMed

    Menzel, Tila R; Weldegergis, Berhane T; David, Anja; Boland, Wilhelm; Gols, Rieta; van Loon, Joop J A; Dicke, Marcel

    2014-09-01

    Jasmonic acid (JA) plays a central role in induced plant defence e.g. by regulating the biosynthesis of herbivore-induced plant volatiles that mediate the attraction of natural enemies of herbivores. Moreover, exogenous application of JA can be used to elicit plant defence responses similar to those induced by biting-chewing herbivores and mites that pierce cells and consume their contents. In the present study, we used Lima bean (Phaseolus lunatus) plants to explore how application of a low dose of JA followed by minor herbivory by spider mites (Tetranychus urticae) affects transcript levels of P. lunatus (E)-β-ocimene synthase (PlOS), emission of (E)-β-ocimene and nine other plant volatiles commonly associated with herbivory. Furthermore, we investigated the plant's phytohormonal response. Application of a low dose of JA increased PlOS transcript levels in a synergistic manner when followed by minor herbivory for both simultaneous and sequential infestation. Emission of (E)-β-ocimene was also increased, and only JA, but not SA, levels were affected by treatments. Projection to latent structures-discriminant analysis (PLS-DA) of other volatiles showed overlap between treatments. Thus, a low-dose JA application results in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defence after herbivore infestation.

  7. Comparative Modeling and Molecular Dynamics Simulation of Substrate Binding in Human Fatty Acid Synthase: Enoyl Reductase and β-Ketoacyl Reductase Catalytic Domains

    PubMed Central

    John, Arun; Krishnakumar, Subramanian

    2015-01-01

    Fatty acid synthase (FASN, EC 2.3.1.85), is a multi-enzyme dimer complex that plays a critical role in lipogenesis. This lipogenic enzyme has gained importance beyond its physiological role due to its implications in several clinical conditions-cancers, obesity, and diabetes. This has made FASN an attractive pharmacological target. Here, we have attempted to predict the theoretical models for the human enoyl reductase (ER) and β-ketoacyl reductase (KR) domains based on the porcine FASN crystal structure, which was the structurally closest template available at the time of this study. Comparative modeling methods were used for studying the structure-function relationships. Different validation studies revealed the predicted structures to be highly plausible. The respective substrates of ER and KR domains-namely, trans-butenoyl and β-ketobutyryl-were computationally docked into active sites using Glide in order to understand the probable binding mode. The molecular dynamics simulations of the apo and holo states of ER and KR showed stable backbone root mean square deviation trajectories with minimal deviation. Ramachandran plot analysis showed 96.0% of residues in the most favorable region for ER and 90.3% for the KR domain, respectively. Thus, the predicted models yielded significant insights into the substrate binding modes of the ER and KR catalytic domains and will aid in identifying novel chemical inhibitors of human FASN that target these domains. PMID:25873848

  8. The metastasis inducer CCN1 (CYR61) activates the fatty acid synthase (FASN)-driven lipogenic phenotype in breast cancer cells

    PubMed Central

    Menendez, Javier A.; Vellon, Luciano; Espinoza, Ingrid; Lupu, Ruth

    2016-01-01

    The angiogenic inducer CCN1 (Cysteine-rich 61, CYR61) is differentially activated in metastatic breast carcinomas. However, little is known about the precise mechanisms that underlie the pro-metastatic actions of CCN1. Here, we investigated the impact of CCN1 expression on fatty acid synthase (FASN), a metabolic oncogene thought to provide cancer cells with proliferative and survival advantages. Forced expression of CCN1 in MCF-7 cells robustly up-regulated FASN protein expression and also significantly increased FASN gene promoter activity 2- to 3-fold, whereas deletion of the sterol response element-binding protein (SREBP) binding site in the FASN promoter completely abrogated CCN1-driven transcriptional activation. Pharmacological blockade of MAPK or PI-3'K activation similarly prevented the ability of CCN1 to induce FASN gene activation. Pharmacological inhibition of FASN activity with the mycotoxin cerulenin or the small compound C75 reversed CCN1-induced acquisition of estrogen independence and resistance to hormone therapies such as tamoxifen and fulvestrant in anchorage-independent growth assays. This study uncovers FASNdependent endogenous lipogenesis as a new mechanism controlling the metastatic phenotype promoted by CCN1. Because estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer, this previously unrecognized CCN1-driven lipogenic phenotype represents a novel metabolic target to clinically manage metastatic disease progression. PMID:27713913

  9. Mulberry leaf polyphenol extract induced apoptosis involving regulation of adenosine monophosphate-activated protein kinase/fatty acid synthase in a p53-negative hepatocellular carcinoma cell.

    PubMed

    Yang, Tzi-Peng; Lee, Huei-Jane; Ou, Ting-Tsz; Chang, Ya-Ju; Wang, Chau-Jong

    2012-07-11

    The polyphenols in mulberry leaf possess the ability to inhibit cell proliferation, invasion, and metastasis of tumors. It was reported that the p53 status plays an important role in switching apoptosis and the cell cycle following adenosine monophosphate-activated protein kinase (AMPK) activation. In this study, we aimed to detect the effect of the mulberry leaf polyphenol extract (MLPE) on inducing cell death in p53-negative (Hep3B) and p53-positive (Hep3B with transfected p53) hepatocellular carcinoma cells and also to clarify the role of p53 in MLPE-treated cells. After treatment of the Hep3B cells with MLPE, apoptosis was induced via the AMPK/PI3K/Akt and Bcl-2 family pathways. Transient transfection of p53 into Hep3B cells led to switching autophagy instead of apoptosis by MLPE treatment. We demonstrated that acridine orange staining and protein expressions of LC-3 and beclin-1 were increased in p53-transfected cells. These results implied induction of apoptosis or autophagy in MLPE-treated hepatocellular carcinoma cells can be due to the p53 status. We also found MLPE can not only activate AMPK but also diminish fatty acid synthase, a molecular target for cancer inhibition. At present, our results indicate MLPE can play an active role in mediating the cell death of hepatocellular carcinoma cells and the p53 might play an important role in regulating the death mechanisms.

  10. Effects of a subconvulsive dose of kainic acid on the gene expressions of the arginine vasopressin, oxytocin and neuronal nitric oxide synthase in the rat hypothalamus.

    PubMed

    Yoshimura, Mitsuhiro; Ohkubo, Jun-ichi; Hashimoto, Hirofumi; Matsuura, Takanori; Maruyama, Takashi; Onaka, Tatsushi; Suzuki, Hideaki; Ueta, Yoichi

    2015-10-01

    Arginine vasopressin (AVP) synthesis in the hypothalamo-neurohypophysial system (HNS) is up-regulated by kainic acid (KA)-induced seizure in rats. However, it remains unknown whether a subconvulsive dose of KA affects the HNS. Here we examined the effects of subcutaneous (s.c.) administration of a low dose of KA (4 mg/kg) on the gene expressions of the AVP, oxytocin (OXT) and neuronal nitric oxide synthase (nNOS) in the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus, using in situ hybridization histochemistry. The expression of the AVP gene in the SON and PVN was judged to be up-regulated in KA-treated rats in comparison with saline-treated rats as controls. Next, the expression of the OXT gene was significantly increased in the SON at 6-24h and in the PVN at 6 and 12h after s.c. administration of KA. Finally, the expression of the nNOS gene was significantly increased in the SON and PVN at 3 and 6h after s.c. administration of KA. These results suggest that up-regulation of the gene expressions of the AVP, OXT and nNOS in the rat hypothalamus may be differentially affected by peripheral administration of a subconvulsive dose of KA.

  11. Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy

    PubMed Central

    Silva Miranda, Mayra; Rodríguez, Kendy Wek; Martínez Cordero, Erasmo; Rojas-Espinosa, Oscar

    2006-01-01

    Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression. PMID:17222216

  12. Tomato allene oxide synthase and fatty acid hydroperoxide lyase, two cytochrome P450s involved in oxylipin metabolism, are targeted to different membranes of chloroplast envelope.

    PubMed

    Froehlich, J E; Itoh, A; Howe, G A

    2001-01-01

    Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are related cytochrome P450s that metabolize a common fatty acid hydroperoxide substrate to different classes of bioactive oxylipins within chloroplasts. Here, we report the use of in vitro import assays to investigate the targeting of tomato (Lycopersicon esculentum) AOS (LeAOS) and HPL (LeHPL) to isolated chloroplasts. LeAOS, which contains a typical N-terminal transit peptide, was targeted to the inner envelope membrane by a route that requires both ATP and proteinase-sensitive components on the surface of chloroplasts. Imported LeAOS was peripherally associated with the inner envelope; the bulk of the protein facing the stroma. LeHPL, which lacks a typical chloroplast-targeting sequence, was targeted to the outer envelope by an ATP-independent and protease-insensitive pathway. Imported LeHPL was integrated into the outer envelope with most of the protein exposed to the inter-membrane space. We conclude that LeAOS and LeHPL are routed to different envelope membranes by distinct targeting pathways. Partitioning of AOS and HPL to different envelope membranes suggests differences in the spatial organization of these two branches of oxylipin metabolism.

  13. Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission

    PubMed Central

    Menzel, Tila R.; Weldegergis, Berhane T.; David, Anja; Boland, Wilhelm; Gols, Rieta; van Loon, Joop J. A.; Dicke, Marcel

    2014-01-01

    Jasmonic acid (JA) plays a central role in induced plant defence e.g. by regulating the biosynthesis of herbivore-induced plant volatiles that mediate the attraction of natural enemies of herbivores. Moreover, exogenous application of JA can be used to elicit plant defence responses similar to those induced by biting-chewing herbivores and mites that pierce cells and consume their contents. In the present study, we used Lima bean (Phaseolus lunatus) plants to explore how application of a low dose of JA followed by minor herbivory by spider mites (Tetranychus urticae) affects transcript levels of P. lunatus (E)-β-ocimene synthase (PlOS), emission of (E)-β-ocimene and nine other plant volatiles commonly associated with herbivory. Furthermore, we investigated the plant’s phytohormonal response. Application of a low dose of JA increased PlOS transcript levels in a synergistic manner when followed by minor herbivory for both simultaneous and sequential infestation. Emission of (E)-β-ocimene was also increased, and only JA, but not SA, levels were affected by treatments. Projection to latent structures-discriminant analysis (PLS-DA) of other volatiles showed overlap between treatments. Thus, a low-dose JA application results in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defence after herbivore infestation. PMID:25318119

  14. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    SciTech Connect

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  15. Thrombospondin-1-induced smooth muscle cell chemotaxis and proliferation are dependent on transforming growth factor-β2 and hyaluronic acid synthase.

    PubMed

    Stein, Jeffrey J; Iwuchukwu, Chinenye; Maier, Kristopher G; Gahtan, Vivian

    2013-12-01

    Angioplasty causes local vascular injury, leading to the release of thrombospondin-1 (TSP-1), which stimulates vascular smooth muscle cell (VSMC) migration and proliferation, important steps in the development of intimal hyperplasia. Transforming growth factor beta 2 (TGF-β2) and hyaluronic acid synthase (HAS) are two pro-stenotic genes upregulated in VSMCs by TSP-1. We hypothesized that inhibition of TGF-β2 or HAS would inhibit TSP-1-induced VSMC migration, proliferation, and TSP-1 signaling. Our data demonstrate that Inhibition of either TGF-β2 or HAS inhibited TSP-1-induced VSMC migration and proliferation. Activation of ERK 1 was decreased by TGF-β2 inhibition and unaffected by HAS inhibition. TGF-β2 and HAS are not implicated in TSP-1-induced thbs1 expression, while they are each implicated in TSP-1-induced expression of their own gene. In summary, TSP-1-induced VSMC migration and proliferation rely on intact TGF-β2 signaling and HAS function. TSP-1 activation of ERK 1 is dependent on TGF-β2. These data further expand our understanding of the complexity of TSP-1 cellular signaling and the involvement of TGF-β2 and HAS.

  16. Effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf and MAPK signal transduction pathway on inducible nitric oxide synthase expression in alveolar macrophage of chronic bronchitis rats.

    PubMed

    Huang, Y; Li, J; Meng, X M; Jiang, G L; Li, H; Cao, Q; Yu, S C; Lv, X W; Cheng, W M

    2009-01-01

    The goal of this study was to investigate the possible therapy mechanism of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. Leaf (TAL) in alveolar macrophage (AM) of chronic bronchitis (CB) rats. CB model was established by injection of bacillus calmette guein (BCG) plus lipopolisacharide (LPS) in rats. TAL significantly inhibited the increased NO concentration, iNOS expression and phosphorylation of p38 MAPK in alveolar macrophages (AMs) of CB rats. Using in vivo test, we found that SB203580, a p38 MAPK inhibitor, (10 muM) significantly inhibited inducible nitric oxide synthase (iNOS) mRNA expression in AM. This data indicate that TAL highly decreases excessive iNOS expression and NO induction, and p38 MAPK signal transduction participates in iNOS expression and NO induction in AM of CB rats. The effect of TAL on iNOS expression in AM may be related to its inhibition of p38 MAPK signal transduction. PMID:19938219

  17. A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the FAS1 gene product.

    PubMed

    Wenz, P; Schwank, S; Hoja, U; Schüller, H J

    2001-11-15

    The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are transcriptionally co-regulated by general transcription factors (such as Reb1, Rap1 and Abf1) and by the phospholipid-specific heterodimeric activator Ino2/Ino4, acting via their corresponding upstream binding sites. Here we provide evidence for a positive autoregulatory influence of FAS1 on FAS2 expression. Even with a constant FAS2 copy number, a 10-fold increase of FAS2 transcript amount was observed in the presence of FAS1 in multi-copy, compared to a fas1 null mutant. Surprisingly, the first 66 nt of the FAS2 coding region turned out as necessary and sufficient for FAS1-dependent gene expression. FAS2-lacZ fusion constructs deleted for this region showed high reporter gene expression even in the absence of FAS1, arguing for a negatively-acting downstream repression site (DRS) responsible for FAS1-dependent expression of FAS2. Our data suggest that the FAS1 gene product, in addition to its catalytic function, is also required for the coordinate biosynthetic control of the yeast FAS complex. An excess of uncomplexed Fas1 may be responsible for the deactivation of an FAS2-specific repressor, acting via the DRS. PMID:11713312

  18. Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium[S

    PubMed Central

    Rudolph, Michael C.; Wellberg, Elizabeth A.; Lewis, Andrew S.; Terrell, Kristina L.; Merz, Andrea L.; Maluf, N. Karl; Serkova, Natalie J.; Anderson, Steven M.

    2014-01-01

    Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. PMID:24771867

  19. Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens.

    PubMed

    Umeda, F; Nishikawa, T; Miyasaka, H; Maeda, I; Kawase, M; Yagi, K

    2001-11-01

    Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens. PMID:11916262

  20. Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe) Varieties

    PubMed Central

    Ghasemzadeh, Ali; Jaafar, Hawa Z. E.; Karimi, Ehsan

    2012-01-01

    The effect of foliar application of salicylic acid (SA) at different concentrations (10−3 M and 10−5 M) was investigated on the production of secondary metabolites (flavonoids), chalcone synthase (CHS) activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231) in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC) analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin) decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS) enzyme activity (involving flavonoid synthesis) and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10−5 M SA treatment. As the SA concentration was decreased from 10−3 M to 10−5 M, the free radical scavenging power (FRAP) increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 μg mL−1, the DPPH antioxidant activity recorded the highest value of 58.30%–72.90% with the 10−5 M SA treatment followed by the 10−3 M SA (52.14%–63.66%) treatment. The lowest value was recorded in the untreated control plants (42.5%–46.7%). These results indicate that SA can act not only as an inducer but also as an inhibitor of secondary metabolites. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines was observed for H. Bara extracts treated with 10−5 M SA with values of 61.53 and 59.88%, respectively. The results suggest that the high anticancer activity in these varieties may be related to the high concentration of potent anticancer components including fisetin and anthocyanin. The results thus indicate that the synthesis of flavonoids in

  1. Involvement of salicylic acid on antioxidant and anticancer properties, anthocyanin production and chalcone synthase activity in ginger (Zingiber officinale Roscoe) varieties.

    PubMed

    Ghasemzadeh, Ali; Jaafar, Hawa Z E; Karimi, Ehsan

    2012-01-01

    The effect of foliar application of salicylic acid (SA) at different concentrations (10-3 M and 10-5 M) was investigated on the production of secondary metabolites (flavonoids), chalcone synthase (CHS) activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231) in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC) analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin) decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS) enzyme activity (involving flavonoid synthesis) and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10-5 M SA treatment. As the SA concentration was decreased from 10-3 M to 10-5 M, the free radical scavenging power (FRAP) increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 μg mL-1, the DPPH antioxidant activity recorded the highest value of 58.30%-72.90% with the 10-5 M SA treatment followed by the 10-3 M SA (52.14%-63.66%) treatment. The lowest value was recorded in the untreated control plants (42.5%-46.7%). These results indicate that SA can act not only as an inducer but also as an inhibitor of secondary metabolites. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines was observed for H. Bara extracts treated with 10-5 M SA with values of 61.53 and 59.88%, respectively. The results suggest that the high anticancer activity in these varieties may be related to the high concentration of potent anticancer components including fisetin and anthocyanin. The results thus indicate that the synthesis of flavonoids in ginger can be increased

  2. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius.

    PubMed

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-01-01

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT-PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress. PMID:24865400

  3. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius

    PubMed Central

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-01-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance. PMID:24309561

  4. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    PubMed Central

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-01-01

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress. PMID:24865400

  5. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

    PubMed

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-11-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

  6. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius.

    PubMed

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-06-25

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT-PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.

  7. Theanaphthoquinone inhibits fatty acid synthase expression in EGF-stimulated human breast cancer cells via the regulation of EGFR/ErbB-2 signaling

    SciTech Connect

    Weng, M.-S.; Ho, C.-T.; Ho, Y.-S.; Lin, J.-K. . E-mail: jklin@ha.mc.ntu.edu.tw

    2007-01-15

    Fatty acid synthase (FAS) is a major lipogenic enzyme catalyzing the synthesis of long-chain saturated fatty acids. Most breast cancers require lipogenesis for growth. Here, we demonstrated the effects of theanaphthoquinone (TNQ), a member of the thearubigins generated by the oxidation of theaflavin (TF-1), on the expression of FAS in human breast cancer cells. TNQ was found to suppress the EGF-induced expression of FAS mRNA and FAS protein in MDA-MB-231 cells. Expression of FAS has previously been shown to be regulated by the SREBP family of transcription factors. In this study, we demonstrated that the EGF-induced nuclear translocation of SREBP-1 was blocked by TNQ. Moreover, TNQ also modulated EGF-induced ERK1/2 and Akt phosphorylation. Treatment of MDA-MB-231 cells with PI 3-kinase inhibitors, LY294002 and Wortmannin, inhibited the EGF-induced expression of FAS and nuclear translocation of SREBP-1. Treatment with TNQ inhibited EGF-induced EGFR/ErbB-2 phosphorylation and dimerization. Furthermore, treatment with kinase inhibitors of EGFR and ErbB-2 suggested that EGFR/ErbB-2 activation was involved in EGF-induced FAS expression. In constitutive FAS expression, TNQ inhibited FAS expression and Akt autophosphorylation in BT-474 cells. The PI 3-kinase inhibitors and tyrosine kinase inhibitors of EGFR and ErbB-2 also reduced constitutive FAS expression. In addition, pharmacological blockade of FAS by TNQ decreased cell viability and induced cell death in BT-474 cells. In summary, our findings suggest that TNQ modulates FAS expression by the regulation of EGFR/ErbB-2 pathways and induces cell death in breast cancer cells.

  8. Deficiency in a Very-Long-Chain Fatty Acid β-Ketoacyl-Coenzyme A Synthase of Tomato Impairs Microgametogenesis and Causes Floral Organ Fusion1[W

    PubMed Central

    Smirnova, Anna; Leide, Jana; Riederer, Markus

    2013-01-01

    Previously, it was shown that β-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C28 in tomato (Solanum lycopersicum) fruits and C26 in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C27 or greater and of anteiso-alkanes with chain lengths of C28 or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis. PMID:23144186

  9. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    PubMed

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  10. Identification and Characterization of FabA from the Type II Fatty Acid Synthase of Streptomyces coelicolor.

    PubMed

    Singh, Renu; Reynolds, Kevin A

    2016-01-22

    FabA is proposed to catalyze the dehydration step of chain elongation in fatty acid and undecylprodiginine biosynthesis in Streptomyces coelicolor. Analysis of the S. coelicolor genome has revealed a fabA gene (SCO4636-SCO4637, encoding a heterodimer 3-hydroxyacyl-ACP dehydratase). Herein, we report the identification and characterization of the corresponding gene products. Kinetic analysis has demonstrated that FabA is capable of utilizing various chain lengths of straight- and branched-chain 3-hydroxyacyl-NAC substrates. Additionally, FabA does not discriminate between acyl carrier proteins (ACPs) from primary and secondary metabolism. These data provide the first experimental evidence that FabA has 3-hydroxyacyl-ACP dehydratase activity and processes intermediates for both biosynthetic pathways.

  11. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

    PubMed

    Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2014-09-01

    In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast. PMID:24752598

  12. Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition.

    PubMed

    Leng, Yan; Liang, Min-Huei; Ren, Ming; Marinova, Zoya; Leeds, Peter; Chuang, De-Maw

    2008-03-01

    Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 alpha and beta isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3alpha small interfering RNA (siRNA) and/or GSK-3beta siRNA mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated beta-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

  13. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

    PubMed

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R Douglas; Powles, Stephen B

    2015-04-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. PMID:25717039

  14. Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance1

    PubMed Central

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R. Douglas; Powles, Stephen B.

    2015-01-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. PMID:25717039

  15. Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case.

    PubMed

    Hansen, Bjarne G; Salomonsen, Bo; Nielsen, Morten T; Nielsen, Jakob B; Hansen, Niels B; Nielsen, Kristian F; Regueira, Torsten B; Nielsen, Jens; Patil, Kiran R; Mortensen, Uffe H

    2011-05-01

    Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid. PMID:21398493

  16. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    PubMed Central

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  17. Downregulation of inducible nitric oxide synthase (iNOS) expression is implicated in the antiviral activity of acetylsalicylic acid in HCV-expressing cells.

    PubMed

    Ríos-Ibarra, Clara Patricia; Lozano-Sepulveda, Sonia; Muñoz-Espinosa, Linda; Rincón-Sánchez, Ana Rosa; Cordova-Fletes, Carlos; Rivas-Estilla, Ana María G

    2014-12-01

    Previously, we described that acetylsalicylic acid (ASA) decreases HCV expression, but the mechanisms involved have not been clearly established. We evaluated the participation of inducible nitric oxide synthase (iNOS) in the regulation of HCV-RNA induced by ASA. Huh7 cells expressing non-structural HCV proteins were exposed to 4 mM ASA and incubated at the same times we reported HCV downregulation (24-72 h), and iNOS mRNA and protein levels were then measured by real-time PCR and Western blot, respectively. Nitric oxide levels were measured at the same time. Inhibition of iNOS mRNA by small interfering RNAs (siRNA) and activation of the iNOS gene promoter by ASA treatment were evaluated. In Huh7 replicon cells treated with ASA, we found decreased levels of iNOS mRNA, iNOS protein and nitrosylated protein levels at 48-72 h. ASA exposure also reduced the transactivation of the iNOS promoter in HCV replicon cells at 48 h, and this was partly due to the decrease in the affinity of transcription factor C/EBP-β for its binding site in the iNOS promoter. siRNA silencing of iNOS decreased HCV-RNA expression (65 %) and potentiated the antiviral effect (80 %) of ASA compared with control cells. ASA reduces iNOS expression by downregulating promoter activity, mRNA and protein levels at the same time that it decreases HCV expression. These findings suggest that the antiviral activity of ASA is mediated partially through the modulation of iNOS.

  18. Dual Fatty Acid Synthase and HER2 Signaling Blockade Shows Marked Antitumor Activity against Breast Cancer Models Resistant to Anti-HER2 Drugs

    PubMed Central

    Blancafort, Adriana; Giró-Perafita, Ariadna; Oliveras, Glòria; Palomeras, Sònia; Turrado, Carlos; Campuzano, Òscar; Carrión-Salip, Dolors; Massaguer, Anna; Brugada, Ramon; Palafox, Marta; Gómez-Miragaya, Jorge; González-Suárez, Eva; Puig, Teresa

    2015-01-01

    Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies. PMID:26107737

  19. 1-Aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC synthase expression in soybean roots, root tips, and soybean cyst nematode (Heterodera glycines)-infected roots.

    PubMed

    Tucker, Mark L; Xue, Ping; Yang, Ronghui

    2010-01-01

    Colonization of plant roots by root knot and cyst nematodes requires a functional ethylene response pathway. However, ethylene plays many roles in root development and whether its role in nematode colonization is direct or indirect, for example lateral root initiation or root hair growth, is not known. The temporal requirement for ethylene and localized synthesis of ethylene during the life span of soybean cyst nematode (SCN) on soybean roots was further investigated. Although a significant increase in ethylene evolution was not detected from SCN-colonized roots, the concentration of the immediate precursor to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), was higher in SCN-colonized root pieces and root tips than in other parts of the root. Moreover, expression analysis of 17 ACC synthase (ACS) genes indicated that a select set of ACS genes is expressed in SCN-colonized root pieces that is clearly different from the set of genes expressed in non-colonized roots or root tips. Semi-quantitative real-time PCR indicated that ACS transcript accumulation correlates with the high concentration of ACC in root tips. In addition, an ACS-like sequence was found in the public SCN nucleotide database. Acquisition of a full-length sequence for this mRNA (accession GQ389647) and alignment with transcripts for other well-characterized ACS proteins indicated that the nematode sequence is missing a key element required for ACS activity and therefore probably is not a functional ACS. Moreover, no significant amount of ACC was found in any growth stage of SCN that was tested.

  20. Fatty acid synthase/oxidized low-density lipoprotein as metabolic oncogenes linking obesity to colon cancer via NF-kappa B in Egyptians.

    PubMed

    Keshk, Walaa Arafa; Zineldeen, Doaa Hussein; Wasfy, Rania E L-sayed; El-Khadrawy, Osama Helmy

    2014-10-01

    Obesity is a major health problem which heightens the risk of several chronic illnesses including cancer development particularly colon cancer. The underlying pathophysiology of obesity associated colon cancer remains to be elucidated. The purpose of this current study was to determine fatty acid synthase (FASN) activity/expression, oxidized low-density lipoprotein (ox-LDL) level and redox status under the context of anthropometric measurements and lipid profile to find their potential role as interacting biomarkers relating obesity to colon cancer initiation and progression via nuclear factor kappa-B (NF-κB) signaling. This study was conducted upon Egyptian individuals; 30 obese subjects with colon cancer, 11 nonobese and 11 obese subjects without colon cancer. FASN gene expression, NF-κB immunoreactivity, and serum ox-LDL level were estimated by real-time PCR, immunohistochemistry and immunoassay, respectively. FASN activity, glycemic status, obesity, and oxidative stress indices were also assessed. It was found that FASN expression and activity were statistically increased in obese with colon cancer (P=0.021 and 0.018, respectively), with statistically significant increase in patients with advanced grading. Moreover, NF-κB immunoreactivity and serum ox-LDL level were significantly increased in obese colon cancer patients with significantly higher levels in those with advanced grading (all P<0.05). Dyslipidemia, insulin resistance, and oxidative stress indices were worsened in obese patients with colon cancer. These results revealed that FASN and ox-LDL as well as oxidative stress may increase the risk of obesity related colon cancer, particularly via NF-κB signaling and could be used as potential predictive and prognostic biomarkers for obesity complicated with colon cancer.

  1. Dual fatty acid synthase and HER2 signaling blockade shows marked antitumor activity against breast cancer models resistant to anti-HER2 drugs.

    PubMed

    Blancafort, Adriana; Giró-Perafita, Ariadna; Oliveras, Glòria; Palomeras, Sònia; Turrado, Carlos; Campuzano, Òscar; Carrión-Salip, Dolors; Massaguer, Anna; Brugada, Ramon; Palafox, Marta; Gómez-Miragaya, Jorge; González-Suárez, Eva; Puig, Teresa

    2015-01-01

    Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies.

  2. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    PubMed

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening. PMID:22419220

  3. DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements.

    PubMed Central

    Foufelle, F; Lepetit, N; Bosc, D; Delzenne, N; Morin, J; Raymondjean, M; Ferré, P

    1995-01-01

    We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7772036

  4. Fetal and neonatal exposure to nicotine leads to augmented hepatic and circulating triglycerides in adult male offspring due to increased expression of fatty acid synthase

    SciTech Connect

    Ma, Noelle; Nicholson, Catherine J.; Wong, Michael; Holloway, Alison C.; Hardy, Daniel B.

    2014-02-15

    While nicotine replacement therapy is assumed to be a safer alternative to smoking during pregnancy, the long-term consequences for the offspring remain elusive. Animal studies now suggest that maternal nicotine exposure during perinatal life leads to a wide range of adverse outcomes for the offspring including increased adiposity. The focus of this study was to investigate if nicotine exposure during pregnancy and lactation leads to alterations in hepatic triglyceride synthesis. Female Wistar rats were randomly assigned to receive daily subcutaneous injections of saline (vehicle) or nicotine bitartrate (1 mg/kg/day) for two weeks prior to mating until weaning. At postnatal day 180 (PND 180), nicotine exposed offspring exhibited significantly elevated levels of circulating and hepatic triglycerides in the male offspring. This was concomitant with increased expression of fatty acid synthase (FAS), the critical hepatic enzyme in de novo triglyceride synthesis. Given that FAS is regulated by the nuclear receptor Liver X receptor (LXRα), we measured LXRα expression in both control and nicotine-exposed offspring. Nicotine exposure during pregnancy and lactation led to an increase in hepatic LXRα protein expression and enriched binding to the putative LXRE element on the FAS promoter in PND 180 male offspring. This was also associated with significantly enhanced acetylation of histone H3 [K9,14] surrounding the FAS promoter, a hallmark of chromatin activation. Collectively, these findings suggest that nicotine exposure during pregnancy and lactation leads to an increase in circulating and hepatic triglycerides long-term via changes in the transcriptional and epigenetic regulation of the hepatic lipogenic pathway. - Highlights: • Our data reveals the links nicotine exposure in utero and long-term hypertriglyceridemia. • It is due to nicotine-induced augmented expression of hepatic FAS and LXRα activity. • Moreover, this involves nicotine-induced enhanced

  5. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

    PubMed

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R Douglas; Powles, Stephen B

    2015-04-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.

  6. DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements.

    PubMed

    Foufelle, F; Lepetit, N; Bosc, D; Delzenne, N; Morin, J; Raymondjean, M; Ferré, P

    1995-06-01

    We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation.

  7. The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit.

    PubMed

    Starrett, D A; Laties, G G

    1991-03-01

    When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A(+)) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.

  8. Serous tubal intraepithelial carcinoma upregulates markers associated with high-grade serous carcinomas including Rsf-1 (HBXAP), cyclin E and fatty acid synthase.

    PubMed

    Sehdev, Ann Smith; Kurman, Robert J; Kuhn, Elisabetta; Shih, Ie-Ming

    2010-06-01

    Serous tubal intraepithelial carcinoma (STIC) has been proposed as a precursor for many pelvic high-grade serous carcinomas. Our previous analysis of the ovarian cancer genome identified several genes with oncogenic potential that are amplified and/or overexpressed in the majority of high-grade serous carcinomas. Determining whether these genes are upregulated in STICs is important in further elucidating the relationship of STICs to high-grade serous carcinomas and is fundamental in understanding the molecular pathogenesis of high-grade serous carcinomas. In this study, 37 morphologically defined STICs were obtained from 23 patients with stage IIIC/IV high-grade serous carcinomas. Both STICs and the high-grade serous carcinomas were analyzed for expression of Rsf-1 (HBXAP), cyclin E, fatty acid synthase (FASN) and mucin-4. In addition, they were examined for expression of established markers including p53, Ki-67 and p16. We found that diffuse nuclear p53 and p16 immunoreactivity was observed in 27 (75%) of 36 and 18 (55%) of 33 STICs, respectively, whereas an elevated Ki-67 labeling index (>or=10%) was detected in 29 (78%) of 37 STICs. Cyclin E nuclear staining was seen in 24 (77%) of 35 STICs, whereas normal tubal epithelial cells were all negative. Increased Rsf-1 and FASN immunoreactivity occurred in 63%, and 62% of STICs, respectively, compared with adjacent normal-appearing tubal epithelium. Interestingly, only one STIC showed increased mucin-4 immunoreactivity. Carcinomas, when compared with STICs, overexpressed p16, Rsf-1, cyclin E and FASN in a higher proportion of cases. In conclusion, STICs express several markers including Rsf-1, cyclin E and FASN in high-grade serous carcinomas. In contrast, mucin-4 immunoreactivity either did not change or was reduced in most STICs. These results suggest that overexpression of Rsf-1, cyclin E and FASN occurs early in tumor progression.

  9. Persistent induction of nitric oxide synthase in tumours from mice treated with the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid.

    PubMed

    Moilanen, E; Thomsen, L L; Miles, D W; Happerfield, D W; Knowles, R G; Moncada, S

    1998-01-01

    An anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) induced nitric oxide synthase (NOS) in the tumour, spleen, thymus and small intestine, but not in the lung, liver, kidney, heart or skeletal muscle in B6D2F1 mice bearing subcutaneous colon 38 tumours. This pattern of induction is distinct from that caused by agents such as endotoxin, muramyl dipeptide or Corynebacterium parvum. The induction of NOS (iNOS) in the tumour was more persistent (maximal at 3 days) than in other tissues (maximal at 12 h). Immunohistochemical staining suggested that iNOS was located in macrophages and endothelial cells within and around the tumour. Treatment with 5,6-MeXAA also caused substantial increases in plasma nitrite and nitrate (NOx) concentrations that peaked at 8-12 h after 5,6-MeXAA. The increase in plasma NOx was prevented by a NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), indicating that it was due to enhanced production of NO. Tumour-bearing mice were more responsive than controls to 5,6-MeXAA both in their plasma NOx increase and in their lower maximally tolerated dose. L-NIO was unable to prevent the complete tumour necrosis and regression caused by 5,6-MeXAA at a dose that substantially inhibited the increase of plasma NOx. In conclusion, the experimental anti-tumour agent 5,6-MeXAA induced NO synthesis in tumour-associated macrophages and in immunologically active tissues in parallel with its effects on tumour growth. The experiments with a non-selective NOS inhibitor L-NIO, however, suggest that NO is not a significant component in the mechanism of the anti-tumour action of 5,6-MeXAA in this particular model. PMID:9472639

  10. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    PubMed Central

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2010-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to the extraordinary structural diversity of naturally occurring polyketides. PMID:19362634

  11. Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

    SciTech Connect

    Wu Defeng; Cederbaum, Arthur . E-mail: arthur.cederbaum@mssm.edu

    2006-10-15

    Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N {sup G}-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 {+-} 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 {+-} 5%, while, SNAP or DETA-NONO increased viability to 66 {+-} 8 or 71 {+-} 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA

  12. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  13. Divinyl ether synthase gene and protein, and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  14. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  15. OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

    PubMed

    Wakuta, Shinji; Suzuki, Erika; Saburi, Wataru; Matsuura, Hideyuki; Nabeta, Kensuke; Imai, Ryozo; Matsui, Hirokazu

    2011-06-17

    The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice. PMID:21619871

  16. OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

    PubMed

    Wakuta, Shinji; Suzuki, Erika; Saburi, Wataru; Matsuura, Hideyuki; Nabeta, Kensuke; Imai, Ryozo; Matsui, Hirokazu

    2011-06-17

    The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice.

  17. Upregulation of UGT2B4 Expression by 3′-Phosphoadenosine-5′-Phosphosulfate Synthase Knockdown: Implications for Coordinated Control of Bile Acid Conjugation

    PubMed Central

    Barrett, Kathleen G.; Fang, Hailin; Cukovic, Daniela; Dombkowski, Alan A.; Kocarek, Thomas A.

    2015-01-01

    During cholestasis, the bile acid–conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 3′-phosphoadenosine-5′-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 5′-flanking region, which included a response element for the bile acid–sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5′-flanking region indicated the presence of distal regulatory elements between nucleotides −10090 and −10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3′-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid–conjugating activity. PMID:25948711

  18. Modulation of nitric oxide synthase activity in brain, liver, and blood vessels of spontaneously hypertensive rats by ascorbic acid: protection from free radical injury.

    PubMed

    Newaz, M A; Yousefipour, Z; Nawal, N N A

    2005-08-01

    End organ damage in essential hypertension has been linked to increased oxygen free radical generation, reduced antioxidant defense, and/or attenuation of nitric oxide synthase (NOS) activity. Ascorbic acid (AA), a water-soluble antioxidant, has been reported as a strong defense against free radicals in both aqueous and nonaqueous environment. In this study we examined the hypothesis that antioxidant ascorbic acid may confer protection from increased free radical activity in brain, liver, and blood vessels of spontaneously hypertensive rats (SHR). Male SHRs were divided into groups: SHR + AA (treated with AA, 1 mg/rat/day; for 12 weeks) or SHR (untreated). Wister-Kyoto rats (WKY) served as the control. Mean systolic blood pressure (SBP) in treated and untreated SHR was 145 +/- 7 mmHg and 142 +/- 8 mmHg, respectively. AA treatment prevented the increase in systolic blood pressure in SHR by 37 +/- 1% (p < 0.05). NOS activity in the brain, liver, and blood vessels of WKY rat was 1.82 +/- 0.02, 0.14 +/- 0.003, and 1.54 +/- 0.06 pmol citruline/mg protein, respectively. In SHR, total NOS activity was significantly reduced by 52 +/- 1%, 21 +/- 3%, and 44 +/- 4%, respectively. AA increased NOS activity in brain, liver, and blood vessels of SHR from 0.87 +/-.03, 0.11 +/-.01, and 0.87 +/-.08 pmol citruline/mg protein to 0.93 +/- 0.01, 0.13 +/- 0.001, and 1.11 +/- 0.03 pmol citruline/mg protein (p < 0.05), respectively. Lipid peroxides in the brain, liver, and blood vessels from WKY rats were 0.87 +/- 0.06, 0.11 +/- 0.005, and 0.47 +/- 0.04 nmol MDA equiv/mg protein, respectively. In SHR, lipid peroxides in brain, liver, and blood vessels were significantly increased by 40 +/- 3%, 64 +/- 3%, and 104 +/- 13%, respectively. AA reduced lipid peroxidation in liver and blood vessels by 17 +/- 1% and 34 +/- 3% but not in brain. Plasma lipid peroxides were almost doubled in SHR (p < 0.01) together with a reduction in total antioxidant status (6 +/- 0.1%; p < 0.05), nitrite (53 +/- 2

  19. Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Colomer, Ramon; Lupu, Ruth

    2005-05-20

    The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the

  20. Physiological function of mycobacterial mtFabD, an essential malonyl-CoA:AcpM transacylase of type 2 fatty acid synthase FASII, in yeast mct1Delta cells.

    PubMed

    Gurvitz, Aner

    2009-01-01

    Mycobacterium tuberculosis mtFabD is an essential malonyl-CoA:AcpM transacylase and is important for vital protein-protein interactions within type 2 fatty acid synthase FASII. mtFabD contacts KasA, KasB, FabH, InhA, and possibly also HadAB, HadBC, and FabG1/MabA. Disruption of mtFabD's interactions during FASII has been proposed for drug development. Here, the gene for a mitochondrially targeted mtFabD was ectopically expressed in Saccharomyces cerevisiae mct1Delta mutant cells lacking the corresponding mitochondrial malonyl-CoA transferase Mct1p, allowing the mutants to recover their abilities to respire on glycerol and synthesize lipoic acid. Hence, mtFabD could physiologically function in an environment lacking holo-AcpM or other native interaction partners. PMID:19859569

  1. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    PubMed

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  2. Palmitic acid exerts pro-inflammatory effects on vascular smooth muscle cells by inducing the expression of C-reactive protein, inducible nitric oxide synthase and tumor necrosis factor-α.

    PubMed

    Wu, Di; Liu, Juntian; Pang, Xiaoming; Wang, Shuyue; Zhao, Jingjing; Zhang, Xiaolu; Feng, Liuxin

    2014-12-01

    Atherosclerosis is a chronic inflammatory disease in the vessel, and inflammatory cytokines play an important role in the inflammatory process of atherosclerosis. A high level of free fatty acids (FFAs) produced in lipid metabolism disorders are known to participate in the formation of atherosclerosis through multiple bioactivities. As the main saturated fatty acid in FFAs, palmitic acid stimulates the expression of inflammatory cytokines in macrophages. However, it is unclear whether palmitic acid exerts a pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe the effect of palmitic acid on the expression of C-reactive protein (CRP), tumor necrosis factor α (TNF-α) and inducible nitric oxide synthase (iNOS) in VSMCs. Rat VSMCs were cultured, and palmitic acid was used as a stimulant for CRP, TNF-α and iNOS expression. mRNA expression was assayed with reverse transcription-polymerase chain reaction, and protein expression was detected with western blot analysis and immunocytochemistry. The results showed that palmitic acid significantly stimulated mRNA and protein expression of CRP, TNF-α and iNOS in VSMCs in time- and concentration-dependent manners, and therefore, palmitic acid is able to exert a pro-inflammatory effect on VSMCs via stimulating CRP, TNF-α and iNOS expression. The findings provide a novel explanation for the direct pro-inflammatory and atherogenic effects of palmitic acid, and for the association with metabolic syndrome, such as type 2 diabetes mellitus, obesity and atherosclerosis. Therefore, the intervention with anti-inflammatory agents may effectively delay the formation and progression of atherosclerosis in patients with metabolic syndrome.

  3. Identification of a 1-aminocyclopropane-1-carboxylic acid synthase gene linked to the female (F) locus that enhances female sex expression in cucumber.

    PubMed Central

    Trebitsh, T; Staub, J E; O'Neill, S D

    1997-01-01

    Sex determination in cucumber (Cucumis sativus L.) is controlled largely by three genes: F, m, and a. The F and m loci interact to produce monoecious (M_f_) or gynoecious (M_f_) sex phenotypes. Ethylene and factors that induce ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylate (ACC) and auxin, also enhance female sex expression. A genomic sequence (CS-ACS1) encoding ACC synthase was amplified from genomic DNA by a polymerase chain reaction using degenerate oligonucleotide primers. Expression of CS-ACS1 is induced by auxin, but not by ACC, in wounded and intact shoot apices. Southern blo hybridization analysis of near-isogenic gynoecious (MMFF) and monoecious (MMff) lines derived from divers genetic backgrounds revealed the existence of an additional ACC synthase (CS-ACS1G) genomic sequence in the gynoecious lines. Sex phenotype analysis of a segregating F2 population detected a 100% correlation between the CS-ACS1G marker and the presence of the F locus. The CS-ACS1G gene is located in linkage group B coincident with the F locus, and in the population tested there was no recombination between the CS-ACS1G gene and the F locus. Collectively, these data suggest that CS-ACS1G is closely linked to the F locus and may play a pivotal role in the determination of sex in cucumber flowers. PMID:9085580

  4. Inactivation of the inhA-encoded fatty acid synthase II (FASII) enoyl-acyl carrier protein reductase induces accumulation of the FASI end products and cell lysis of Mycobacterium smegmatis.

    PubMed

    Vilchèze, C; Morbidoni, H R; Weisbrod, T R; Iwamoto, H; Kuo, M; Sacchettini, J C; Jacobs, W R

    2000-07-01

    The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, inhA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates inhA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C(26:0)), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the inhA gene of Mycobacterium smegmatis that rendered InhA inactive at 42 degrees C. Thermal inactivation of InhA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C(16:0)) and a concomitant increase of tetracosanoic acid (C(24:0)) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C(16:0), and a concomitant accumulation of C(26:0). Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH. PMID:10869086

  5. Farnesyl pyrophosphate synthase enantiospecificity with a chiral risedronate analog, [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501): Synthetic, structural, and modeling studies.

    PubMed

    Deprèle, Sylvine; Kashemirov, Boris A; Hogan, James M; Ebetino, Frank H; Barnett, Bobby L; Evdokimov, Artem; McKenna, Charles E

    2008-05-01

    The complex formed from crystallization of human farnesyl pyrophosphate synthase (hFPPS) from a solution of racemic [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501, 8), a chiral analog of the anti-osteoporotic drug risedronate, contained the R enantiomer in the enzyme active site. This enantiospecificity was assessed by computer modeling of inhibitor-active site interactions using Autodock 3, which was also evaluated for predictive ability in calculations of the known configurations of risedronate, zoledronate, and minodronate complexed in the active site of hFPPS. In comparison with these structures, the 8 complex exhibited certain differences, including the presence of only one Mg(2+), which could contribute to its 100-fold higher IC(50). An improved synthesis of 8 is described, which decreases the number of steps from 12 to 8 and increases the overall yield by 17-fold.

  6. Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases.

    PubMed

    Aaron, Julie A; Christianson, David W

    2010-01-01

    Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster not only serves to bind and orient the flexible isoprenoid substrate in the precatalytic Michaelis complex, but it also triggers the departure of the diphosphate leaving group to generate a carbocation that initiates catalysis. Additional conserved hydrogen bond donors assist the metal cluster in this function. Crystal structure analysis reveals that the constellation of three metal ions required for terpenoid synthase catalysis is generally identical among all class I terpenoid synthases of known structure.

  7. Anthranilate synthase from Ruta graveolens. Duplicated AS alpha genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.

    PubMed Central

    Bohlmann, J; Lins, T; Martin, W; Eilert, U

    1996-01-01

    Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the biosynthetic precursor of both tryptophan and numerous secondary metabolites, including inducible plant defense compounds. The higher plant Ruta graveolens produces tryptophan and elicitor-inducible, anthranilate-derived alkaloids by means of two differentially expressed nuclear genes for chloroplast-localized AS alpha subunits, AS alpha 1 and AS alpha 2. Mechanisms that partition chorismate between tryptophan and inducible alkaloids thus do not entail chloroplast/cytosol separation of AS isoenzymes and yet might involve differential feedback regulation of pathway-specific AS alpha subunits. The two AS alpha isoenzymes of R. graveolens were expressed as glutathione S-transferase fusion proteins in Escherichia coli deletion mutants defective in AS activity and were purified to homogeneity. Differential sensitivity of the transformed E. coli strains toward 5-methyltryptophan, a false-feedback inhibitor of AS, was demonstrated. Characterization of affinity-purified AS alpha isoenzymes revealed that the noninducible AS alpha 2 of R. graveolens is strongly feedback inhibited by 10 microns tryptophan. In contrast, the elicitor-inducible AS alpha 1 isoenzyme is only slightly affected even by tryptophan concentrations 10-fold higher than those observed in planta. These results are consistent with the hypothesis that chorismate flux into biosynthesis of tryptophan and defense-related alkaloid biosynthesis in R. graveolens is regulated at the site of AS alpha isoenzymes at both genetic and enzymatic levels. PMID:8787026

  8. Peroxisomal and mitochondrial citrate synthase in CAM plants.

    PubMed

    Zafra, M F; Segovia, J L; Alejandre, M J; García-Peregrín, E

    1981-12-01

    Citrate synthase wa studied for the first time in peroxisomes and mitochondria of crassulacean acid metabolism plants. Cellular organelles were isolated from Agave americana leaves by sucrose density gradient centrifugation and characterized by the use of catalase and cytochrome oxidase as marker enzymes, respectively. 48,000 X g centrifugation caused the breakdown of the cellular organelles. The presence of a glyoxylate cycle enzyme (citrate synthase) and a glycollate pathway enzyme (catalase) in the same organelles, besides the absence of another glyoxalate cycle enzyme (malate synthase) is reported for the first time, suggesting that peroxisomal and glyoxysomal proteins are synthesized at the same time and housed in he same organelle.

  9. NADPH Oxidase NOX5-S and Nuclear Factor κB1 Mediate Acid-Induced Microsomal Prostaglandin E Synthase-1 Expression in Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Zhou, Xiaoxu; Li, Dan; Resnick, Murray B.; Wands, Jack

    2013-01-01

    The mechanisms of progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not known. Cycloxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has been shown to be important in esophageal tumorigenesis. We have shown that COX-2 mediates acid-induced PGE2 production. The prostaglandin E synthase (PGES) responsible for acid-induced PGE2 production in BE, however, is not known. We found that microsomal PGES1 (mPGES1), mPGES2, and cytosolic PGES (cPGES) were present in FLO EA cells. Pulsed acid treatment significantly increased mPGES1 mRNA and protein levels but had little or no effect on mPGES2 or cPGES mRNA. Knockdown of mPGES1 by mPGES1 small interfering RNA (siRNA) blocked acid-induced increase in PGE2 production and thymidine incorporation. Knockdown of NADPH oxidase, NOX5-S, a variant lacking calcium-binding domains, by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a nuclear factor κB (NF-κB) in vivo activation reporter plasmid pNF-κB-Luc. Knockdown of NF-κB1 p50 by p50 siRNA significantly decreased acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Two novel NF-κB binding elements, GGAGTCTCCC and CGGGACACCC, were identified in the mPGES1 gene promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. PMID:23439561

  10. Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

    PubMed Central

    Eberhardt, S; Korn, S; Lottspeich, F; Bacher, A

    1997-01-01

    Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. PMID:9139911

  11. Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

    PubMed

    Kingsbury, Joanne M; McCusker, John H

    2010-03-01

    The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal

  12. Evaluation of epigallocatechin gallate and related plant polyphenols as inhibitors of the FabG and FabI reductases of bacterial type II fatty-acid synthase.

    PubMed

    Zhang, Yong-Mei; Rock, Charles O

    2004-07-23

    Epigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial, antiviral, and antitumor activity. Our study focused on validating the inhibition of the bacterial type II fatty acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols. EGCG and the related tea catechins potently inhibited both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC(50) values between 5 and 15 microm. The presence of the galloyl moiety was essential for activity, and EGCG was a competitive inhibitor of FabI and a mixed type inhibitor of FabG demonstrating that EGCG interfered with cofactor binding in both enzymes. EGCG inhibited acetate incorporation into fatty acids in vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened for FabG/FabI inhibition and antibacterial activity. Most of these inhibited both reductase steps, possessed antibacterial activity, and inhibited cellular fatty acid synthesis. The ability of the plant secondary metabolites to interfere with the activity of multiple NAD(P)-dependent cellular processes must be taken into account when assessing the specificity of their effects.

  13. Role of cysteine residues in pseudouridine synthases of different families.

    PubMed

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  14. Rare structural variants of human and murine uroporphyrinogen I synthase.

    PubMed Central

    Meisler, M H; Carter, M L

    1980-01-01

    An isoelectric focusing method for detection of structural variants of the enzyme uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] in mammalian tissues has been developed. Mouse and human erythrocytes contain one or two major isozymes of uroporphyrinogen I synthase, respectively. Other tissues contain a set of more acidic isozymes that are encoded by the same structural gene as the erythrocyte isozymes. Mouse populations studied with this method were monomorphic for uroporphyrinogen I synthase, with the exception of one feral mouse population. The pedigree of a human family with a rare structural variant is consistent with autosomal linkage of the structural gene. This system provides a convenient isozyme marker for genetic studies and will facilitate determination of the chromosomal location of the uroporphyrinogen I synthase locus. Images PMID:6930671

  15. Homology modeling and docking studies of FabH (β-ketoacyl-ACP synthase III) enzyme involved in type II fatty acid biosynthesis of Chlorella variabilis: a potential algal feedstock for biofuel production.

    PubMed

    Misra, Namrata; Patra, Mahesh Chandra; Panda, Prasanna Kumar; Sukla, Lala Bihari; Mishra, Barada Kanta

    2013-03-01

    The concept of using microalgae as an alternative renewable source of biofuel has gained much importance in recent years. However, its commercial feasibility is still an area of concern for researchers. Unraveling the fatty acid metabolic pathway and understanding structural features of various key enzymes regulating the process will provide valuable insights to target microalgae for augmented oil content. FabH (β-ketoacyl-acyl carrier protein synthase; KAS III) is a condensing enzyme catalyzing the initial elongation step of type II fatty acid biosynthetic process and acyl carrier protein (ACP) facilitates the shuttling of the fatty acyl intermediates to the active site of the respective enzymes in the pathway. In the present study, a reliable three-dimensional structure of FabH from Chlorella variabilis, an oleaginous green microalga was modeled and subsequently the key residues involved in substrate binding were determined by employing protein-protein docking and molecular dynamics (MD) simulation protocols. The FabH-ACP complex having the lowest docking energy score showed the binding of ACP to the electropositive FabH surface with strong hydrogen bond interactions. The MD simulation results indicated that the substrate-complexed FabH adopted a more stable conformation than the free enzyme. Further, the FabH structure retained its stability throughout the simulation although noticeable displacements were observed in the loop regions. Molecular simulation studies suggested the importance of crucial hydrogen bonding of the conserved Arg(91) of FabH with Glu(53) and Asp(56) of ACP for exhibiting high affinity between the enzyme and substrate. The molecular modeling results are consistent with available experimental results on the flexibility of FabH and the present study provides first in silico insights into the structural and dynamical aspect of catalytic mechanism of FabH, which could be used for further site-specific mutagenic experiments to develop

  16. Benzophenone synthase from Garcinia mangostana L. pericarps.

    PubMed

    Nualkaew, Natsajee; Morita, Hiroyuki; Shimokawa, Yoshihiko; Kinjo, Keishi; Kushiro, Tetsuo; De-Eknamkul, Wanchai; Ebizuka, Yutaka; Abe, Ikuro

    2012-05-01

    The cDNA of a benzophenone synthase (BPS), a type III polyketide synthase (PKS), was cloned and the recombinant protein expressed from the fruit pericarps of Garcinia mangostana L., which contains mainly prenylated xanthones. The obtained GmBPS showed an amino acid sequence identity of 77-78% with other plant BPSs belonging to the same family (Clusiaceae). The recombinant enzyme produced 2,4,6-trihydroxybenzophenone as the predominant product with benzoyl CoA as substrate. It also accepted other substrates, such as other plant PKSs, and used 1-3 molecules of malonyl CoA to form various phloroglucinol-type and polyketide lactone-type compounds. Thus, providing GmBPS with various substrates in vivo might redirect the xanthone biosynthetic pathway.

  17. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

    PubMed

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

    2015-12-01

    Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

  18. Non-enzymatic modifications of prostaglandin H synthase 1 affect bifunctional enzyme activity - Implications for the sensitivity of blood platelets to acetylsalicylic acid.

    PubMed

    Kassassir, Hassan; Siewiera, Karolina; Talar, Marcin; Stec-Martyna, Emilia; Pawlowska, Zofia; Watala, Cezary

    2016-06-25

    Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1

  19. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, E.

    1998-12-15

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

  20. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, Eran

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  1. Artemisia capillaris formula inhibits hepatic steatosis via an miR‑122‑induced decrease in fatty acid synthase expression in vivo and in vitro.

    PubMed

    Liu, Liya; Zhao, Jinyan; Li, Ying; Wan, Yun; Lin, Jiumao; Shen, Aling; Xu, Wei; Li, Huang; Zhang, Yuchen; Xu, Jianfeng; Peng, Jun; Hong, Zhenfeng

    2016-06-01

    treatment decreased the expression levels of fatty acid synthase (FASN) and increased miR‑122 in vivo and in vitro. In conclusion, these results suggested that ACF may inhibit hepatic steatosis via miR‑122‑induced downregulation of FASN in vivo and in vitro.

  2. A C. elegans Model for Mitochondrial Fatty Acid Synthase II: The Longevity-Associated Gene W09H1.5/mecr-1 Encodes a 2-trans-Enoyl-Thioester Reductase

    PubMed Central

    Gurvitz, Aner

    2009-01-01

    Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17β-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated

  3. Terminalia paniculata bark extract attenuates non-alcoholic fatty liver via down regulation of fatty acid synthase in high fat diet-fed obese rats

    PubMed Central

    2014-01-01

    Background This study was performed to understand the possible therapeutic activity of Terminalia paniculata ethanolic extract (TPEE) on non alcoholic fatty liver in rats fed with high fat diet. Methods Thirty six SD rats were divided into 6 groups (n = 6): Normal control (NC), high fat diet (HFD), remaining four groups were fed on HFD along with different doses of TPEE (100,150 and 200 mg/kg b.wt) or orlistat, for ten weeks. Liver tissue was homogenized and analyzed for lipid profiles, activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content. Further, the expression levels of FAS and AMPK-1α were also studied in addition to histopathology examination of liver tissue in all the groups. Results HFD significantly increased hepatic liver total cholesterol (TC), triglycerides (TG), free fatty acids (FFA) and MDA but decreased the activities of SOD and CAT which were subsequently reversed by supplementation with TPEE in a dose-dependent manner. In addition, TPEE administration significantly down regulated hepatic mRNA expression of FAS but up regulated AMPK-1α compared to HFD alone fed group. Furthermore, western blot analysis of FAS has clearly demonstrated decreased expression of FAS in HFD + TPEE (200 mg/kg b.wt) treated group when compared to HFD group at protein level. Conclusions Our biochemical studies on hepatic lipid profiles and antioxidant enzyme activities supported by histological and expression studies suggest a potential therapeutic role for TPEE in regulating obesity through FAS. PMID:24678767

  4. Citrate synthase from the liver fluke Fasciola hepatica.

    PubMed

    Zinsser, Veronika L; Moore, Catherine M; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2013-06-01

    Citrate synthase catalyses the first step of the Krebs' tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

  5. The female-specific Cs-ACS1G gene of cucumber. A case of gene duplication and recombination between the non-sex-specific 1-aminocyclopropane-1-carboxylate synthase gene and a branched-chain amino acid transaminase gene.

    PubMed

    Knopf, Ronit Rimon; Trebitsh, Tova

    2006-09-01

    Cucumber (Cucumis sativus L.) is a monoecious plant in which female sex expression (gynoecy) is controlled by the Female (F) locus that can be modified by other sex-determining genes as well as by environmental and hormonal factors. As in many other cucurbits, ethylene is the major plant hormone regulating female sex expression. Previously we isolated the Cs-ACS1 (ACS, 1-aminocyclopropane-1-carboxylate synthase) gene that encodes the rate-limiting enzyme in the ethylene biosynthetic pathway. We proposed that Cs-ACS1 is present in a single copy in monoecious (ffMM) plants whereas gynoecious plants (FFMM) contain an additional copy Cs-ACS1G that was mapped to the F locus. To study the origin of Cs-ACS1G, we cloned and analyzed both the gynoecious-specific Cs-ACS1G gene and the non-sex-specific Cs-ACS1 gene. Our results indicate that Cs-ACS1G is the result of a relatively recent gene duplication and recombination, between Cs-ACS1 and a branched-chain amino acid transaminase (BCAT) gene. Taking into consideration that the Cs-ACS1G gene was mapped to the F locus, we propose that this duplication event gave rise to the F locus and to gynoecious cucumber plants. Computer analysis of the 1 kb region upstream of the transcription initiation site revealed several putative cis-acting regulatory elements that can potentially confer the responsiveness of Cs-ACS1G to developmental and hormonal factors and thereby control female sex determination in cucumber. These findings lead us to a model explaining the action of Cs-ACS1 and Cs-ACS1G in cucumber floral sex determination. PMID:16887844

  6. Identification of avian wax synthases

    PubMed Central

    2012-01-01

    Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms. Results Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. Conclusions We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities. PMID:22305293

  7. CYP4 Enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities

    PubMed Central

    Edson, Katheryne Z.; Rettie, Allan E.

    2014-01-01

    The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20-hydroxyeicosatetraenoic acid or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases. PMID:23688133

  8. CYP4 enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities.

    PubMed

    Edson, Katheryne Z; Rettie, Allan E

    2013-01-01

    The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20- hydroxyeicosatetraenoic acid, or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases.

  9. Molecular and chemical characterization of the biosynthesis of the 6-MSA-derived meroterpenoid yanuthone D in Aspergillus niger.

    PubMed

    Holm, Dorte K; Petersen, Lene M; Klitgaard, Andreas; Knudsen, Peter B; Jarczynska, Zofia D; Nielsen, Kristian F; Gotfredsen, Charlotte H; Larsen, Thomas O; Mortensen, Uffe H

    2014-04-24

    Secondary metabolites in filamentous fungi constitute a rich source of bioactive molecules. We have deduced the genetic and biosynthetic pathway of the antibiotic yanuthone D from Aspergillus niger. Our analyses show that yanuthone D is a meroterpenoid derived from the polyketide 6-methylsalicylic acid (6-MSA). Yanuthone D formation depends on a cluster composed of ten genes including yanA and yanI, which encode a 6-MSA polyketide synthase and a previously undescribed O-mevalon transferase, respectively. In addition, several branching points in the pathway were discovered, revealing five yanuthones (F, G, H, I, and J). Furthermore, we have identified another compound (yanuthone X1) that defines a class of yanuthones that depend on several enzymatic activities encoded by genes in the yan cluster but that are not derived from 6-MSA.

  10. Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

    PubMed Central

    Eldar-Finkelman, H; Argast, G M; Foord, O; Fischer, E H; Krebs, E G

    1996-01-01

    In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8816781

  11. Conversion of anthranilate synthase into isochorismate synthase: implications for the evolution of chorismate-utilizing enzymes.

    PubMed

    Plach, Maximilian G; Löffler, Patrick; Merkl, Rainer; Sterner, Reinhard

    2015-09-14

    Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free-living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.

  12. Identification of cystathionine γ-synthase and threonine synthase from Cicer arietinum and Lens culinaris.

    PubMed

    Morneau, Dominique J K; Jaworski, Allison F; Aitken, Susan M

    2013-04-01

    In plants, cystathionine γ-synthase (CGS) and threonine synthase (TS) compete for the branch-point metabolite O-phospho-L-homoserine. These enzymes are potential targets for metabolic engineering studies, aiming to alter the flux through the competing methionine and threonine biosynthetic pathways, with the goal of increasing methionine production. Although CGS and TS have been characterized in the model organisms Escherichia coli and Arabidopsis thaliana, little information is available on these enzymes in other, particularly plant, species. The functional CGS and TS coding sequences from the grain legumes Cicer arietinum (chickpea) and Lens culinaris (lentil) identified in this study share approximately 80% amino acid sequence identity with the corresponding sequences from Glycine max. At least 7 active-site residues of grain legume CGS and TS are conserved in the model bacterial enzymes, including the catalytic base. Putative processing sites that remove the targeting sequence and result in functional TS were identified in the target species.

  13. Engineering of chimeric class II polyhydroxyalkanoate synthases.

    PubMed

    Niamsiri, Nuttawee; Delamarre, Soazig C; Kim, Young-Rok; Batt, Carl A

    2004-11-01

    PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic

  14. β-Carboline alkaloids from Galianthe ramosa inhibit malate synthase from Paracoccidioides spp.

    PubMed

    de Freitas, Carla S; Kato, Lucilia; de Oliveira, Cecília M A; Queiroz, Luiz H K; Santana, Mábio J; Schuquel, Ivânia T; Delprete, Piero G; da Silva, Roosevelt A; Quintino, Guilherme O; da Silva Neto, Benedito R; Soares, Célia M A; Pereira, Maristela

    2014-12-01

    As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.

  15. Homocysteine homeostasis in the rat is maintained by compensatory changes in cystathionine β-synthase, betaine-homocysteine methyltransferase, and phosphatidylethanolamine N-methyltransferase gene transcription occurring in response to maternal protein and folic acid intake during pregnancy and fat intake after weaning.

    PubMed

    Chmurzynska, Agata; Malinowska, Anna M

    2011-07-01

    The reactions of the methionine/homocysteine pathway are mediated by several enzymes, including phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase. Homocysteine homeostasis is regulated by these enzymes. We hypothesized here that the protein and folic acid content in the maternal diet affects methionine/homocysteine metabolism in the progeny. To test this hypothesis, pregnant rats were fed a diet with normal protein and normal folic acid levels (a modified casein-based AIN-93G diet), a protein-restricted and normal folic acid diet, a protein-restricted and folic acid-supplemented diet, or a normal protein and folic acid-supplemented diet. The progeny were fed either the modified AIN-93G diet or a high-fat lard-based diet. Progeny were analyzed for expression of the phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase genes in the liver and for serum homocysteine concentration. Interactions between prenatal and postnatal nutrition were also determined. The progeny of the dams fed the diets supplemented with folic acid showed decreased expression of all 3 genes (P < .001). An interaction effect between the protein and folic acid content in the maternal diet contributed to this down-regulation (P < .001), and the postweaning diet modified these effects. Serum homocysteine concentrations were approximately 15% higher in the male rats (P < .01), but neither prenatal nutrition nor the postweaning diet affected it significantly. We conclude that maternal diet during gestation has an important effect on the transcription level of these 3 genes, but changes in gene expression were not associated with significant changes in progeny homocysteine concentrations.

  16. Assessing the allelotypic effect of two aminocyclopropane carboxylic acid synthase-encoding genes MdACS1 and MdACS3a on fruit ethylene production and softening in Malus.

    PubMed

    Dougherty, Laura; Zhu, Yuandi; Xu, Kenong

    2016-01-01

    Phytohormone ethylene largely determines apple fruit shelf life and storability. Previous studies demonstrated that MdACS1 and MdACS3a, which encode 1-aminocyclopropane-1-carboxylic acid synthases (ACS), are crucial in apple fruit ethylene production. MdACS1 is well-known to be intimately involved in the climacteric ethylene burst in fruit ripening, while MdACS3a has been regarded a main regulator for ethylene production transition from system 1 (during fruit development) to system 2 (during fruit ripening). However, MdACS3a was also shown to have limited roles in initiating the ripening process lately. To better assess their roles, fruit ethylene production and softening were evaluated at five time points during a 20-day post-harvest period in 97 Malus accessions and in 34 progeny from 2 controlled crosses. Allelotyping was accomplished using an existing marker (ACS1) for MdACS1 and two markers (CAPS866 and CAPS870) developed here to specifically detect the two null alleles (ACS3a-G289V and Mdacs3a) of MdACS3a. In total, 952 Malus accessions were allelotyped with the three markers. The major findings included: The effect of MdACS1 was significant on fruit ethylene production and softening while that of MdACS3a was less detectable; allele MdACS1-2 was significantly associated with low ethylene and slow softening; under the same background of the MdACS1 allelotypes, null allele Mdacs3a (not ACS3a-G289V) could confer a significant delay of ethylene peak; alleles MdACS1-2 and Mdacs3a (excluding ACS3a-G289V) were highly enriched in M. domestica and M. hybrid when compared with those in M. sieversii. These findings are of practical implications in developing apples of low and delayed ethylene profiles by utilizing the beneficial alleles MdACS1-2 and Mdacs3a. PMID:27231553

  17. Assessing the allelotypic effect of two aminocyclopropane carboxylic acid synthase-encoding genes MdACS1 and MdACS3a on fruit ethylene production and softening in Malus

    PubMed Central

    Dougherty, Laura; Zhu, Yuandi; Xu, Kenong

    2016-01-01

    Phytohormone ethylene largely determines apple fruit shelf life and storability. Previous studies demonstrated that MdACS1 and MdACS3a, which encode 1-aminocyclopropane-1-carboxylic acid synthases (ACS), are crucial in apple fruit ethylene production. MdACS1 is well-known to be intimately involved in the climacteric ethylene burst in fruit ripening, while MdACS3a has been regarded a main regulator for ethylene production transition from system 1 (during fruit development) to system 2 (during fruit ripening). However, MdACS3a was also shown to have limited roles in initiating the ripening process lately. To better assess their roles, fruit ethylene production and softening were evaluated at five time points during a 20-day post-harvest period in 97 Malus accessions and in 34 progeny from 2 controlled crosses. Allelotyping was accomplished using an existing marker (ACS1) for MdACS1 and two markers (CAPS866 and CAPS870) developed here to specifically detect the two null alleles (ACS3a-G289V and Mdacs3a) of MdACS3a. In total, 952 Malus accessions were allelotyped with the three markers. The major findings included: The effect of MdACS1 was significant on fruit ethylene production and softening while that of MdACS3a was less detectable; allele MdACS1–2 was significantly associated with low ethylene and slow softening; under the same background of the MdACS1 allelotypes, null allele Mdacs3a (not ACS3a-G289V) could confer a significant delay of ethylene peak; alleles MdACS1–2 and Mdacs3a (excluding ACS3a-G289V) were highly enriched in M. domestica and M. hybrid when compared with those in M. sieversii. These findings are of practical implications in developing apples of low and delayed ethylene profiles by utilizing the beneficial alleles MdACS1-2 and Mdacs3a. PMID:27231553

  18. Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte.

    PubMed Central

    Charles, I G; Palmer, R M; Hickery, M S; Bayliss, M T; Chubb, A P; Hall, V S; Moss, D W; Moncada, S

    1993-01-01

    Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species. Images Fig. 1 PMID:7504305

  19. Incremental truncation of PHA synthases results in altered product specificity.

    PubMed

    Wang, Qian; Xia, Yongzhen; Chen, Quan; Qi, Qingsheng

    2012-05-10

    PHA synthase is the key enzyme involved in the biosynthesis of microbial polymers, polyhydroxyalkanoates (PHA). In this study, we created a hybrid library of PHA synthase gene with different crossover points by an incremental truncation method between the C-terminal fragments of the phaC(Cn) (phaC from Cupriavidus necator) and the N-terminal fragments of the phaC1(Pa) (phaC from Pseudomonas aeruginosa). As the truncation of the hybrid enzyme increased, the in vivo PHB synthesis ability of the hybrids declined gradually. PHA synthase PhaC(Cn) with a deletion on N-terminal up to 83 amino acid residues showed no synthase activity. While with the removal of up to 270 amino acids from the N-terminus, the activity of the truncated PhaC(Cn) could be complemented by the N-terminus of PhaC1(Pa). Three of the hybrid enzymes W188, W235 and W272 (named by the deleted nucleic acid number) were found to have altered product specificities. PMID:22500895

  20. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    PubMed

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  1. Development and Binding Mode Assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid (BGC 945), a Novel Thymidylate Synthase Inhibitor that Targets Tumor Cells

    PubMed Central

    Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O’Connell, Joseph D.; Griner, Sarah; Finer-Moore, Janet S.; Stroud, Robert M.

    2013-01-01

    N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in Phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor glycoprotein, which is over expressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2’-deoxyuridine-5’-monophosphate, and a model for a similar complex with human TS. PMID:23710599

  2. Genetics Home Reference: GM3 synthase deficiency

    MedlinePlus

    ... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... diagnosis or management of GM3 synthase deficiency: American Epilepsy Society: Find a Doctor Clinic for Special Children ( ...

  3. Structure of Aminodeoxychorismate Synthase from Stenotrophomonas maltophilia†

    PubMed Central

    Bera, Asim K.; Atanasova, Vesna; Dhanda, Anjali; Ladner, Jane E.; Parsons, James F.

    2012-01-01

    PabB, aminodeoxychorismate synthase, is the chorismic acid binding component of the heterodimeric PabAB complex that converts chorismic acid to 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folic acid in microorganisms. The second component, a glutamine amidotransferase subunit, PabA, generates ammonia that is channeled to the PabB active site where it attacks the C4 carbon of a chorismate derived intermediate that is covalently bound, through C2, to an active site lysine residue. The presence of a PIKGT motif was, until recently, believed to be discriminate PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active site motif and does not form a covalent enzyme-substrate intermediate with chorismate. A subclass of PabB enzymes that employ an alternative mechanism requiring two equivalents of ammonia from glutamine and that feature a noncovalently bound 2-amino-2-deoxyisochorismate intermediate was recently identified. Here we report the 2.25 Å crystal structure of PabB from the emerging pathogen Stenotrophomonas maltophilia. It is the first reported structure of a PabB that features the PIAGT motif. Surprisingly, no dedicated pabA is evident in the genome of S. maltophilia suggesting that another cellular amidotransferase is able to fulfill the role of PabA in this organism. Evaluation of the ammonia-dependent aminodeoxychorismate synthase activity of S. maltophilia PabB alone revealed that it is virtually inactive. However, in the presence of a heterologous PabA surrogate, typical levels of activity were observed using either glutamine or ammonia as the nitrogen source. Additionally, the structure suggests that a key segment of the polypeptide can remodel itself to interact with a nonspecialized or shared amidotransferase partner in vivo. The structure and mass spectral analysis further suggest that S. maltophilia PabB, like Escherichia coli PabB, binds tryptophan in a vestigial regulatory site

  4. Mapping a kingdom-specific functional domain of squalene synthase.

    PubMed

    Linscott, Kristin B; Niehaus, Thomas D; Zhuang, Xun; Bell, Stephen A; Chappell, Joe

    2016-09-01

    Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi.

  5. Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11 beta-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production.

    PubMed

    Tremblay, A; Parker, K L; Lehoux, J G

    1992-06-01

    Increasing evidence indicates that the adrenal cortex of most mammalian species expresses distinct forms of cytochrome P-450(11 beta), a steroidogenic enzyme that catalyses the terminal steps in the biosynthesis of both glucocorticoids and mineralocorticoids. In the human, mouse, and rat, two genes have been isolated, designated CYP11B1 and CYP11B2. The product of CYP11B2 (aldosterone synthase) is required for the successive 11 beta-, 18-hydroxylations and 18-oxidation of deoxycorticosterone that lead to the production of aldosterone in the zona glomerulosa. In contrast, the product of CYP11B1 (11 beta-hydroxylase) mediates only the 11 beta-hydroxylation of deoxycorticosterone and 11-deoxycortisol. The recent identification of these two P-450(11 beta) isozymes mandates further analysis of their expression in different zones of the adrenal cortex, both under basal conditions and in response to conditions known to alter mineralocorticoid biosynthesis. To evaluate the expression of the two isozymes in different adrenocortical zones, we performed Northern blotting analyses with specific oligonucleotide probes that discriminated between the two forms of rat P-450(11 beta). The transcripts detected by the two probes were of similar size (2.7 kilobase), but differed in their zonal distribution: aldosterone synthase P-450 messenger RNA (mRNA) was detected only in zona glomerulosa, whereas 11 beta-hydroxylase P-450 was expressed in both zona fasciculata-reticularis and zona glomerulosa. Next, we analyzed the response of these two genes to various physiological and pharmacological interventions known to affect aldosterone biosynthesis. High potassium or low sodium diet given to rats for 1 week increased aldosterone synthase P-450 mRNA levels by approximately 5- and 6-fold, respectively. These increases, moreover, were significantly attenuated by treatment with captopril, an inhibitor of angiotensin-converting enzyme. In contrast, neither dietary manipulation significantly

  6. Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold.

    PubMed

    Kaya, Yusuf; Del Campo, Mark; Ofengand, James; Malhotra, Arun

    2004-04-30

    TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine. It has little sequence homology with the other 10 pseudouridine synthases in E. coli which themselves have been grouped into four related protein families. Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

  7. Plasticity and Evolution of (+)-3-Carene Synthase and (−)-Sabinene Synthase Functions of a Sitka Spruce Monoterpene Synthase Gene Family Associated with Weevil Resistance*

    PubMed Central

    Roach, Christopher R.; Hall, Dawn E.; Zerbe, Philipp; Bohlmann, Jörg

    2014-01-01

    The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (−)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. PMID:25016016

  8. Inhibition of rabbit erythroid 15-lipoxygenase and sheep vesicular gland prostaglandin H synthase by gallic esters.

    PubMed

    Luther, H; Jordanov, D; Ludwig, P; Schewe, T

    1991-02-01

    Gallic acid esters possessing a varying chain length of their alcohol moiety were tested for their inhibitory potencies on 15-lipoxygenase from rabbit reticulocytes and prostaglandin H synthase from sheep vesicular glands. Octyl gallate and decyl gallate proved to be the most powerful inhibitors of both enzymes showing concentrations of half-inhibition of about 0.25 mumol/l for the reticulocyte lipoxygenase and of about 25 mumol/l for the prostaglandin H synthase.

  9. A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

    PubMed

    Sato-Masumoto, Naoko; Ito, Michiho

    2014-06-01

    Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group.

  10. Molecular cloning and expression profile of ß-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tung tree (Vernicia fordii) is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid). Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a ...

  11. Lipoxin synthase activity of human platelet 12-lipoxygenase.

    PubMed Central

    Romano, M; Chen, X S; Takahashi, Y; Yamamoto, S; Funk, C D; Serhan, C N

    1993-01-01

    Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity. Images Figure 1 PMID:8250832

  12. Radiolabeling of a wound-inducible pyridoxal phosphate utilizing protein from tomato: evidence for its identification as ACC synthase

    SciTech Connect

    Privalle, L.S.; Graham, J.S.; Caughey, P.A.

    1986-05-01

    Aminocyclopropane 1-carboxylic acid (ACC) synthase, a pyridoxal phosphate utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate limiting step in the biosynthesis of the plant hormone, ethylene. Ethylene, besides being involved in normal plant growth processes, is also produced in response to stress, e.g. wounding, pathogen infection, etc. The authors report the partial purification (400 fold) of ACC synthase from wounded pink tomato pericarp by classical techniques including ammonium sulfate precipitation, ion exchange and phenyl sepharose chromatography. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme and the low levels present in plant tissue. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase enriched fraction was achieved. Evidence that this radiolabeled protein is ACC synthase will be presented. Amino acid sequence determination of putative ACC synthase-derived peptides is underway.

  13. Cloning and characterization of a mammalian pseudouridine synthase.

    PubMed Central

    Chen, J; Patton, J R

    1999-01-01

    This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame. PMID:10094309

  14. The pseudouridine synthases: revisiting a mechanism that seemed settled.

    PubMed

    Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2004-10-13

    RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.

  15. Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

    PubMed

    Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

    2014-01-01

    In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender. PMID:24078339

  16. Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus.

    PubMed

    Sthapit, Basundhara; Oh, Tae-Jin; Lamichhane, Rajan; Liou, Kwangkyoung; Lee, Hei Chan; Kim, Chun-Gyu; Sohng, Jae Kyung

    2004-05-21

    Enediyne antibiotics are known for their potent antitumor activities. One such enediyne, neocarzinostatin (NCS), consists of a 1:1 complex of non-peptide chromophore (1a), and peptide apoprotein. The structurally diverse non-peptide chromophore is responsible for its biological activity. One of its structural components, the naphthoic acid moiety (2,7-dihydroxy-5-methyl-1-naphthoic acid, 1d) is synthesized by a polyketide synthase (PKS) pathway through condensing six intact acetate units. The 5.45 kb iterative type I PKS, neocarzinostatin naphthoate synthase (NNS), responsible for naphthoic acid moiety biosynthesis, shares sequence homology with 6-methyl salicylic acid synthase of fungi and orsellinic acid synthases (AviM and CalO5) of Streptomyces origin. Cultures of S. lividans TK24 and S. coelicolor YU105 containing plasmids with NNS were able to produce 2-hydroxy-5-methyl-1-naphthoic acid (2a), a key intermediate of naphthoic acid moiety in NCS. In addition to 2a, a novel product, 2-hydroxy-5-hydroxymethyl-1-naphthoic acid (2d) was isolated. This is the first report of a bacterial iterative type I PKS from an enediyne producer which enables the biosynthesis of bicyclic aromatic compounds. PMID:15147895

  17. Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

    PubMed

    Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W V; Sivaraman, J

    2015-11-01

    ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

  18. Stabilization of the second oxyanion intermediate by 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase of the menaquinone pathway: spectroscopic evidence of the involvement of a conserved aspartic acid.

    PubMed

    Chen, Minjiao; Jiang, Ming; Sun, Yueru; Guo, Zu-Feng; Guo, Zhihong

    2011-07-01

    1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small

  19. Ozone stress induces the expression of ACC synthase in potato plants

    SciTech Connect

    Schlagnhaufer, C.D.; Arteca, R.N.; Pell, E.J. )

    1993-05-01

    When potato plants (Solanum tuberosum L. cv Norland) are subjected to oxone stress ethylene is emitted. Increases in ethylene production are often the result of increased expression of the enzyme ACC synthase. We used the polymerase chain reaction (PCR) to clone a cDNA encoding an ozone-induced ACC synthase. After treating potato plants with 300 ppb ozone for 4 h, RNA was extracted using a guanidinium isothiocyanate method. Using degenerate oligonucleotides corresponding to several conserved regions of ACC synthase sequences reported from different plant tissues as primers, we were able to reverse transcribe the RNA and amplify a cDNA for ACC synthase. The clone is 1098 bp in length encoding for 386 amino acids comprising [approximately]80% of the protein. Computer analysis of the deduced amino acid sequence showed that our clone is 50-70% homologous with ACC synthase genes cloned from other plant tissues. Using the cDNA as a probe in northern analysis we found that there is little or no expression in control tissue: however there is a large increase in the expression of the ACC synthase message in response to ozone treatment.

  20. Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

    PubMed

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases. PMID:25230152

  1. Architecture of the polyketide synthase module: surprises from electron cryo-microscopy

    PubMed Central

    Smith, Janet L; Skiniotis, Georgios; Sherman, David H

    2015-01-01

    Modular polyketide synthases produce a vast array of bioactive molecules that are the basis of many highly valued pharmaceuticals. The biosynthesis of these compounds is based on ordered assembly lines of multi-domain modules, each extending and modifying a specific chain-elongation intermediate before transfer to the next module for further processing. The first 3D structures of a full polyketide synthase module in different functional states were obtained recently by electron cryo-microscopy. The unexpected module architecture revealed a striking evolutionary divergence of the polyketide synthase compared to its metazoan fatty acid synthase homolog, as well as remarkable conformational rearrangements dependent on its biochemical state during the full catalytic cycle. The design and dynamics of the module are highly optimized for both catalysis and fidelity in the construction of complex, biologically active natural products. PMID:25791608

  2. Tight linkage of genes that encode the two glutamate synthase subunits of Escherichia coli K-12.

    PubMed Central

    Lozoya, E; Sanchez-Pescador, R; Covarrubias, A; Vichido, I; Bolivar, F

    1980-01-01

    A hybrid deoxyribonucleic acid molecule, plasmid pRSP20, which was isolated from the Clarke and Carbon Escherichia coli gene bank, was shown to complement the gltB31 mutation, which affects the synthesis of glutamate synthase in E. coli strain PA340. We present evidence which demonstrates that plasmid pRSP20 carries an 8-megadalton E. coli chromosomal fragment, including the genes encoding the two unequal glutamate synthase subunits. Polypeptides with molecular weights of about 135,000 and 53,000, which comigrated with purified E. coli glutamate synthase subunit polypeptides and immunoprecipitated with antibodies to E. coli glutamate synthase, were synthesized by minicells carrying the pRSP20 plasmid. Images PMID:6107287

  3. Pseudouridines and pseudouridine synthases of the ribosome.

    PubMed

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  4. Polymorphism and expression of isoflavone synthase genes from soybean cultivars.

    PubMed

    Kim, Hyo-Kyoung; Jang, Yun-Hee; Baek, Il-Sun; Lee, Jeong-Hwan; Park, Min Joo; Chung, Young-Soo; Chung, Jong-Il; Kim, Jeong-Kook

    2005-02-28

    Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings. PMID:15750342

  5. Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor

    PubMed Central

    Kawasaki, Toshiyuki; Marumo, Toshiyuki; Shirakami, Keiko; Mori, Tomoko; Doi, Hisashi; Suzuki, Masaaki; Watanabe, Yasuyoshi; Chaki, Shigeyuki; Nakazato, Atsuro; Ago, Yukio; Hashimoto, Hitoshi; Matsuda, Toshio; Baba, Akemichi; Onoe, Hirotaka

    2012-01-01

    20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a 11C-labeled specific 20-HETE synthase inhibitor, N′(4-dimethylaminohexyloxy)phenyl imidazole ([11C]TROA). Autoradiographic study showed that [11C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [11C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [11C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [11C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia. PMID:22669478

  6. Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro

    PubMed Central

    Della-Cioppa, Guy; Bauer, S. Christopher; Klein, Barbara K.; Shah, Dilip M.; Fraley, Robert T.; Kishore, Ganesh M.

    1986-01-01

    5-enolPyruvylshikimate-3-phosphate synthase (EPSP synthase; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of the shikimate pathway in plants. This enzyme is the target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). We have previously isolated a full-length cDNA clone of EPSP synthase from Petunia hybrida. DNA sequence analysis suggested that the enzyme is synthesized as a cytosolic precursor (pre-EPSP synthase) with an amino-terminal transit peptide. Based on the known amino terminus of the mature enzyme, and the 5′ open reading frame of the cDNA, the transit peptide of pre-EPSP synthase would be maximally 72 amino acids long. To confirm this prediction and to assay directly for translocation of pre-EPSP synthase into chloroplasts in vitro, we cloned the full-length cDNA into an SP6 transcription system to produce large amounts of mRNA for in vitro translation. The translation products, when analyzed by NaDodSO4/PAGE autoradiography, indicate a relative molecular mass for pre-EPSP synthase of ≈55 kDa. Uptake studies with intact chloroplasts, in vitro, indicate that pre-EPSP synthase was rapidly taken up into chloroplasts and proteolytically cleaved to the mature ≈48-kDa enzyme. The transit peptide was shown to be essential for import of the precursor enzyme into the chloroplast. To our knowledge, post-translational import into chloroplasts of a precursor enzyme involved in amino acid biosynthesis has not been reported previously. Furthermore, enzymatic analysis of translation products indicates that pre-EPSP synthase is catalytically active and has a similar sensitivity to the herbicide glyphosate as the mature enzyme. To our knowledge, pre-EPSP synthase represents the only example of a catalytically competent chloroplast-precursor enzyme. Images PMID:16593759

  7. CJ-15,183, a new inhibitor of squalene synthase produced by a fungus, Aspergillus aculeatus.

    PubMed

    Watanabe, S; Hirai, H; Ishiguro, M; Kambara, T; Kojima, Y; Matsunaga, T; Nishida, H; Suzuki, Y; Sugiura, A; Harwood, H J; Huang, L H; Kojima, N

    2001-11-01

    A new squalene synthase (SSase) inhibitor, CJ-15,183 (I) was isolated from the fermentation broth of a fungus, Aspergillus aculeatus CL38916. The compound potently inhibited rat liver and Candida albicans microsomal SSases and also inhibited the human enzyme. It also showed antifungal activities against filamentous fungi and a yeast. The structure was determined to be an aliphatic tetracarboxylic acid compound consisting of an alkyl gamma-lactone, malic acid and isocitric acid moieties by spectroscopic studies.

  8. Translocation of the potato 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase into isolated spinach chloroplasts

    SciTech Connect

    Zhao, Jianmin; Weaver, L.M.; Herrmann, K.M. )

    1990-05-01

    A cDNA for potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, encodes a 56 KD polypeptide whose amino terminus resembles a chloroplast transit sequence. The cDNA was placed downstream of the phage T7 polymerase recognition sequence in plasmid pGEM-3Z. DNA of the resulting plasmid pGEM-DWZ directed T7 polymerase to synthesize potato DAHP synthase mRNA in vitro. The mRNA was used in wheat germ and rabbit reticulocyte lysates for the synthesis of {sup 35}S-labeled pro-DAHP synthase. The predominant translation product is a 59 KD polypeptide that can be immunoprecipitated by rabbit polyclonal antibodies raised against the 53 KD DAHP synthase purified from potato tubers. Isolated spinach chloroplasts process the 59 KD pro-DAHP synthase to a 50 KD polypeptide. The processed polypeptide is protected from protease degradation, suggesting uptake of the enzyme into the cell organelle. Fractionation of reisolated chloroplasts after import of pro-DAHP synthase showed mature enzyme in the stroma. The uptake and processing of DAHP synthase is inhibited by antibodies raised against the mature enzyme. Our results are consistent with the assumption that potato contains a nuclear DNA encoded DAHP synthase that is synthesized as a proenzyme and whose mature form resides in the chloroplasts. Our data provide further evidence that green plants synthesize aromatic amino acids in plastids.

  9. Trichodiene synthase. Substrate specificity and inhibition.

    PubMed

    Cane, D E; Yang, G; Xue, Q; Shim, J H

    1995-02-28

    The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70. PMID:7873526

  10. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

    PubMed

    Grundy, Daniel J; Chen, Mengbin; González, Verónica; Leoni, Stefano; Miller, David J; Christianson, David W; Allemann, Rudolf K

    2016-04-12

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues

  11. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    PubMed Central

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  12. 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

  13. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  14. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site.

  15. Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits

    SciTech Connect

    Olson, D.C.; White, J.A.; Edelman, L.; Kende, H. ); Harkins, R.N. )

    1991-06-15

    1-Aminocyclopropane-1-carboxylate synthase is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits. The authors report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5{prime} and 3{prime} ends. Gene-specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes.

  16. Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

    PubMed

    Sutton, Kristin A; Breen, Jennifer; Russo, Thomas A; Schultz, L Wayne; Umland, Timothy C

    2016-03-01

    The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. PMID:26919521

  17. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    SciTech Connect

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  18. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    DOEpatents

    Somerville, Chris R.; Scheible, Wolf

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  19. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes

    PubMed Central

    Mott, David M.; Stone, Karen; Gessel, Mary C.; Bunt, Joy C.; Bogardus, Clifton

    2008-01-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = −0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism. PMID:18056794

  20. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes.

    PubMed

    Mott, David M; Stone, Karen; Gessel, Mary C; Bunt, Joy C; Bogardus, Clifton

    2008-02-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

  1. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    NASA Astrophysics Data System (ADS)

    Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

    2014-07-01

    The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  2. Human isoprenoid synthase enzymes as therapeutic targets

    PubMed Central

    Park, Jaeok; Matralis, Alexios N.; Berghuis, Albert M.; Tsantrizos, Youla S.

    2014-01-01

    In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies. PMID:25101260

  3. Characterization of Methylglyoxal Synthase from Clostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

    PubMed Central

    Huang, Ke-xue; Rudolph, Frederick B.; Bennett, George N.

    1999-01-01

    A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km and Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min−1 μg−1, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced. PMID:10388730

  4. Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin.

    PubMed

    Parry, R J; Hoyt, J C

    1997-02-01

    Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD+ for activity but lacked rigid substrate specificity, since analogs of both NAD+ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5'-monophosphate dehydrogenase. PMID:9023226

  5. Crystal structure of riboflavin synthase

    SciTech Connect

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  6. Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp.

    PubMed Central

    Schendel, F J; August, P R; Anderson, C R; Hanson, R S; Flickinger, M C

    1992-01-01

    The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes. PMID:1311544

  7. Altering small and medium alcohol selectivity in the wax ester synthase.

    PubMed

    Barney, Brett M; Ohlert, Janet M; Timler, Jacobe G; Lijewski, Amelia M

    2015-11-01

    The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.

  8. Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice

    PubMed Central

    Scherthan, Harry; Horsch, Marion; Beckers, Johannes; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Ford, Steven J.; Burton, Neal C.; Razansky, Daniel; Trümbach, Dietrich; Aichler, Michaela; Walch, Axel Karl; Calzada-Wack, Julia; Neff, Frauke; Wurst, Wolfgang; Hartmann, Tobias; Floss, Thomas

    2016-01-01

    Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility. PMID:27788151

  9. Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences

    PubMed Central

    Carroll, Andrew; Specht, Chelsea D.

    2011-01-01

    The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments. PMID:22629257

  10. Aromatic polyketide synthases from 127 Fusarium: pas de deux for chemical diversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium species collectively cause disease on almost all crop plants and produce numerous natural products (NPs), including mycotoxins, of great concern. Many Fusarium NPs are derived from polyketide synthases (PKSs), large enzymes that catalyze the condensation of simple carboxylic acids. To gain ...

  11. Synthesis and antibacterial activity of alaremycin derivatives for the porphobilinogen synthase.

    PubMed

    Iwai, Noritaka; Nakayama, Kyosuke; Oku, Jumpei; Kitazume, Tomoya

    2011-05-15

    The preparation and the antibacterial activity of alaremycin derivatives such as their CF(3)-derivatives and (R)- and (S)-4-oxo-5-acetylaminohexanoic acid for the porphobilinogen synthase (PBGS), were described. The IC(50) values of the antibacterial activity of the prepared materials for the inhibitor of PBGS, were determined using PBGS assay. PMID:21514151

  12. Light- and metabolism-related regulation of the chloroplast ATP synthase has distinct mechanisms and functions.

    PubMed

    Kohzuma, Kaori; Dal Bosco, Cristina; Meurer, Jörg; Kramer, David M

    2013-05-01

    The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

  13. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    SciTech Connect

    Bagautdinov, Bagautdin Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The crystal structure of 3-oxoacyl-(acyl-carrier protein) synthase II from T. thermophilus HB8 has been determined at 2.0 Å resolution and compared with the structures of β-keto-ACP synthases from other sources. The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain.

  14. Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth, Sphingolipid Metabolism, Programmed Cell Death, and Mycotoxin Resistance.

    PubMed

    Luttgeharm, Kyle D; Chen, Ming; Mehra, Amit; Cahoon, Rebecca E; Markham, Jonathan E; Cahoon, Edgar B

    2015-10-01

    Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB1, whereas LOH1-overexpressing plants showed no increase in FB1 resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB1. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance

  15. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    PubMed

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  16. The β-ketoacyl-ACP synthase from Mycobacterium tuberculosis as potential drug targets.

    PubMed

    Singh, V; Mani, I; Chaudhary, D K; Somvanshi, P

    2011-01-01

    The continuous preventive measures and control of tuberculosis are often hampered by re-emergence of multi-drug-resistant (MDR) strains of Mycobacterium tuberculosis. A novel drug approach is desperately needed to combat the global threat posed by MDR strains. In spite of current advancement in biological techniques viz. microarray and proteomics data for tuberculosis, no such potent drug has been developed in the past decades yet. Therefore, mycolic acid is an essential constituent which is involved in the formation of cell wall of Mycobacterium species. The biosynthesis of mycolic acid is involved in two fatty acid synthase systems, the multifunctional polypeptide fatty acid synthase I (FASI) which performs de novo fatty acid synthesis and dissociate FASII system. FASII system consists of monofunctional enzymes and acyl carrier protein (ACP), elongating FASI products to long chain mycolic acid precursor. In this review, the β-ketoacyl-ACP synthases (fadH, kasA and kasB) are distinct and play a vital role in mycolic acid synthesis, cell wall synthesis, biofilm formation and also pathogenesis. On the basis of substantial observation we suggest that these enzymes may be used as promising and attractive targets for novel anti-TB drugs designing and discovery. PMID:21370994

  17. Identification of a cellulose synthase-associated protein required for cellulose biosynthesis.

    PubMed

    Gu, Ying; Kaplinsky, Nick; Bringmann, Martin; Cobb, Alex; Carroll, Andrew; Sampathkumar, Arun; Baskin, Tobias I; Persson, Staffan; Somerville, Chris R

    2010-07-20

    Cellulose synthase-interactive protein 1 (CSI1) was identified in a two-hybrid screen for proteins that interact with cellulose synthase (CESA) isoforms involved in primary plant cell wall synthesis. CSI1 encodes a 2,150-amino acid protein that contains 10 predicted Armadillo repeats and a C2 domain. Mutations in CSI1 cause defective cell elongation in hypocotyls and roots and reduce cellulose content. CSI1 is associated with CESA complexes, and csi1 mutants affect the distribution and movement of CESA complexes in the plasma membrane. PMID:20616083

  18. Identification of a novel gene coding for neoxanthin synthase from Solanum tuberosum.

    PubMed

    Al-Babili, S; Hugueney, P; Schledz, M; Welsch, R; Frohnmeyer, H; Laule, O; Beyer, P

    2000-11-24

    The polymerase chain reaction analysis of potato plants, transformed with capsanthin capsorubin synthase ccs, revealed the presence of a highly related gene. The cloned cDNA showed at the protein level 89.6% identity to CCS. This suggested that the novel enzyme catalyzes a mechanistically similar reaction. Such a reaction is represented by neoxanthin synthase (NXS), forming the xanthophyll neoxanthin, a direct substrate for abscisic acid formation. The function of the novel enzyme could be proven by transient expression in plant protoplasts and high performance liquid chromatography analysis. The cloned NXS was imported in vitro into plastids, the compartment of carotenoid biosynthesis.

  19. [Intraspecific polymorphism of the sucrose synthase genes in Russian and Kazakhstan potato cultivars].

    PubMed

    Slugina, M A; Boris, K V; Kakimzhanova, A A; Kochieva, E Z

    2014-06-01

    In 12 different Russian and Kazakhstan potato cultivars, the polymorphism of the glycosyltransferase domain of the sucrose synthase gene was first examined, as well as the polymorphism of the sucrose synthase domain fragment of the same gene in the potato cultivars of Kazakhstan breed. It was demonstrated that the examined sequences contained point mutations, as well as insertions and deletions, including those not described earlier. Amino acid substitutions specific to heat- and drought-tolerant varieties were also identified and could be associated with the development of abiotic stress resistance. PMID:25715458

  20. Unique animal prenyltransferase with monoterpene synthase activity

    NASA Astrophysics Data System (ADS)

    Gilg, Anna B.; Tittiger, Claus; Blomquist, Gary J.

    2009-06-01

    Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.

  1. Nitric oxide synthases in pregnant rat uterus.

    PubMed

    Farina, M; Ribeiro, M L; Franchi, A

    2001-03-01

    The conversion of [14C]arginine into [14C]citrulline as an indicator of nitric oxide synthesis was studied in uteri isolated from rats on different days of gestation, after labour and during dioestrus. Nitric oxide synthesis was present in uterine tissues isolated at each stage of gestation and also in tissues collected during dioestrus and after labour. Expression of neuronal nitric oxide synthase was not detectable at any of the stages studied. Endothelial nitric oxide synthase was present at all the stages studied, but there was a significant increase on day 13 of gestation and a decrease thereafter, with the lowest expression recorded on the day after labour. Inducible nitric oxide synthase expression in rat uteri increased substantially during pregnancy, with the highest expression on day 13 of gestation; expression decreased at term and after labour. The changes in expression of inducible nitric oxide synthase were coincident with the changes in nitric oxide synthase activity in uteri treated with aminoguanidine. Thus, these findings indicate that an increase in expression of inducible nitric oxide synthase in the uterus may be important for maintenance of uterine quiescence during pregnancy and its decrease near the time of labour could have an effect on the start of uterine contractility. PMID:11226066

  2. Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

    PubMed

    Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

    2016-04-01

    Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase. PMID:26880289

  3. Characterization of α-humulene synthases responsible for the production of sesquiterpenes induced by methyl jasmonate in Aquilaria cell culture.

    PubMed

    Kumeta, Yukie; Ito, Michiho

    2016-07-01

    The resinous portions of Aquilaria and Gyrinops plants are known as 'agarwood' and have a distinctive fragrance. To examine the biosynthesis of these fragrant compounds, we previously established cell cultures of Aquilaria crassna in which the production of three sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) could be induced by methyl jasmonate (MJ), and showed that cloned δ-guaiene synthase from MJ-treated cells is involved in the synthesis of these three compounds, although only very small amounts of α-humulene are produced. In the present study, cDNAs encoding α-humulene synthases were also isolated. Three putative sesquiterpene synthase clones (AcHS1-3) isolated from the MJ-treated cells had very similar amino acid sequences and shared 52 % identity with δ-guaiene synthases. The recombinant enzymes catalyzed the formation of α-humulene as a major product. Expression of transcripts of the α-humulene synthase and δ-guaiene synthase genes in cultured cells increased after treatment with MJ. These results revealed that these α-humulene and δ-guaiene synthases are involved in the synthesis of three sesquiterpenes induced by MJ treatment. PMID:27180085

  4. Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

    PubMed

    Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

    2016-04-01

    Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase.

  5. Malate synthase a membrane protein

    SciTech Connect

    Chapman, K.D.; Turley, R.B.; Hermerath, C.A.; Carrapico, F.; Trelease, R.N.

    1987-04-01

    Malate synthase (MS) is generally regarded as a peripheral membrane protein, and believed by some to be ontogenetically associated with ER. However, immuno- and cyto-chemical in situ localizations show MS throughout the matrix of cotton (and cucumber) glyoxysomes, not specifically near their boundary membranes, nor in ER. Only a maximum of 50% MS can be solubilized from cotton glyoxysomes with 1% Triton X-100, 2mM Zwittergen 14, or 10mM DOC +/- salts. Cotton MS does not incorporate /sup 3/H-glucosamine in vivo, nor does it react with Con A on columns or blots. Cotton MS banded with ER in sucrose gradients (20-40%) in Tricine after 3h, but not after 22h in Tricine or Hepes, or after 3h in Hepes or K-phosphate. Collectively the authors data are inconsistent with physiologically meaningful MS-membrane associations in ER or glyoxysomes. It appears that experimentally-induced aggregates of MS migrate in ER gradients and occur in isolated glyoxysomes. These data indicate that ER is not involved in synthesis or modification of cottonseed MS prior to its import into the glyoxysomal matrix.

  6. Dihydrodipicolinate synthase from Thermotoga maritima.

    PubMed

    Pearce, F Grant; Perugini, Matthew A; McKerchar, Hannah J; Gerrard, Juliet A

    2006-12-01

    DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 degrees C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage. PMID:16872276

  7. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning Fo portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil γ-subunit. This acts as a rotating `cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the γ shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility - supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump - can also be explained by our model.

  8. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    DOEpatents

    Croteau, Rodney Bruce; Crock, John E.

    2005-01-25

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  9. Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

    1999-01-01

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  10. Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

    PubMed Central

    Niyogi, K K; Fink, G R

    1992-01-01

    Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes. PMID:1392592

  11. Nitric oxide synthases: structure, function and inhibition.

    PubMed Central

    Alderton, W K; Cooper, C E; Knowles, R G

    2001-01-01

    This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated. PMID:11463332

  12. Tertiary model of a plant cellulose synthase

    PubMed Central

    Sethaphong, Latsavongsakda; Haigler, Candace H.; Kubicki, James D.; Zimmer, Jochen; Bonetta, Dario; DeBolt, Seth; Yingling, Yaroslava G.

    2013-01-01

    A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose–synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs. PMID:23592721

  13. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

    PubMed

    Schönitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M

    2011-12-01

    Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

  14. Characterization of a sabinene synthase gene from rough lemon (Citrus jambhiri).

    PubMed

    Kohzaki, Keisuke; Gomi, Kenji; Yamasaki-Kokudo, Yumiko; Ozawa, Rika; Takabayashi, Junji; Akimitsu, Kazuya

    2009-10-15

    We previously isolated two putative monoterpene synthase genes, RlemTPS1 and RlemTPS2, from rough lemon (Citrus jambhiri) and showed that gene expression of RlemTPS2 was induced by microbial attack. The protein product of RlemTPS2 was obtained using a prokaryotic expression system, and GC and GC-MS of monoterpene synthesis by RlemTPS2 determined that RlemTPS2 encodes a sabinene synthase. Sabinene has antifungal activity toward Alternaria alternata. Furthermore, site-directed mutagenesis identified one amino acid, Ile, located at the front of the metal ion binding motif as an important residue for the product specificity of sabinene synthase.

  15. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway

    PubMed Central

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. PMID:26971881

  16. Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua

    PubMed Central

    Ruan, Ju-Xin; Li, Jian-Xu; Fang, Xin; Wang, Ling-Jian; Hu, Wen-Li; Chen, Xiao-Ya; Yang, Chang-Qing

    2016-01-01

    Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography–mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant–environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant. PMID:27242840

  17. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    SciTech Connect

    Schoenitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  18. Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua.

    PubMed

    Ruan, Ju-Xin; Li, Jian-Xu; Fang, Xin; Wang, Ling-Jian; Hu, Wen-Li; Chen, Xiao-Ya; Yang, Chang-Qing

    2016-01-01

    Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg(2+) and Mn(2+) were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn(2+) for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant. PMID:27242840

  19. Terpene synthases are widely distributed in bacteria

    PubMed Central

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  20. Identification and characterization of a second isogene encoding γ-terpinene synthase in Thymus caespititius.

    PubMed

    Mendes, Marta D; Barroso, José G; Oliveira, M Margarida; Trindade, Helena

    2014-07-15

    Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high polymorphism. They consist mostly of mono- and sesquiterpene, some of them with interest for the pharmaceutical and food industries. The search for terpene synthase genes was performed in three in vitro T. caespititius genotypes. For these plants, the expression of a previously described γ-terpinene synthase gene, Tctps2, was confirmed, occurring concomitantly with a new gene encoding an enzyme with similar activity, named Thymus caespititius terpene synthase 4 (Tctps4). The two isogenes were isolated and functionally characterized in the three plant genotypes. Alignment of the two Tctps revealed a transit peptide much shorter in Tctps4 than in Tctps2 (3-4 amino acids instead of 47). The Tctps4 open reading frame is shorter than Tctps2 (1665 bp versus 1794 bp). The amino acid sequence of both γ-terpinene synthases shared an 88% pairwise identity. The fact that T. caespititius carries two isogenes for γ-terpinene synthases, suggests gene duplication along the evolutionary process, followed by mutations leading to the differentiation of both genes. These mutations didn't compromise protein activity. A high accumulation of transcripts from both genes was found in shoots of in vitro plantlets, while in roots they could not be detected. Still, γ-terpinene levels in aerial parts were reduced, probably due to fast conversion into carvacrol and thymol, the main components from T. caespititius essential oils. This study is a contribution to the identification of terpene synthase genes in Lamiaceae.

  1. Primary structure of a cerulenin-binding. beta. -ketoacyl-(acyl carrier protein) synthase from barley chloroplasts

    SciTech Connect

    Siggaard-Andersen, M.; Kauppinen, S. ); von Wettstein-Knowles, P. Univ. of Copenhagen )

    1991-05-15

    The radioactively labeled {beta}-ketoacyl thioester synthase inhibitor ({sup 3}H)cerulenin was used to tag three dimeric barley chloroplast proteins ({alpha}{alpha}, {alpha}{beta}, and {beta}{beta}) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the {beta} subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the {beta}-ketoacyl-(acyl carrier protein) (ACP) synthase I (3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41) of Escherichia coli. Under analogous experimental conditions ({sup 3}H)cerulenin tagged a single dimeric protein from spinach chloroplasts.

  2. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

  3. Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy.

    PubMed Central

    Mehmann, B; Brunner, I; Braus, G H

    1994-01-01

    DNA sequences of single-copy genes coding for chitin synthases (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase; EC 2.4.1.16) were used to characterize ectomycorrhizal fungi. Degenerate primers deduced from short, completely conserved amino acid stretches flanking a region of about 200 amino acids of zymogenic chitin synthases allowed the amplification of DNA fragments of several members of this gene family. Different DNA band patterns were obtained from basidiomycetes because of variation in the number and length of amplified fragments. Cloning and sequencing of the most prominent DNA fragments revealed that these differences were due to various introns at conserved positions. The presence of introns in basidiomycetous fungi therefore has a potential use in identification of genera by analyzing PCR-generated DNA fragment patterns. Analyses of the nucleotide sequences of cloned fragments revealed variations in nucleotide sequences from 4 to 45%. By comparison of the deduced amino acid sequences, the majority of the DNA fragments were identified as members of genes for chitin synthase class II. The deduced amino acid sequences from species of the same genus differed only in one amino acid residue, whereas identity between the amino acid sequences of ascomycetous and basidiomycetous fungi within the same taxonomic class was found to be approximately 43 to 66%. Phylogenetic analysis of the amino acid sequence of class II chitin synthase-encoding gene fragments by using parsimony confirmed the current taxonomic groupings. In addition, our data revealed a fourth class of putative zymogenic chitin synthesis. Images PMID:7944356

  4. Nuclear genetic defects of mitochondrial ATP synthase.

    PubMed

    Hejzlarová, K; Mráček, T; Vrbacký, M; Kaplanová, V; Karbanová, V; Nůsková, H; Pecina, P; Houštěk, J

    2014-01-01

    Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis.

  5. An investigation into eukaryotic pseudouridine synthases.

    PubMed

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  6. 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

  7. Exploring biosynthetic diversity with trichodiene synthase.

    PubMed

    Vedula, L Sangeetha; Zhao, Yuxin; Coates, Robert M; Koyama, Tanetoshi; Cane, David E; Christianson, David W

    2007-10-15

    Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP(i)). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.85A resolution X-ray crystal structure of the complex with Mg(2+) (3)-PP(i) and the benzyl triethylammonium cation, which is a bulkier mimic of the bisabolyl carbocation intermediate in catalysis. Taken together, these findings show that the active site of trichodiene synthase is sufficiently flexible to accommodate bulkier and electronically-diverse substrates and intermediates, which could indicate additional potential for the biosynthetic utility of this terpenoid cyclase. PMID:17678871

  8. Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

    PubMed

    Culbertson, Justin E; Chung, Dong hee; Ziebart, Kristin T; Espiritu, Eduardo; Toney, Michael D

    2015-04-14

    The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS. PMID:25710100

  9. Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699

    SciTech Connect

    Greenspan, M.D.; Yudkovitz, J.B.; Lo, C.Y.L.; Chen, J.S.; Alberts, A.W.; Hunt, V.M.; Chang, M.N.; Yang, S.S.; Thompson, K.L.; Chiang, Y.C.P.; Chabala, J.C.

    1987-11-01

    A ..beta..-lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase from rat liver. The structure of this ..beta..-lactone, termed L-659,699, is (E,E)-11-(3-hydroxymethyl)-4-oxo-2-oxytanyl)-3,5,7-trimethyl-2,4-undecadienenoic acid. A partially purified preparation of cytoplasmic HMG-CoA synthase from rat liver was inhibited by L-659,699 with an IC/sub 50/ of 0.12 ..mu..M. The enzymes HMG-CoA reductase, ..beta..-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, an fatty acid synthase were not inhibited to any extent by this compound. In cultured Hep G2 cells, the compound inhibited the incorporation of (/sup 14/C)acetate into sterols with an IC/sub 50/ of 6 ..mu..M, while incorporation of (/sup 3/H)mevalonate into sterols in these cells was not affected. The activity of HMG-CoA reductase in the cultured Hep G2 cells was induced in a dose-dependent manner by incubation with L-659,699. A 37-fold increase in reductase was observed after a 24-hr incubation with 62 ..mu..M L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed.

  10. Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.

    PubMed

    Picaud, Sarah; Olsson, Mikael E; Brodelius, Maria; Brodelius, Peter E

    2006-08-01

    A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis. PMID:16839518

  11. Biochemical and Structural Study of the Atypical Acyltransferase Domain from the Mycobacterial Polyketide Synthase Pks13*

    PubMed Central

    Bergeret, Fabien; Gavalda, Sabine; Chalut, Christian; Malaga, Wladimir; Quémard, Annaïk; Pedelacq, Jean-Denis; Daffé, Mamadou; Guilhot, Christophe; Mourey, Lionel; Bon, Cécile

    2012-01-01

    Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction. PMID:22825853

  12. Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog.

    PubMed Central

    Watanabe, K; Fujii, Y; Nakayama, K; Ohkubo, H; Kuramitsu, S; Kagamiyama, H; Nakanishi, S; Hayaishi, O

    1988-01-01

    Cloned cDNA sequences specific for prostaglandin F (PGF) synthase have been isolated from a cDNA library of bovine lung mRNA sequences. Nucleotide-sequence analyses of cloned cDNA inserts have revealed that PGF synthase consists of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a Mr of 36,666. The sequence analysis indicates that bovine lung PGF synthase shows 62% identical plus conservative substitutions compared with human liver aldehyde reductase [Wermuth, B., Omar, A., Forster, A., Francesco, C., Wolf, M., Wartburg, J.P., Bullock, B. & Gabbay, K.H. (1987) in Enzymology and Molecular Biology of Carbonyl Metabolism: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase, eds. Weiner, H. & Flynn, T.G. (Liss, New York), pp. 297-307], which is similar to PGF synthase in molecular weight and substrate specificity. However, comparison of the amino acid sequence of PGF synthase with the National Biomedical Research Foundation protein data base reveals that the sequences of 225 amino acids from C termini of epsilon-crystallin of the European common frog (Rana temporaria) [Tomarev, S.I., Zinovieva, R.D., Dolgilevich, S.M., Luchin, S.V., Krayev, A.S., Skryabin, K.G. & Gause, G.G. (1984) FEBS Lett. 171, 297-302] and of PGF synthase show 77% identical and conservative substitutions without deletions/additions. The result suggests that European common frog lens epsilon-crystallin is identical to bovine lung PGF synthase. Images PMID:2829166

  13. Genetic characterization of the poly(hydroxyalkanoate) synthases of various Pseudomonas oleovorans strains.

    PubMed

    Solaiman, Daniel K Y; Ashby, Richard D

    2005-06-01

    We identified the poly(hydroxyalkanoate) synthase (PHAS) genes of three strains of Pseudomonas oleovorans by using polymerase chain reaction (PCR)-based detection methods. P. oleovorans NRRL B-14682 contains Class I PHA synthase gene (phaC), NRRL B-14683 harbors Class II phaC1 and phaC2 genes, and NRRL B-778 contain both the Class I and II PHA synthase genes. Inverse-PCR and chromosomal walking techniques were employed to obtain the complete sequences of the Class I phaCs of NRRL B-778 (phbC778; 1698 bps) and B-14682 (phbC14682; 1899 bps). BLAST search indicated that these genes are new and had not been previously cloned. The gene product of phbC778 (i.e., PhbC778; 566 amino acid residues) is homologous to the Class I PHA synthases of Pseudomonas sp. HJ-2 and Pseudomonas sp. strain 61-3, and that of phbC14682 (PhbC14682; 632 amino acids) is homologous to PHAS of Delftia acidovorans. The PhbC14682 contains an extra sequence of 33 amino acids in its conserved alpha/beta-hydrolase domain, making it only the second Class I PHA synthase found to contain this cellular proteolytic sequence. Consistent with their Pseudomonas origin, the codon-usage profiles of PhbC778 and PhbC14682 are similar to those of Pseudomonas Class II PHASs. These new Pseudomonas Class I phbC genes provide valuable addition to the gene pool for the construction of novel PHASs through gene shuffling. PMID:15968501

  14. ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity1[OPEN

    PubMed Central

    Kimberlin, Athen N.; Chen, Ming; Dunn, Teresa M.

    2016-01-01

    Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network. PMID:27506241

  15. Optimization of heterologous production of the polyketide 6-MSA in Saccharomyces cerevisiae.

    PubMed

    Wattanachaisaereekul, Songsak; Lantz, Anna Eliasson; Nielsen, Michael Lynge; Andrésson, Olafur S; Nielsen, Jens

    2007-07-01

    Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.

  16. Cellulose Synthase Complexes: Composition and Regulation

    PubMed Central

    Lei, Lei; Li, Shundai; Gu, Ying

    2012-01-01

    Live cell imaging has greatly advanced our knowledge on the molecular mechanism by which cellulose is deposited. Both the actin and microtubule cytoskeleton are involved in assuring the proper distribution, organization, and dynamics of cellulose synthase complexes (CSCs). This review is an update on the most recent progress on the characterization of the composition, regulation, and trafficking of CSCs. With the newly identified cellulose synthase interactive protein 1 (CSI1) on hand, we begin to unveil the mystery of an intimate relationship between cellulose microfibrils and microtubules. PMID:22639663

  17. Chromosomal localization of the human and mouse hyaluronan synthase genes

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Seldin, M.F.

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  18. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    SciTech Connect

    Napolitano, H. B.; Sculaccio, S. A.; Thiemann, O. H.; Oliva, G.

    2005-01-01

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa. Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P2{sub 1}2{sub 1}2 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 Å. The crystals diffract to a maximum resolution of 3.3 Å and a complete data set to 3.5 Å resolution was collected and analysed.

  19. The crystal structure of human GDP-L-fucose synthase.

    PubMed

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  20. Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages.

    PubMed Central

    Jorens, P. G.; van Overveld, F. J.; Bult, H.; Vermeire, P. A.; Herman, A. G.

    1992-01-01

    1. The synthesis of nitrite and citrulline from L-arginine by immune-stimulated rat alveolar macrophages and the modulation of this synthesis were studied. 2,4-Diamino-6-hydroxypyrimidine (DAHP), 6R-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-sepiapterin were potent inhibitors of the recombinant interferon-gamma induced production of nitrogen oxides in intact cultured cells with I50 values for BH4 and L-sepiapterin of approximately 10 microM. They were equally effective in inhibiting the induced production of citrulline. This inhibitory effect was concentration-dependent for all three modulators investigated. 2. The inhibitory effects were not dependent on incubation times of either 24 or 48 h, on the immune-stimulus used (lipopolysaccharide, interferon-gamma), or whether these stimuli were added during or after the induction period. 3. Pterin-6-carboxylic acid (PCA), which cannot be converted into BH4, and methotrexate (MTX), which inhibits dihydrofolatereductase but not de novo biosynthesis of BH4, did not change the production of nitrite. 4. The data indicate that DAHP, an inhibitor of the de novo biosynthesis of the co-factor BH4, blocks the nitric oxide synthase activity in intact cells. Since the pterins BH4 and L-sepiapterin blocked the L-arginine dependent production of nitrite and citrulline, the activity of nitric oxide synthase in phagocytic cells may be regulated by metabolic endproducts of the de novo biosynthesis of BH4. PMID:1281717

  1. Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins.

    PubMed Central

    Reiser, C O; Lanz, T; Hofmann, F; Hofer, G; Rupprecht, H D; Goppelt-Struebe, M

    1998-01-01

    During inflammatory processes of the kidney, lesions of the glomerulus lead to aggregation of thrombocytes and infiltration of macrophages, which can release bioactive mediators. One of these important signalling molecules is lysophosphatidic acid (LPA). Incubation of rat mesangial cells with LPA induced mRNA and protein expression of the early-response genes pghs-2 (for prostaglandin G/H synthase-2/cyclo-oxygenase-2) and egr-1. As shown by antisense experiments, induction of egr-1 was related to the strong mitogenic effect of LPA. LPA-mediated gene expression was inhibited by pertussis toxin, indicating coupling to G-proteins of the Gi family. Specific inhibition of proteins of the small G-protein subfamily Rho with toxin B from Clostridium difficile led to changes in mesangial cell morphology without induction of apoptosis. LPA-mediated expression of pghs-2 and egr-1 was reduced to base-line levels by toxin B, indicating a role for Rho proteins in LPA-mediated gene induction. Of the two mitogen-activated protein kinase (MAPK) pathways investigated, the MAPK kinase-extracellular signal-regulated kinase pathway was involved in the induction of both pghs-2 and egr-1 mRNA expression, as shown by the inhibitory effect of PD98059. Activation of the MAPK p38, however, was only related to pghs-2 expression, whereas egr-1 expression was not affected by treatment of mesangial cells with the specific inhibitor SB203580. Taken together our data provide evidence that LPA-mediated activation of MAPK kinase and Rho proteins leads to the induction of the functionally distinct early-response genes pghs-2 and egr-1, whereas activation of MAPK p38 revealed considerable differences between the regulation of these two genes. PMID:9494074

  2. Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat.

    PubMed

    Curry, T E; Bryant, C; Clark, M R

    1991-05-01

    The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.

    PubMed

    Sato, Tsutomu; Yamaga, Hiroaki; Kashima, Shoji; Murata, Yusuke; Shinada, Tetsuro; Nakano, Chiaki; Hoshino, Tsutomu

    2013-05-10

    Basic enzyme: The tetraprenyl-β-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-β-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. PMID:23554321

  4. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    PubMed

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  5. Cloning and analysis of valerophenone synthase gene expressed specifically in lupulin gland of hop (Humulus lupulus L.).

    PubMed

    Okada, Y; Ito, K

    2001-01-01

    Resin and essential oil derived from hop (Humulus lupulus L.) cones are very important compounds for beer brewing, and they specifically accumulate in the lupulin gland of hop cones. In order to identify the genes responsible for the biosynthetic pathway of these compounds and use the identified genes for hop breeding using Marker Assisted Selection and transformation techniques, genes expressed specifically in the lupulin gland were cloned and sequenced. One of them was suggested to be similar to the chalcone synthase gene from the DNA sequence. The translation product of the gene had the activity of valerophenone synthase, which catalyzes a part of the synthesis reaction of alpha-acid and beta-acid. Northern analysis showed that the valerophenone synthase gene seemed to be expressed specifically in the lupulin gland.

  6. Cloning and analysis of valerophenone synthase gene expressed specifically in lupulin gland of hop (Humulus lupulus L.).

    PubMed

    Okada, Y; Ito, K

    2001-01-01

    Resin and essential oil derived from hop (Humulus lupulus L.) cones are very important compounds for beer brewing, and they specifically accumulate in the lupulin gland of hop cones. In order to identify the genes responsible for the biosynthetic pathway of these compounds and use the identified genes for hop breeding using Marker Assisted Selection and transformation techniques, genes expressed specifically in the lupulin gland were cloned and sequenced. One of them was suggested to be similar to the chalcone synthase gene from the DNA sequence. The translation product of the gene had the activity of valerophenone synthase, which catalyzes a part of the synthesis reaction of alpha-acid and beta-acid. Northern analysis showed that the valerophenone synthase gene seemed to be expressed specifically in the lupulin gland. PMID:11272819

  7. Suppression of vascular smooth muscle cell responses induced by TNF-α in GM3 synthase gene transfected cells.

    PubMed

    Park, Sung-Suk; Kim, Wun-Jae; Moon, Sung-Kwon

    2011-01-01

    The natural accumulation of ganglioside GM3 (N-glycolylneuraminic acid) on atherosclerotic lesions is a common theory. The present study is the first to examine the effects of the GM3 synthase gene on the responses of vascular smooth muscle cells (VSMC) to tumor necrosis factor-α (TNF-α). We found that overexpression of the GM3 synthase gene inhibited DNA synthesis and ERK1/2 activity induced by TNF-α in VSMC, whereas the basal levels of DNA synthesis and ERK1/2 activity remained unchanged. In addition, GM3 synthase gene transfectants significantly reduced the migration and invasion of VSMC following TNF-α treatment, compared with empty vector transfectants. Furthermore, TNF-α-induced matrix metalloproteinase-9 (MMP-9) expression and promoter activity were also decreased in GM3 synthase gene transfectants. GM3 synthase gene expression markedly suppressed the TNF-α-stimulated transcriptional activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), which are the controlling factors of MMP-9 expression. Consistent with these results, the addition of anti-GM3 antibody into the GM3 synthase gene transfectants blocked inhibition of DNA synthesis, ERK1/2 activity, migration and invasion. Finally, GM3 synthase gene transfectants treated with anti-GM3 antibody reversed the suppression of MMP-9 expression by reducing AP-1 and NF-κB binding activity. These results suggest regulatory roles for the GM3 synthase gene in VSMC proliferation and migration during the formation of atherosclerotic lesions.

  8. Lessons from 455 Fusarium polyketide synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In fungi, polyketide synthases (PKSs) synthesize a structurally diverse array of secondary metabolites (SMs) with a range of biological activities. The most studied SMs are toxic to animals and/or plants, alter plant growth, have beneficial pharmaceutical activities, and/or are brightly colored pigm...

  9. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering.

    PubMed

    Zerbe, Philipp; Bohlmann, Jörg

    2015-07-01

    Plants produce thousands of diterpenoid natural products; some of which are of significant industrial value as biobased pharmaceuticals (taxol), fragrances (sclareol), food additives (steviosides), and commodity chemicals (diterpene resin acids). In nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. While some diTPSs also form oxygenated compounds, more commonly, oxygenation is achieved by cytochrome P450-dependent mono-oxygenases. Recent genome-, transcriptome-, and metabolome-guided gene discovery and enzyme characterization identified novel diTPS functions that form the core of complex modular pathway systems. Insights into diterpene metabolism may translate into the development of new bioengineered microbial and plant-based production systems.

  10. Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase

    SciTech Connect

    Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephan W.; Lee, Richard E.

    2010-01-12

    Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

  11. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    SciTech Connect

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W.

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  12. Catalysis and sulfa drug resistance in dihydropteroate synthase.

    PubMed

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M Brett; Ferreira, Antonio M; Lee, Richard E; Bashford, Donald; White, Stephen W

    2012-03-01

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  13. Catalytic Roles of βLys87 in Tryptophan Synthase: 15N Solid State NMR Studies

    PubMed Central

    Caulkins, Bethany G.; Yang, Chen; Hilario, Eduardo; Fan, Li; Dunn, Michael F.; Mueller, Leonard J.

    2015-01-01

    The proposed mechanism for tryptophan synthase shows βLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4′ for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis). ε-15N-lysine TS was prepared to access the protonation state of βLys87 using 15N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the β-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. PMID:25688830

  14. Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp

    SciTech Connect

    Schendel, F.J.; August, P.R.; Anderson, C.R.; Flickinger, M.C. ); Hanson, R.S. )

    1992-01-01

    Acetate salts are emerging as potentially attractive bulk chemicals for a variety of environmental applications, for example, as catalysts to facilitate combustion of high-sulfur coal by electrical utilities and as the biodegradable noncorrosive highway deicing salt calcium magnesium acetate. The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (M{sub r}, 84,500) with a sub unit with an M{sub r} of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.

  15. The Primary Diterpene Synthase Products of Picea abies Levopimaradiene/Abietadiene Synthase (PaLAS) Are Epimers of a Thermally Unstable Diterpenol*

    PubMed Central

    Keeling, Christopher I.; Madilao, Lina L.; Zerbe, Philipp; Dullat, Harpreet K.; Bohlmann, Jörg

    2011-01-01

    The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC-MS of this and orthologous conifer diterpene synthases over the past 15 years suggested that these diterpenes may not be the initial enzyme assay products but are rather the products of dehydration of an unstable alcohol. We have identified epimers of the thermally unstable allylic tertiary alcohol 13-hydroxy-8(14)-abietene as the products of PaLAS. The identification of these compounds, not previously described in conifers, as the initial products of PaLAS has considerable implications for our understanding of the complexity of the biosynthetic pathway of the structurally diverse diterpene resin acids of conifer defense. PMID:21518766

  16. Re-Citrate Synthase from Clostridium kluyveri Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to Si-Citrate Synthase† ▿

    PubMed Central

    Li, Fuli; Hagemeier, Christoph H.; Seedorf, Henning; Gottschalk, Gerhard; Thauer, Rudolf K.

    2007-01-01

    The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity. PMID:17400742

  17. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  18. Haplofungins, new inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 II. Structure elucidation.

    PubMed

    Ohnuki, Takashi; Yano, Tatsuya; Takatsu, Toshio

    2009-10-01

    Eight new inositol phosphorylceramide synthase inhibitors: haplofungin A, B, C, D, E, F, G and H, were discovered in a culture broth of the fungus Lauriomyces bellulus SANK 26899. The planar structures for these haplofungins were elucidated by various spectroscopic analyses and a GC/MS analysis of their degradation products. All eight compounds were found to comprise an arabinonic acid moiety linked through an ester bond to a modified long alkyl chain.

  19. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning

    PubMed Central

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

    2014-01-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H+-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H+-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H+-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H+-ATP synthase as a target to prevent neuronal cell death. PMID:24521670

  20. Characterisation of a Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes.

    PubMed

    Frister, Thore; Hartwig, Steffen; Alemdar, Semra; Schnatz, Katharina; Thöns, Laura; Scheper, Thomas; Beutel, Sascha

    2015-08-01

    The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential. PMID:26100386

  1. Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.

    PubMed

    Funa, Nobutaka; Ozawa, Hiroki; Hirata, Aiko; Horinouchi, Sueharu

    2006-04-18

    Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an ars operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The ars operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.

  2. Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA.

    PubMed

    Huang, L; Ku, J; Pookanjanatavip, M; Gu, X; Wang, D; Greene, P J; Santi, D V

    1998-11-10

    Several putative Escherichia coli pseudouridine (Psi) synthases have been identified by iterative searching of genomic databases for ORFs homologous to known Psi synthases [Gustafsson et al. (1996) Nucleic Acids Res. 24, 3756-3762]. Of these, yceC and yfiI were proposed to encode Psi synthases which modify 23S rRNA. In the present work, yceC and yfiI were cloned and overexpressed in E. coli, and the encoded enzymes, YceC and YfiI, were purified to homogeneity. Both proteins converted Urd residues of rRNA to Psi, thus confirming their identities as Psi synthases. However, in in vitro experiments both enzymes extensively modified Urd residues of both 23S rRNA and 16S rRNA. Gene-disruption of yceCresulted in the absence of Psi modification at positions U955, 2504, and 2580 of 23S RNA, thus identifying these sites as in vivo targets for YceC. Likewise, yfiI disruption resulted in the absence of Psi modification at positions U1911, 1917, and possibly 1915 of 23S RNA. Disruption of yceC did not affect the growth under the conditions tested, whereas yfiI-disrupted cells showed a dramatic decrease in growth rate. Since YceC and YfiI hypermodify RNA in vitro, factors in addition to ribonucleotide sequence must contribute to the in vivo specificity of these enzymes.

  3. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

    PubMed

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

    2014-04-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

  4. A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase.

    PubMed

    Lee, Eun-Jin; Pontes, Mauricio H; Groisman, Eduardo A

    2013-07-01

    Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein.

  5. Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

    SciTech Connect

    Mogelson, S.; Lange, L.G.

    1984-08-28

    Fatty acyl ethyl esters arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of myocardium, as assayed by the rate of synthesis of ethyl (/sup 14/C)oleate from 0.4 mM (/sup 14/C)oleic acid and 0.2 M ethanol, was 31 nmol/ (g x h), and was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M0 separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was purified over 5000-fold to homogeneity. Gel electrophoresis showed a single polypeptide with M/sub r/ 26,000, and gel permeation chromatography under nondenaturing conditions indicated a M/sub r/ of 50,000 for the active enzyme. Kinetic analyses indicated that greatest rates of synthesis were observed with unsaturated octadecanoic fatty acid substrates. K/sub m/'s for these fatty acids were essentially identical and equal to 0.2 mM; substrate specificity resulted from varying K/sub m/'s for methanol, ethanol, 1-propanol, and 1-butanol, while V/sub max/ was constant at approximately 1.5 nmol/(mg x s). The amino acid analysis of this synthase distinguishes it from typical cholesterol esterases. When the enzyme is maximally active with respect to ethyl ester synthesis, it does not hydrolyze cholesterol oleate. Fatty acid ethyl esters are synthesized in myocardium primarily by a soluble dimeric enzyme comprised of two nearly identical subunits which esterifies free fatty acids with ethanol to produce a nonoxidative metabolite.

  6. Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth, Sphingolipid Metabolism, Programmed Cell Death, and Mycotoxin Resistance1[OPEN

    PubMed Central

    Luttgeharm, Kyle D.; Chen, Ming; Mehra, Amit; Cahoon, Rebecca E.; Markham, Jonathan E.; Cahoon, Edgar B.

    2015-01-01

    Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB1, whereas LOH1-overexpressing plants showed no increase in FB1 resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB1. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance

  7. Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1.

    PubMed

    Koeberle, Andreas; Northoff, Hinnak; Werz, Oliver

    2009-08-01

    Prostaglandin E(2) (PGE(2)) plays a crucial role in the apparent link between tumor growth and chronic inflammation. Cyclooxygenase (COX)-2 and microsomal PGE(2) synthase-1, which are overexpressed in many cancers, are functionally coupled and thus produce massive PGE(2) in various tumors. Curcumin, a polyphenolic beta-diketone from tumeric with anti-carcinogenic and anti-inflammatory activities, was shown to suppress PGE(2) formation and to block the expression of COX-2 and of microsomal PGE(2) synthase-1. Here, we identified microsomal PGE(2) synthase-1 as a molecular target of curcumin and we show that inhibition of microsomal PGE(2) synthase-1 activity is the predominant mechanism of curcumin to suppress PGE(2) biosynthesis. Curcumin reversibly inhibited the conversion of PGH(2) to PGE(2) by microsomal PGE(2) synthase-1 in microsomes of interleukin-1beta-stimulated A549 lung carcinoma cells with an IC(50) of 0.2 to 0.3 micromol/L. Closely related polyphenols (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic acid) failed in this respect, and isolated ovine COX-1 and human recombinant COX-2 were not inhibited by curcumin up to 30 micromol/L. In lipopolysaccharide-stimulated human whole blood, curcumin inhibited COX-2-derived PGE(2) formation from endogenous or from exogenous arachidonic acid, whereas the concomitant formation of COX-2-mediated 6-keto PGF(1)alpha and COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was suppressed only at significant higher concentrations. Based on the key function of PGE(2) in inflammation and carcinogenesis, inhibition of microsomal PGE(2) synthase-1 by curcumin provides a molecular basis for its anticarcinogenic and anti-inflammatory activities.

  8. Building-block selectivity of polyketide synthases.

    PubMed

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  9. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    PubMed

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  10. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  11. All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity[S

    PubMed Central

    Ding, Tingbo; Kabir, Inamul; Li, Yue; Lou, Caixia; Yazdanyar, Amirfarbod; Xu, Jiachen; Dong, Jibin; Zhou, Hongwen; Park, Taesik; Boutjdir, Mohamed; Li, Zhiqiang; Jiang, Xian-Cheng

    2015-01-01

    Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo. PMID:25605874

  12. Haplofungins, novel inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 I. Taxonomy, fermentation, isolation and biological activities.

    PubMed

    Ohnuki, Takashi; Yano, Tatsuya; Ono, Yasunori; Kozuma, Shiho; Suzuki, Toshihiro; Ogawa, Yasumasa; Takatsu, Toshio

    2009-10-01

    In the course of screening for antifungal agents, we have discovered eight novel compounds, haplofungin A, B, C, D, E, F, G and H, from a culture broth of the fungus strain Lauriomyces bellulus SANK 26899. Haplofungins are composed of an arabinonic acid moiety linked through an ester to a modified long alkyl chain and show potent inhibitory activities against fungal inositol phosphorylceramide (IPC) synthase. Haplofungin A inhibited the activity of IPC synthase from Saccharomyces cerevisiae with an IC(50) value of 0.0015 microg ml(-1). This inhibitor also suppressed the growth of Candida glabrata at the MIC value of 0.5 microg ml(-1).

  13. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  14. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  15. Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis).

    PubMed

    Schmiderer, Corinna; Grausgruber-Gröger, Sabine; Grassi, Paolo; Steinborn, Ralf; Novak, Johannes

    2010-07-01

    Common sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, composed mainly of the monoterpenes 1,8-cineole, alpha-thujone, beta-thujone and camphor, is responsible for some of these effects. Gibberellins regulate diverse physiological processes in plants, such as seed germination, shoot elongation and cell division. In this study, we analyzed the effect of exogenously applied plant growth regulators, namely gibberellic acid (GA(3)) and daminozide, on leaf morphology and essential oil formation of two leaf stages during the period of leaf expansion. Essential oil content increased with increasing levels of gibberellins and decreased when gibberellin biosynthesis was blocked with daminozide. With increasing levels of gibberellins, 1,8-cineole and camphor contents increased. Daminozide blocked the accumulation of alpha- and beta-thujone. GA(3) at the highest level applied also led to a significant decrease of alpha- and beta-thujone. Monoterpene synthases are a class of enzymes responsible for the first step in monoterpene biosynthesis, competing for the same substrate geranylpyrophosphate. The levels of gene expression of the three most important monoterpene synthases in sage were investigated, 1,8-cineole synthase leading directly to 1,8-cineole, (+)-sabinene synthase responsible for the first step in the formation of alpha- and beta-thujone, and (+)-bornyl diphosphate synthase, the first step in camphor biosynthesis. The foliar application of GA(3) increased, while daminozide significantly decreased gene expression of the monoterpene synthases. The amounts of two of the end products, 1,8-cineole and camphor, were directly correlated with the levels of gene expression of the respective monoterpene synthases, indicating transcriptional control, while the formation of alpha- and beta

  16. Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target.

    PubMed

    González-Ibarra, Joaquín; Milewski, Sławomir; Villagómez-Castro, Julio C; Cano-Canchola, Carmen; López-Romero, Everardo

    2010-02-01

    The first committed step of the biosynthetic pathway leading to uridine-5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is catalyzed by glucosamine-6-phosphate synthase (GlcN-6-P synthase), an enzyme proposed as a potential antifungal chemotherapy target. Here, we describe the purification and biochemical characterization of the native enzyme from the dimorphic pathogenic fungus Sporothrix schenckii. The availability of the pure protein facilitated its biochemical characterization. The enzyme exhibited subunit and native molecular masses of 79 and 350+/-5 kDa, respectively, suggesting a homotetrameric structure. Isoelectric point was 6.26 and K(m) values for fructose-6-phosphate and L-glutamine were 1.12+/-0.3 and 2.2+/-0.7 mM, respectively. Inhibition of activity by UDP-GlcNAc was enhanced by Glc-6-P and phosphorylation stimulated GlcN-6-P synthase activity without affecting the enzyme sensitivity to the aminosugar. A glutamine analogue, FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid] was a more potent inhibitor of activity than ADMP (2-Amino-2-deoxy-D-mannitol-6-phosphate) but the latter was a stronger inhibitor of growth in two culture media. To our knowledge, this is the first report on the purification and biochemical characterization of a non-recombinant GlcN-6-P synthase from a true dimorphic fungus. Inhibition of enzyme activity and fungal growth by specific inhibitors of GlcN-6-P synthase strongly reinforces the role of this enzyme as a potential target for antifungal chemotherapy. PMID:19353425

  17. Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa.

    PubMed Central

    Donald, L J; Molgat, G F; Duckworth, H W

    1989-01-01

    The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted. Images PMID:2507528

  18. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

    PubMed

    Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

    2014-09-01

    Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. PMID:24856135

  19. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

    PubMed

    Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

    2014-09-01

    Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

  20. The metabolite alpha-ketoglutarate extends lifespan by inhibiting the ATP synthase and TOR

    PubMed Central

    Chin, Randall M.; Fu, Xudong; Pai, Melody Y.; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Diep, Simon; Lomenick, Brett; Meli, Vijaykumar S.; Monsalve, Gabriela C.; Hu, Eileen; Whelan, Stephen A.; Wang, Jennifer X.; Jung, Gwanghyun; Solis, Gregory M.; Fazlollahi, Farbod; Kaweeteerawat, Chitrada; Quach, Austin; Nili, Mahta; Krall, Abby S.; Godwin, Hilary A.; Chang, Helena R.; Faull, Kym F.; Guo, Feng; Jiang, Meisheng; Trauger, Sunia A.; Saghatelian, Alan; Braas, Daniel; Christofk, Heather R.; Clarke, Catherine F.; Teitell, Michael A.; Petrascheck, Michael; Reue, Karen; Jung, Michael E.; Frand, Alison R.; Huang, Jing

    2014-01-01

    Metabolism and ageing are intimately linked. Compared to ad libitum feeding, dietary restriction (DR) or calorie restriction (CR) consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms1,2. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits3,4. Recently, several metabolites have been identified that modulate ageing5,6 with largely undefined molecular mechanisms. Here we show that the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (α-KG) extends the lifespan of adult C. elegans. ATP synthase subunit beta is identified as a novel binding protein of α-KG using a small-molecule target identification strategy called DARTS (drug affinity responsive target stability)7. The ATP synthase, also known as Complex V of the mitochondrial electron transport chain (ETC), is the main cellular energy-generating machinery and is highly conserved throughout evolution8,9. Although complete loss of mitochondrial function is detrimental, partial suppression of the ETC has been shown to extend C. elegans lifespan10–13. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit beta and is dependent on the target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased upon starvation and α-KG does not extend the lifespan of DR animals, indicating that α-KG is a key metabolite that mediates longevity by DR. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator, and DR in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases. PMID:24828042

  1. Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

    PubMed

    Manhas, Reetika; Gowri, Venkatraman Subramanian; Madhubala, Rentala

    2016-01-15

    The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different. PMID:26586914

  2. Identification and characterization of the Populus sucrose synthase gene family.

    PubMed

    An, Xinmin; Chen, Zhong; Wang, Jingcheng; Ye, Meixia; Ji, Lexiang; Wang, Jia; Liao, Weihua; Ma, Huandi

    2014-04-10

    In this study, we indentified 15 sucrose synthase (SS) genes in Populus and the results of RT-qPCR revealed that their expression patterns were constitutive and partially overlapping but diverse. The release of the most recent Populus genomic data in Phytozome v9.1 has revealed the largest SS gene family described to date, comprising 15 distinct members. This information will now enable the analysis of transcript expression profiles for those that have not been previously reported. Here, we performed a comprehensive analysis of SS genes in Populus by describing the gene structure, chromosomal location and phylogenetic relationship of each family member. A total of 15 putative SS gene members were identified in the Populus trichocarpa (Torr. & Gray) genome using the SS domain and amino acid sequences from Arabidopsis thaliana as a probe. A phylogenetic analysis indicated that the 15 members could be classified into four groups that fall into three major categories: dicots, monocots & dicots 1 (M & D 1), and monocots & dicots 2 (M & D 2). In addition, the 15 SS genes were found to be unevenly distributed on seven chromosomes. The two conserved domains (sucrose synthase and glycosyl transferase) were found in this family. Meanwhile, the expression profiles of all 15 gene members in seven different organs were investigated in Populus tomentosa (Carr.) by using RT-qPCR. Additional analysis indicated that the poplar SS gene family is also involved in response to water-deficit. The current study provides basic information that will assist in elucidating the functions of poplar SS family. PMID:24508272

  3. Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor

    SciTech Connect

    Chemler, Joseph A.; Buchholz, Tonia J.; Geders, Todd W.; Akey, David L.; Rath, Christopher M.; Chlipala, George E.; Smith, Janet L.; Sherman, David H.

    2012-08-10

    Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 {angstrom} germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.