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Sample records for synthetase methionine synthase

  1. Nucleotide variation at the methionine synthase locus in an ...

    African Journals Online (AJOL)

    Nucleotide variation at the methionine synthase (MetE) locus within and among populations of an endangered forest tree Fokienia hodginsii in Vietnam was investigated in the present study. A total of 12 populations were sampled across Vietnam. The length of the sequenced locus varied from 1567 to 1559 bp. A total of 42 ...

  2. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    Science.gov (United States)

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  3. [Methionine sulfoximine and phosphinothricin--glutamine synthetase inhibitors and activators and their herbicidal activity (A review)].

    Science.gov (United States)

    Evstigneeva, Z G; Solov'eva, N A; Sidel'nikova, L I

    2003-01-01

    Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairments of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in a concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.

  4. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    Science.gov (United States)

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  5. Insights into the reactivation of cobalamin-dependent methionine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Datta, Supratim; Pattridge, Katherine A.; Smith, Janet L.; Matthews, Rowena G.; (Michigan)

    2009-12-10

    Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every {approx}2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH{sup CT}) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH{sup CT} ({sub s-s}MetH{sup CT}) that offer further insight into the reactivation of MetH. The structure of {sub s-s}MetH{sup CT} with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of {sub s-s}MetH{sub CT} with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.

  6. Methionine synthase A2756G and reduced folate carrier1 A80G ...

    African Journals Online (AJOL)

    Aim of the study: To analyze the effect of methionine synthase (MTR) A2756G, and reduced folate carrier (RFC1) A80G gene polymorphisms on the maternal risk for DS. Patients: This study was conducted in the Medical Genetics Center, Ain-Shams University hospitals, on a total of 170 mothers of children, diagnosed with ...

  7. Methionine synthase A2756G and reduced folate carrier1 A80G ...

    African Journals Online (AJOL)

    Background: Polymorphisms of genes encoding enzymes involved in folate metabolism have long been hypothesized to be maternal risk factors for Down syndrome, however, results are conflicting and inconclusive. Aim of the study: To analyze the effect of methionine synthase (MTR) A2756G, and reduced folate carrier ...

  8. Quick guide to polyketide synthase and nonribosomal synthetase genes in Fusarium

    DEFF Research Database (Denmark)

    Hansen, Jørgen T.; Sørensen, Jens L.; Giese, Henriette

    2012-01-01

    for future polyketide synthases (PKSs) and nonribosomal peptides synthetases (NRPSs) nomenclature assignment and classification. Sequence similarities of the adenylation and ketosynthase domain sequences were used to group the identified NRPS and PKS genes. We present the current state of knowledge of PKS......Fusarium species produce a plethora of bioactive polyketides and nonribosomal peptides that give rise to health problems in animals and may have drug development potential. Using the genome sequences for Fusarium graminearum, F. oxysporum, F. solani and F. verticillioides we developed a framework...

  9. Overexpression of S-adenosyl-L-methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism.

    Science.gov (United States)

    Gong, Biao; Li, Xiu; VandenLangenberg, Kyle M; Wen, Dan; Sun, Shasha; Wei, Min; Li, Yan; Yang, Fengjuan; Shi, Qinghua; Wang, Xiufeng

    2014-08-01

    S-adenosyl-L-methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1-overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na(+) absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269.

    Science.gov (United States)

    Huang, Sha; Romanchuk, Gail; Pattridge, Katherine; Lesley, Scott A; Wilson, Ian A; Matthews, Rowena G; Ludwig, Martha

    2007-08-01

    The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome-wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin-dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E. coli MetH. The fourth module of E. coli MetH is required for reductive remethylation of the cob(II)alamin form of the cofactor and binds the methyl donor for this reactivation, S-adenosylmethionine (AdoMet). Measurements of the rates of methionine formation in the presence and absence of TM0269 and AdoMet demonstrate that both TM0269 and AdoMet are required for reactivation of the inactive cob(II)alamin form of TM0268. These activity measurements confirm the structure-based assignment of the function of the TM0269 gene product. In the presence of TM0269, AdoMet, and reductants, the measured activity of T. maritima MetH is maximal near 80 degrees C, where the specific activity of the purified protein is approximately 15% of that of E. coli methionine synthase (MetH) at 37 degrees C. Comparisons of the structures and sequences of TM0269 and the reactivation domain of E. coli MetH suggest that AdoMet may be bound somewhat differently by the homologous proteins. However, the conformation of a hairpin that is critical for cobalamin binding in E. coli MetH, which constitutes an essential structural element, is retained in the T. maritima reactivation protein despite striking divergence of the sequences.

  11. Purification of soluble cytochrome b5 as a component of the reductive activation of porcine methionine synthase.

    Science.gov (United States)

    Chen, Z; Banerjee, R

    1998-10-02

    In mammals, methionine synthase plays a central role in the detoxification of the rogue metabolite homocysteine. It catalyzes a transmethylation reaction in which a methyl group is transferred from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The vitamin B12 cofactor cobalamin plays a direct role in this reaction by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homocysteine). The reactivity of the cofactor intermediate cob(I)alamin renders the enzyme susceptible to oxidative damage. The oxidized enzyme may be returned to the catalytic turnover cycle via a reductive methylation reaction that requires S-adenosylmethionine as a methyl group donor, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methionine synthase. Two proteins are required for the transfer of electrons from NADPH, one of which is microsomal and the other cytoplasmic. The cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b5. It supports methionine synthase activity in the presence of NADPH and the microsomal component in a saturable manner. In addition, purified microsomal cytochrome P450 reductase and soluble cytochrome b5 reconstitute the activity of the porcine methionine synthase. Identification of soluble cytochrome b5 as a member of the reductive activation system for methionine synthase describes a function for this protein in non-erythrocyte cells. In erythrocytes, soluble cytochrome b5 functions in methemoglobin reduction. In addition, it identifies an additional locus in which genetic polymorphisms may play a role in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.

  12. Genomic insights into the distribution, genetic diversity and evolution of polyketide synthases and nonribosomal peptide synthetases.

    Science.gov (United States)

    Wang, Hao; Sivonen, Kaarina; Fewer, David P

    2015-12-01

    Polyketides and nonribosomal peptides are important secondary metabolites that exhibit enormous structural diversity, have many pharmaceutical applications, and include a number of clinically important drugs. These complex metabolites are most commonly synthesized on enzymatic assembly lines of polyketide synthases and nonribosomal peptide synthetases. Genome-mining studies making use of the recent explosion in the number of genome sequences have demonstrated unexpected enzymatic diversity and greatly expanded the known distribution of these enzyme systems across the three domains of life. The wealth of data now available suggests that genome-mining efforts will uncover new natural products, novel biosynthetic mechanisms, and shed light on the origin and evolution of these important enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Oxidative stress inactivates cobalamin-independent methionine synthase (MetE in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Elise R Hondorp

    2004-11-01

    Full Text Available In nature, Escherichia coli are exposed to harsh and non-ideal growth environments-nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE. We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert

  14. Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

    International Nuclear Information System (INIS)

    Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen; Liang, Yu-He; Su, Xiao-Dong

    2006-01-01

    Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni 2+ -chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P2 1 , with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°

  15. Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen; Liang, Yu-He, E-mail: liangyh@pku.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871 (China); Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871 (China)

    2006-10-01

    Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°.

  16. Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase.

    Science.gov (United States)

    Evans, John C; Huddler, Donald P; Hilgers, Mark T; Romanchuk, Gail; Matthews, Rowena G; Ludwig, Martha L

    2004-03-16

    B(12)-dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH(3)-H(4)folate). Here, we describe crystal structures of a fragment of MetH from Thermotoga maritima comprising the domains that bind Hcy and CH(3)-H(4)folate. These substrate-binding domains are (beta alpha)(8) barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH(3)-H(4)folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by approximately 50 A. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.

  17. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

    International Nuclear Information System (INIS)

    Prasannan, Priya; Suliman, Huda S.; Robertus, Jon D.

    2009-01-01

    Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.

  18. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Prasannan, Priya; Suliman, Huda S. [Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas, Austin, TX 78712 (United States); Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu [Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas, Austin, TX 78712 (United States)

    2009-05-15

    Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.

  19. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  20. Methionine synthase A2756G variation is associated with the risk of retinoblastoma in Iranian children.

    Science.gov (United States)

    Akbari, Mohammad Taghi; Naderi, Asieh; Saremi, Leila; Sayad, Arezou; Irani, Shiva; Ahani, Ali

    2015-12-01

    Association of epigenetic modifications with cancer has been widely studied. Gene-specific hypermethylation and global DNA hypomethylation are the most frequently observed patterns in great number of tumors. The methionine synthase (MTR) gene plays key role in maintaining adequate intracellular folate, methionine and normal homocysteine concentrations and, its polymorphism have been associated with the risk of retinoblastoma and other neoplasms. We evaluated the association of MTR A2756G polymorphism with the risk of retinoblastoma in an Iranian population. Totally, 150 retinoblastoma patients and 300 individuals with no family history of cancer as control were included in this study. Genotyping of the A2756G polymorphism was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using the restriction enzymes HaeIII. Our results showed that the "G" was the minor allele with a frequency of 31.7% and 20.3% in both retinoblastoma and control groups, respectively. The frequency of the 2756GG genotype (P=0.023) and 2756G allele (P=0.0001) were significantly higher in the patients than control group, respectively. Individual with the 2756GG genotype had a 2.99 fold increased risk for retinoblastoma. According to our results, the MTR A2756G polymorphism was associated with the risk of retinoblastoma in Iranian patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Detection of polyketide synthase and nonribosomal peptide synthetase biosynthetic genes from antimicrobial coral-associated actinomycetes.

    Science.gov (United States)

    Li, Jie; Dong, Jun-De; Yang, Jian; Luo, Xiong-Ming; Zhang, Si

    2014-10-01

    The diversity and properties of actinobacteria, predominant residents in coral holobionts, have been rarely documented. In this study, we aimed to explore the species diversity, antimicrobial activities and biosynthetic potential of culturable actinomycetes within the tissues of the scleractinian corals Porites lutea, Galaxea fascicularis and Acropora millepora from the South China Sea. A total of 70 strains representing 13 families and 15 genera of actinobacteria were isolated. The antimicrobial activity and biosynthetic potential of fifteen representative filamentous actinomycetes were estimated. Crude fermentation extracts of 6 strains exhibited comparable or greater activities against Vibrio alginolyticus than ciprofloxacin. Seven of the 15 actinomycetes strains possess type I polyketide synthases (PKS-I) and/or nonribosomal peptide synthetases (NRPS) genes. Nine tested strains possess type II polyketide synthases (PKS-II). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these PKS and NRPS gene screening positive strains belong to genera Nocardiopsis, Pseudonocardia, Streptomyces, Micromonospora, Amycolatopsis and Prauserella. One PKS-I and four NRPS fragments showed actinomycetes to produce bioactive molecules.

  2. Cobalamin-Independent Methionine Synthase Distribution and its Influence on Vitamin B12 Growth Requirements in Marine Diatoms

    Science.gov (United States)

    Ellis, K.; Cohen, N.; Moreno, C.; Marchetti, A.

    2016-02-01

    The requirement for cobalamin (vitamin B12) in microalgae is primarily a function of the type of methionine synthase present within their gene repertoires. This study validates this concept through analysis of the distribution of B12-independent methionine synthase in ecologically relevant diatom genera, including the closely related bloom-forming diatoms Pseudo-nitzschia and Fragilariopsis. Growth and gene expression analysis of the vitamin B12-requiring version of the methionine synthase enzyme, MetH, and the B12-independent version, MetE, demonstrate that it is the presence of the MetE gene which allows Fragilariopsis cylindrus to grow in the absence of B12, while P. granii's lack of a functional MetE gene means that it cannot survive without the vitamin. Through phylogenetic analysis, we further substantiate a lack of obvious grouping in MetE presence among diatom clades. In addition, we also show how this trend may have a biogeographical basis, particularly in High-Nutrient, Low-Chlorophyll (HNLC) regions such as the Southern Ocean where B12 concentrations may be consistently low. These results are paired with field experiments showing patterns of MetE and MetH gene expression in natural phytoplankton communities under a matrix of iron and B12 limitations in the HNLC NE Pacific. Our findings demonstrate the important role vitamins can play in diatom community dynamics within areas where vitamin supply may be variable and limiting.

  3. Methylenetetrahydrofolate reductase and methionine synthase polymorphisms and risk of bladder cancer in a Tunisian population.

    Science.gov (United States)

    Ouerhani, Slah; Oliveira, Elisabete; Marrakchi, Raja; Ben Slama, Mohamed R; Sfaxi, Mohamed; Ayed, Mohsen; Chebil, Mohamed; Amorim, António; El Gaaied, Amel Benammar; Prata, Maria João

    2007-07-01

    Folate insufficiency can induce carcinogenesis by decreasing DNA methylation. It is well known that DNA hypomethylation is a common feature in a number of cancers. Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are enzymes that play central roles in the folate metabolic pathway. Two common polymorphisms in the MTHFR gene (C677T and A1298C) and one in the MS gene (A2756G) are associated with decreased enzymatic activity. In this work, we have conducted a case-control study to assess the role of these three polymorphisms in bladder cancer development in North Tunisia. For MS A2756G, gene and genotypic distributions differed significantly between cases and controls. Furthermore, individuals carrying at least one copy of the variant allele presented a 2.33 times increased risk of developing bladder cancer than their control group [P = 0.001, odds ratio (OR) = 2.33; 95% confidence interval (CI) 1.34-4.06]. Statistically significant odds ratios were also found in patients heterozygous for MTHFR A1298C, who have a 1.8-fold higher risk of developing bladder cancer (P = 0.03, OR = 1.86; CI 95% 1.04-3.33). While the isolated polymorphism C677T did not appear to influence bladder cancer susceptibility, results suggest that it might act with an additive contribution determined by variation at MTHFR A1298C. Identical cumulative effect was detected for the MTHFR A1298C and MS 2756 genotypes. Patients harboring at least one mutant allele for each of the three positions analyzed showed a 4.76-fold increased risk of developing bladder cancer in comparison to their reference group (P = 0.02, OR = 4.76; CI 95% 1.26-17.98).

  4. Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia

    OpenAIRE

    Fofou-Caillierez, Ma'atem B.; Mrabet, Nadir T.; Chéry, Céline; Dreumont, Natacha; Flayac, Justine; Pupavac, Mihaela; Paoli, Justine; Alberto, Jean-Marc; Coelho, David; Camadro, Jean-Michel; Feillet, François; Watkins, David; Fowler, Brian; Rosenblatt, David S.; Guéant, Jean-Louis

    2017-01-01

    The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as ‘cblG-variant'. In four ‘cblG-variant' cases, we observed a decreased conversion of cy...

  5. A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Supratim; Koutmos, Markos; Pattridge, Katherine A.; Ludwig, Martha L.; Matthews, Rowena G. (Michigan)

    2008-07-08

    B{sub 12}-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.

  6. Cobalamin-independent methionine synthase (MetE: a face-to-face double barrel that evolved by gene duplication.

    Directory of Open Access Journals (Sweden)

    Robert Pejchal

    2005-02-01

    Full Text Available Cobalamin-independent methionine synthase (MetE catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH, both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (betaalpha(8 barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys(3Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E.Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  7. Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel that Evolved by Gene Duplication

    Energy Technology Data Exchange (ETDEWEB)

    Pejcha, Robert; Ludwig, Martha L. (Michigan)

    2010-03-08

    Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two ({beta}{alpha}){sub 8} barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys){sub 3}Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E {center_dot} Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  8. Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

    Science.gov (United States)

    Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

    2015-08-01

    Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Individualized supplementation of folic acid according to polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) reduced pregnant complications.

    Science.gov (United States)

    Li, Xiujuan; Jiang, Jing; Xu, Min; Xu, Mei; Yang, Yan; Lu, Wei; Yu, Xuemei; Ma, Jianlin; Pan, Jiakui

    2015-01-01

    This study aimed to detect the genotype distributions and allele frequencies of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and methionine synthase reductase (MTRR) A66G polymorphisms of pregnant women in Jiaodong region in China, and to investigate whether folic acid supplementation affect the pregnancy complications. A total of 7,812 pregnant women from the Jiaodong region in Shandong province in China. By using Taqman-MGB, 2,928 pregnant women (case group) were tested for the genotype distributions and allele frequencies of MTHFR C677T, A1298C and MTRR A66G polymorphisms. Folic acid metabolism ability was ranked at four levels and then pregnant women in different rank group were supplemented with different doses of folic acid. Their pregnancy complications were followed up and compared with 4,884 pregnant women without folic acid supplementation (control group) in the same hospital. The allele frequencies of MTHFR C677T were 49.1 and 50.9%; those of MTHFR A1298C were 80.2 and 19.8%, and those of MTRR A66G were 74.1 and 25.9%. After supplemented with folic acid, the complication rates in different age groups were significantly reduced, especially for gestational diabetes mellitus and hypertension. Periconceptional folic acid supplementation and healthcare following gene polymorphism testing may be a powerful measure to decrease congenital malformations. © 2015 S. Karger AG, Basel.

  10. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes.

    Science.gov (United States)

    Amoutzias, Grigoris D; Chaliotis, Anargyros; Mossialos, Dimitris

    2016-04-16

    Considering that 70% of our planet's surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

  11. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

    Directory of Open Access Journals (Sweden)

    Grigoris D. Amoutzias

    2016-04-01

    Full Text Available Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs and polyketides (PKs are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes and type-I polyketide synthases (PKSes-I, respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

  12. Evidence supporting distinct functions of three cytosolic glutamine synthetases and two NADH-glutamate synthases in rice.

    Science.gov (United States)

    Yamaya, Tomoyuki; Kusano, Miyako

    2014-10-01

    The functions of the three isoenzymes of cytosolic glutamine synthetase (GS1;1, GS1;2, and GS1;3) and two NADH-glutamate synthases (NADH-GOGAT1 and NADH-GOGAT2) in rice (Oryza sativa L.) were characterized using a reverse genetics approach and spatial expression of the corresponding genes. OsGS1;2 and OsNADH-GOGAT1 were mainly expressed in surface cells of rice roots in an NH4 (+)-dependent manner. Disruption of either gene by the insertion of endogenous retrotransposon Tos17 caused reduction in active tiller number and hence panicle number at harvest. Re-introduction of OsGS1;2 cDNA under the control of its own promoter into the knockout mutants successfully restored panicle number to wild-type levels. These results indicate that GS1;2 and NADH-GOGAT1 are important in the primary assimilation of NH4 (+) taken up by rice roots. OsGS1;1 and OsNADH-GOGAT2 were mainly expressed in vascular tissues of mature leaf blades. OsGS1;1 mutants showed severe reduction in growth rate and grain filling, whereas OsNADH-GOGAT2 mutants had marked reduction in spikelet number per panicle. Complementation of phenotypes seen in the OsGS1;1 mutant was successfully observed when OsGS1;1 was re-introduced. Thus, these two enzymes could be important in remobilization of nitrogen during natural senescence. Metabolite profiling data showed a crucial role of GS1;1 in coordinating metabolic balance in rice. Expression of OsGS1:3 was spikelet-specific, indicating that it is probably important in grain ripening and/or germination. Thus, these isoenzymes seem to possess distinct and non-overlapping functions and none was able to compensate for the individual function of another. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. Methylenetetrahydrofolate reductase 677C>T and methionine synthase 2756A>G mutations: no impact on survival, cognitive functioning, or cognitive decline in nonagenarians

    DEFF Research Database (Denmark)

    Bathum, Lise; von Bornemann Hjelmborg, Jacob; Christiansen, Lene

    2007-01-01

    BACKGROUND: Several reports have shown an association between homocysteine, cognitive functioning, and survival among the oldest-old. Two common polymorphisms in the genes coding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and methionine synthase (MTR 2756A>G) have an impact on plasma...... homocysteine level. METHODS: We examined the effect of the MTHFR 677C>T and MTR 2756A>G genotypes on baseline cognitive functioning, cognitive decline over 5 years measured in three assessments, and survival in a population-based cohort of 1581 nonagenarians. Cognitive functioning was assessed by using...

  14. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

    Science.gov (United States)

    Bito, Tomohiro; Yabuta, Yukinori; Ichiyanagi, Tsuyoshi; Kawano, Tsuyoshi; Watanabe, Fumio

    2014-01-01

    In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  15. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  16. Methylenetetrahydrofolate reductase 677C>T and methionine synthase 2756A>G mutations: no impact on survival, cognitive functioning, or cognitive decline in nonagenarians

    DEFF Research Database (Denmark)

    Bathum, Lise; von Bornemann Hjelmborg, Jacob; Christiansen, Lene

    2007-01-01

    BACKGROUND: Several reports have shown an association between homocysteine, cognitive functioning, and survival among the oldest-old. Two common polymorphisms in the genes coding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and methionine synthase (MTR 2756A>G) have an impact on plasma...... homocysteine level. METHODS: We examined the effect of the MTHFR 677C>T and MTR 2756A>G genotypes on baseline cognitive functioning, cognitive decline over 5 years measured in three assessments, and survival in a population-based cohort of 1581 nonagenarians. Cognitive functioning was assessed by using...... three assessments was investigated using regression models for the relationship between cognitive performance and genotype, age, sex, and social group and revealed no significant difference. Furthermore, the MTHFR 677T and MTR 2756A heterozygous and homozygous genotype had no significant impact...

  17. Association of Human Methionine Synthase-A2756G Transition With Prostate Cancer: A Case-Control Study and in Silico Analysis

    Directory of Open Access Journals (Sweden)

    Arezou Ebrahimi

    2017-07-01

    Full Text Available Methionine synthase (MTR is one of the key enzymes of folate pathway, which play a key role in the construction, repair, and methylation of DNA. In this study, an association of MTR A2756G gene transition with prostate cancer in men populations of Kashan-Iran was investigated by a case-control study and an in silico analysis. The 200 samples including 100 patients with prostate cancer, as case group and 100 healthy men, as control group included in this study. MTR-A2756G genotyping was performed by PCR-RFLP technique. Some in silico tools used to evaluate the effects of A2756G transition on the structure and function of MTR. Results showed that the AG genotype (OR: 2.4014, 95% CI: 1.3216-4.3636, P=0.0040, and GG genotype (OR: 3.6324, 95% CI: 1.2629-10.4475, P=0.0167 and G allele (OR: 2.0120, 95% CI: 1.3098-3.0905, P=0.0014 were associated with prostate cancer. In silico analysis showed that polymorphisms of the enzyme protein might change properties of MTR such as relative mutability and flexibility, which leads to alteration of stability and function of the enzyme. Based on the results, an MTR-A2756G polymorphism which changes activity and stability of the methionine synthase associated with prostate cancer in men. It is a preliminary study and is presenting data for future comprehensive study for making a clinical conclusion that this gene transition is a biomarker for susceptibility to prostate cancer.

  18. Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia.

    Science.gov (United States)

    Fofou-Caillierez, Ma'atem B; Mrabet, Nadir T; Chéry, Céline; Dreumont, Natacha; Flayac, Justine; Pupavac, Mihaela; Paoli, Justine; Alberto, Jean-Marc; Coelho, David; Camadro, Jean-Michel; Feillet, François; Watkins, David; Fowler, Brian; Rosenblatt, David S; Guéant, Jean-Louis

    2013-11-15

    The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin  (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.

  19. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  20. Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

    Directory of Open Access Journals (Sweden)

    Mostafa Waly

    2016-01-01

    Full Text Available The folate and cobalamin (Cbl- dependent enzyme methionine synthase (MS is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl or the combination of hydroxocobalamin (OHCbl and S-adenosylmethionine (SAM. OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH but could be rescued by provision of either glutathionylcobalamin (GSCbl or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

  1. Overexpression of S-Adenosyl-l-Methionine Synthetase 2 from Sugar Beet M14 Increased Arabidopsis Tolerance to Salt and Oxidative Stress

    Science.gov (United States)

    Ma, Chunquan; Wang, Yuguang; Gu, Dan; Nan, Jingdong; Chen, Sixue; Li, Haiying

    2017-01-01

    The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH). A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS), is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM), a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H2O2 tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H2O2 stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H2O2 tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet. PMID:28420190

  2. Overexpression of S-Adenosyl-l-Methionine Synthetase 2 from Sugar Beet M14 Increased Arabidopsis Tolerance to Salt and Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Chunquan Ma

    2017-04-01

    Full Text Available The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH. A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS, is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM, a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H2O2 tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H2O2 stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H2O2 tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet.

  3. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    Science.gov (United States)

    Zheng, Desen; Burr, Thomas J

    2016-02-01

    Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

  4. The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

    Science.gov (United States)

    Challis, Gregory L.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

    2012-01-01

    There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism. PMID:23028578

  5. Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species.

    Science.gov (United States)

    Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Takahashi-Nakaguchi, Azusa; Matsuzawa, Tetsuhiro; Suzuki, Ken-ichiro; Fujita, Nobuyuki; Gonoi, Tohru

    2014-04-30

    Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology. Draft genome sequences of Nocardia asteroides NBRC 15531(T), Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402(T), and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text. We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied

  6. Co-ordinate variations in methylmalonyl-CoA mutase and methionine synthase, and the cobalamin cofactors in human glioma cells during nitrous oxide exposure and the subsequent recovery phase.

    Science.gov (United States)

    Riedel, B; Fiskerstrand, T; Refsum, H; Ueland, P M

    1999-07-01

    We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to ordinate distribution of Cbl cofactors during depletion and repletion.

  7. Purification of 1-aminocyclopropane-1-carboxylate synthase from apple fruits using s-adenosyl [3,414C]-methionine (SAM) as a probe

    International Nuclear Information System (INIS)

    Yip, Wingkip; Dong, Jianguo; Yang, Shang Fa

    1989-01-01

    Tomato ACC synthase is inactivated by its substrate SAM, with the moiety of aminobutyrate being covalently linked to ACC synthase during the catalytic reactions. A partial purified ACC synthase (the catalytic activity 100 μmol/h·mg protein) from pellets of apple extract was incubated with [3,4 14 C] SAM. Only one radioactive peak was revealed in a C-4 reverse phase HPLC and one radioactive band on SDS-PAGE with an M.W. of 48 kDa. Apple ACC synthase in native form is resistant to V8, α-chromtrypsin and carboxylpeptidase A digestion, but effectively inactivated by trypsin and ficin, as demonstrated by both the activity assay and SAM labeling. The radioactive protein cut from the SDS-PAGE was injected to three mice, two of the mice showed responses to the protein in western blot analysis. The antibodies from mice is currently under characterization

  8. Progressive cerebral edema associated with high methionine levels and betaine therapy in a patient with cystathionine beta-synthase (CBS) deficiency.

    NARCIS (Netherlands)

    Yaghmai, R.; Kashani, A.H.; Geraghty, M.T.; Okoh, J.; Pomper, M.; Tangerman, A.; Wagner, C.; Stabler, S.P.; Allen, R.; Mudd, S.H.; Braverman, N.E.

    2002-01-01

    Cystathionine beta-synthase (CBS) deficiency, the most common form of homocystinuria, is an autosomal recessive inborn error of homocysteine metabolism. Treatment of B6-nonresponsive patients centers on lowering homocysteine and its disulfide derivatives (tHcy) by adherence to a

  9. Methionine metabolism after portacaval shunt in the rat

    International Nuclear Information System (INIS)

    Benjamin, L.E.; Steele, R.D.

    1985-01-01

    The effect of portacaval shunt (PCS) on methionine metabolism in the rat was investigated. Male Sprague-Dawley rats were subjected to PCS and maintained on an 18% casein diet. Growth curves of operated rats were similar to controls. PCS rats excreted more urinary 35 SO 4 and less [ 35 S]taurine than controls after intraperitoneal injection of 0.3 mmol/100 g [ 35 S]methionine or [ 35 S]cysteine. Total urinary taurine excretion was similar in PCS and control rats after a methionine or cysteine load; however, under basal conditions PCS rats had higher urinary taurine levels than controls, indicating that PCS may cause the taurine pool to be expanded. Hepatic methionine, S-adenosylmethionine, and cysteine pools were significantly decreased in PCS rats, while S-adenosylhomocysteine levels were unchanged. Relative rates of transsulfuration in PCS and control rats were studied by following the decrease in the 3 H-to- 35 S ratio in liver protein after injection of [methyl-3H]methionine and [ 35 S]methionine, and no difference in flux of 35 S from [ 35 S]methionine to [ 35 S]cysteine was found. Similarly, total hepatic activities of methionine adenosyltransferase, cystathionine synthase, and cystathionine gamma-lyase were unchanged in PCS rats. These results indicate that altered methionine metabolism in PCS rats is not explained by changes in conversion of methionine to cysteine via the transsulfuration pathway

  10. Engineering polyketide synthases and nonribosomal peptide synthetases.

    Science.gov (United States)

    Williams, Gavin J

    2013-08-01

    Naturally occurring polyketides and nonribosomal peptides with broad and potent biological activities continue to inspire the discovery of new and improved analogs. The biosynthetic apparatus responsible for the construction of these natural products has been the target of intensive protein engineering efforts. Traditionally, engineering has focused on substituting individual enzymatic domains or entire modules with those of different building block specificity, or by deleting various enzymatic functions, in an attempt to generate analogs. This review highlights strategies based on site-directed mutagenesis of substrate binding pockets, semi-rational mutagenesis, and whole-gene random mutagenesis to engineer the substrate specificity, activity, and protein interactions of polyketide and nonribosomal peptide biosynthetic machinery. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Non-ribosomal peptide synthetases: Identifying the cryptic gene ...

    Indian Academy of Sciences (India)

    2017-01-19

    Jan 19, 2017 ... Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically impor- tant antibiotics and siderophores. Each of the multiple modules of an ...

  12. Non-ribosomal peptide synthetases: Identifying the cryptic gene ...

    Indian Academy of Sciences (India)

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are themajor multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically importantantibiotics and siderophores. Each of the multiple modules of an NRPS activates a ...

  13. Cytosolic glutamine synthetase

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie; Eriksson, Ulf Dennis; Møller, Inge Skrumsager

    2014-01-01

    Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude...

  14. 4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate*

    Science.gov (United States)

    Perche-Letuvée, Phanélie; Kathirvelu, Velavan; Berggren, Gustav; Clemancey, Martin; Latour, Jean-Marc; Maurel, Vincent; Douki, Thierry; Armengaud, Jean; Mulliez, Etienne; Fontecave, Marc; Garcia-Serres, Ricardo; Gambarelli, Serge; Atta, Mohamed

    2012-01-01

    Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed. PMID:23043105

  15. Equilibria and partitioning of complexes in the S-adenosylmethionine synthetase reaction

    International Nuclear Information System (INIS)

    Markham, G.D.

    1987-01-01

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a reaction in which the [enzyme-ATP-methionine] complex reacts to form an intermediate [enzyme-AdoMet-PPPi] complex: hydrolysis of PPPi yields an [enzyme-AdoMet-PPi-Pi] complex from which AdoMet is the last product to dissociate. Analysis of reaction mixtures which were quenched with acid during turnover of E. coli AdoMet synthetase with saturating substrates containing [α - 32 P]ATP showed that PPPi is present in an amount corresponding to 45% of the total enzyme active sites, reflecting the portion of enzyme present in an [enzyme-AdoMet-PPPi] complex. Similar experiments in which excess pyrophosphatase was included (to hydrolyze PPi as it was released from AdoMet synthetase), showed that enzyme-bound PPi is present in an amount corresponding to 22% of the total AdoMet synthetase. The enzyme not present in complexes with PPPi or PPi is probably distributed between the [enzyme-ATP-methionine] and the [enzyme-AdoMet] complexes. AdoMet synthetase forms enzyme-bound 32 PPPi from added 32 PPi and Pi; the equilibrium constant [enzyme-AdoMet-PPi-Pi]/[enzyme-AdoMet-PPPi] is 2.0, greatly displaced from the equilibrium for hydrolysis of free PPPi. Since the ratio of enzyme-bound PPi to PPPi is 0.5 during the steady state, the PPPi hydrolysis step is not at equilibrium during turnover. Formation of [ 32 P]ATP from the [enzyme-AdoMet- 32 PPPi] complex was not detected

  16. Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

    Science.gov (United States)

    Pacana, Tommy; Cazanave, Sophie; Verdianelli, Aurora; Patel, Vaishali; Min, Hae-Ki; Mirshahi, Faridoddin; Quinlivan, Eoin; Sanyal, Arun J.

    2015-01-01

    Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, phomocysteine (25%, phomocysteine remethylation and transsulfuration, was depleted (45%, phomocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation. PMID:26322888

  17. The effects of enhanced methionine synthesis on amino acid and anthocyanin content of potato tubers

    Directory of Open Access Journals (Sweden)

    Bánfalvi Zsófia

    2008-06-01

    Full Text Available Abstract Background Potato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants. Results In higher plants, cystathionine γ-synthase (CgS is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of the Arabidopsis CgS (CgSΔ90, which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein the CgSΔ90 was co-transformed with the methionine-rich 15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgSΔ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL, the enzyme catalysing the first step of anthocyanin synthesis, were decreased. Conclusion Ectopic expression of CgSΔ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgSΔ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgSΔ90 and can not significantly improve the nutritional value of tubers. The level

  18. Rhizobitoxine-induced Chlorosis Occurs in Coincidence with Methionine Deficiency in Soybeans

    Science.gov (United States)

    Okazaki, Shin; Sugawara, Masayuki; Yuhashi, Ken-Ichi; Minamisawa, Kiwamu

    2007-01-01

    Background and Aims Rhizobitoxine, produced by the legume symbiont Bradyrhizobium elkanii, inhibits cystathionine-β-lyase (EC 4·4·1·8) in methionine biosynthesis and 1-aminocyclopropane-1-carboxylate synthase (ACC) in ethylene biosynthesis. Rhizobitoxine production by B. elkanii enhances nodulation of host legumes via the inhibition of ethylene synthesis, but causes foliar chlorosis in susceptible soybeans, though how it does so remains to be investigated. The aim of this study was to examine the physiological basis of rhizobitoxine-induced chlorosis in soybeans. Methods Wild-type B. elkanii and a rhizobitoxine-deficient mutant were inoculated in Glycine max ‘Lee’. Thirty days after inoculation, the upper parts of soybean shoots were analysed for amino acid contents. Chlorotic soybeans inoculated with wild-type B. elkanii were treated with methionine and ACC to assess the effects of the chemicals on the chlorosis. Key Results Chlorotic upper shoots of soybeans inoculated with wild-type B. elkanii had a lower methionine content and higher accumulation of the methionine precursors than those with the rhizobitoxine-deficient mutant. In addition, the foliar chlorosis was alleviated by the application of methionine. Conclusions Rhizobitoxine-induced chlorosis occurs in coincidence with methionine deficiency as a result of cystathione-β-lyase inhibition during methionine biosynthesis. PMID:17525098

  19. S-adenosyl-L-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Brown, James Mike [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States); Kuhlman, Christopher [Southwest Environmental Health Sciences Center, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, AZ (United States); Terneus, Marcus V. [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States); Labenski, Matthew T. [Southwest Environmental Health Sciences Center, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, AZ (United States); Lamyaithong, Andre Benja; Ball, John G. [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States); Lau, Serrine S. [Southwest Environmental Health Sciences Center, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, AZ (United States); Valentovic, Monica A., E-mail: Valentov@marshall.edu [Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Huntington, WV (United States)

    2014-12-01

    Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-L-methionine (SAMe) treatment 1 hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n = 5/group) were divided into the following groups and treated as indicated: Veh (15 ml/kg water, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg), and SAMe given 1 h after APAP (S + A). APAP toxicity was confirmed by an increase (p < 0.05) in plasma ALT (U/l) and liver weight/10 g body weight relative to the Veh, SAMe and S + A groups 4 h following APAP treatment. SAMe administered 1 h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10 g body weights were lower in the S + A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S + A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1. - Highlights: • Acetaminophen (APAP) toxicity protected by S-adenosylmethionine (SAMe) • 4-Hydroxynonenal adducted to sarcosine dehydrogenase • 4-Hydroxynonenal adducted to carbamoyl phosphate synthetase-1 • SAMe reduced APAP mediated CPS-1 mitochondrial leakage.

  20. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

  1. Hepatocytes explanted in the spleen preferentially express carbamoylphosphate synthetase rather than glutamine synthetase

    NARCIS (Netherlands)

    Lamers, W. H.; Been, W.; Charles, R.; Moorman, A. F.

    1990-01-01

    Urea cycle enzymes and glutamine synthetase are essential for NH3 detoxification and systemic pH homeostasis in mammals. Carbamoylphosphate synthetase, the first and flux-determining enzyme of the cycle, is found only in a large periportal compartment, and glutamine synthetase is found only in a

  2. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    International Nuclear Information System (INIS)

    Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

    1986-01-01

    The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6- 3 H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6- 3 H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells

  3. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur

    OpenAIRE

    Stipanuk, Martha H.; Ueki, Iori

    2010-01-01

    Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine β-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when meth...

  4. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  5. Transgenic tobacco plants having a higher level of methionine are more sensitive to oxidative stress.

    Science.gov (United States)

    Hacham, Yael; Matityahu, Ifat; Amir, Rachel

    2017-07-01

    Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ-SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild-type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress-related metabolites. Despite this, FA plants were more sensitive to short- and long-term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non-stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non-stress and stress conditions is important for enabling the plants to cope with stress conditions. © 2017 Scandinavian Plant Physiology Society.

  6. Physical studies of adenylosuccinate synthetase

    International Nuclear Information System (INIS)

    Bass, M.B.

    1987-01-01

    To determine the chemical mechanism of the reaction catalyzed by adenylosuccinate synthetase, positional isotope exchange studies were performed. Positional isotope exchange from the β-γ bridge to the β nonbridge position of [γ- 18 O]GTP was followed using 31 P NMR. The positional isotope exchange was found to occur in the presence of either IMP or IMP and succinate. The exchange did not occur in the presence of asparate. These results support a reaction mechanism which involves formation of a 6-phosphoryl-IMP intermediate with subsequent attack by aspartate to form adenylosuccinate as originally proposed by Lieberman in 1956. In order to resolve the NMR resonances for positional isotope exchange, it was necessary to find a chelator which would limit exchange broadening. trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid was found to be a suitable chelator at neutral and acidic pH. Studies of adenylosuccinate synthetase from Escherichia coli have been limited by the low concentrations of enzyme present in the cell and the difficulty in purifying the enzyme to homogeneity. Overproduction of the enzyme by cloning the purA gene into a runaway replication plasmid allowed the cells to produce a much higher concentration of enzyme. A new purification scheme is reported that takes advantage of the overproduced enzyme. Yields of 75 mg of homogeneous enzyme have been obtained from 76 g of E. coli cell paste

  7. Kinetic isotope effect studies of the S-adenosylmethionine synthetase reaction

    International Nuclear Information System (INIS)

    Markham, G.D.; Parkin, D.W.; Schramm, V.L.

    1986-01-01

    S-adenosylmethionine (AdoMet) synthetase catalyzes a unique substitution reaction at the 5' carbon of MgATP. Kinetic isotope effect (V/K) measurements have been used to investigate the mechanism of AdoMet synthetase from E. coli. Changes in 3 H/ 14 C ratios when AdoMet is formed from a mixture of either ([5'- 14 C]ATP and [5'- 12 C,1'- 3 H]ATP) or ([5'- 3 H]ATP and [5'- 1 H,1'- 14 C]ATP) were examined. The effects of varying the concentrations of the co-substrate methionine and the monovalent cation activator K + were investigated. Substitution of 14 C for 12 C at the 5' position of ATP yields a primary V/K kinetic isotope effect ( 12 C/ 14 C) of 1.128 +/- 0.004 at low K + and methionine concentrations. The observed isotope effect diminishes slightly to 1.107 +/- 0.003 when both K + and methionine are present at saturating concentrations, suggesting that MgATP has only a low commitment to catalysis from at conditions near Vmax. No secondary V/K 3 H isotope effect from [5'- 3 H]ATP was detected ( 1 H/ 3 H) = 0.997 +/- 0.003. The magnitude of the primary 14 C isotope effect and the small secondary 3 H effect demonstrate that AdoMet synthesis occurs with a S/sub N/ 2 transition state which is symmetric with respect to the sulfur nucleophile and the departing tripolyphosphate group

  8. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Roth, G.J.; Machuga, E.T.

    1982-01-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

  9. Increasing levels of dietary crystalline methionine affect plasma methionine profiles, ammonia excretion, and the expression of genes related to the hepatic intermediary metabolism in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Rolland, Marine; Skov, Peter Vilhelm; Larsen, Bodil Katrine

    2016-01-01

    . The diets were fed in excess for six weeks before three sampling campaigns carried out successively to elucidate (i) the hepatic expression of selected genes involved in lipid, glucose and amino acid metabolism; (ii) the postprandial ammonia excretion; and (iii) the postprandial plasma methionine...... concentrations. The transcript levels of enzymes involved in lipid metabolism (fatty acid synthase, glucose 6 phosphate dehydrogenase and carnitine palmitoyl transferase 1 a), gluconeogenesis (fructose-1,6-biphosphatase) and amino acid catabolism (alanine amino transferase and glutamate dehydrogenase) were......Strictly carnivorous fish with high requirements for dietary protein, such as rainbow trout (Oncorhynchus mykiss) are interesting models for studying the role of amino acids as key regulators of intermediary metabolism. Methionine is an essential amino acid for rainbow trout, and works...

  10. Genetics Home Reference: phosphoribosylpyrophosphate synthetase superactivity

    Science.gov (United States)

    ... purines available. In people with the more severe form of PRS superactivity , PRPS1 gene mutations change single protein building blocks ( amino acids ) in the PRPP synthetase 1 enzyme, resulting in ...

  11. Levels of Key Enzymes of Methionine-Homocysteine Metabolism in Preeclampsia

    Directory of Open Access Journals (Sweden)

    Alejandra Pérez-Sepúlveda

    2013-01-01

    Full Text Available Objective. To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM in the physiopathology of preeclampsia (PE. Methods. Plasma and placenta from pregnant women (32 controls and 16 PE patients were analyzed after informed consent. Protein was quantified by western blot. RNA was obtained with RNA purification kit and was quantified by reverse transcritase followed by real-time PCR (RT-qPCR. Identification of the C677T and A1298C methylenetetrahydrofolate reductase (MTHFR single-nucleotide polymorphisms (SNPs and A2756G methionine synthase (MTR SNP was performed using PCR followed by a high-resolution melting (HRM analysis. S-adenosyl methionine (SAM and S-adenosyl homocysteine (SAH were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS. The SNP association analysis was carried out using Fisher’s exact test. Statistical analysis was performed using a Mann-Whitney test. Results. RNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls. Protein, SAM, and SAH levels showed no significant difference between preeclamptic patients and controls. No statistical differences between controls and PE patients were observed with the different SNPs studied. Conclusion. The RNA expression of MTHFR and MTR is elevated in placentas of PE patients, highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in the physiopathology of this disease.

  12. Glutamine Synthetase: Localization Dictates Outcome

    Directory of Open Access Journals (Sweden)

    Alessandra Castegna

    2018-02-01

    Full Text Available Glutamine synthetase (GS is the adenosine triphosphate (ATP-dependent enzyme that catalyses the synthesis of glutamine by condensing ammonium to glutamate. In the circulatory system, glutamine carries ammonia from muscle and brain to the kidney and liver. In brain reduction of GS activity has been suggested as a mechanism mediating neurotoxicity in neurodegenerative disorders. In cancer, the delicate balance between glutamine synthesis and catabolism is a critical event. In vitro evidence, confirmed in vivo in some cases, suggests that reduced GS activity in cancer cells associates with a more invasive and aggressive phenotype. However, GS is known to be highly expressed in cells of the tumor microenvironment, such as fibroblasts, adipocytes and immune cells, and their ability to synthesize glutamine is responsible for the acquisition of protumoral phenotypes. This has opened a new window into the complex scenario of the tumor microenvironment, in which the balance of glutamine consumption versus glutamine synthesis influences cellular function. Since GS expression responds to glutamine starvation, a lower glutamine synthesizing power due to the absence of GS in cancer cells might apply a metabolic pressure on stromal cells. This event might push stroma towards a GS-high/protumoral phenotype. When referred to stromal cells, GS expression might acquire a ‘bad’ significance to the point that GS inhibition might be considered a conceivable strategy against cancer metastasis.

  13. Metabolic Regulation of Methionine Restriction in Diabetes.

    Science.gov (United States)

    Yin, Jie; Ren, Wenkai; Chen, Shuai; Li, Yuying; Han, Hui; Gao, Jing; Liu, Gang; Wu, Xin; Li, Tiejun; Kim, Sung Woo; Yin, Yulong

    2018-03-30

    Although the effects of dietary methionine restriction have been investigated in the physiology of aging and diseases related to oxidative stress, the relationship between methionine restriction and the development of metabolic disorders has not been explored extensively. This review summarizes studies of the possible involvement of dietary methionine restriction in improving insulin resistance, glucose homeostasis, oxidative stress, lipid metabolism, the pentose phosphate pathway, and inflammation, with an emphasis on the fibroblast growth factor 21 and protein phosphatase 2A signals and autophagy in diabetes. Diets deficient in methionine may be a useful nutritional strategy in patients with diabetes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. Radiolabeling of methionine containing proteins and peptides

    International Nuclear Information System (INIS)

    Garlick, R.K.; Jirousek, L.

    1986-01-01

    A process for radiolabeling methionine-containing peptides and proteins is disclosed. The process comprises the steps of oxidizing the protein or peptide, radiolabeling and reducing the radiolabeled protein or peptide. (author)

  15. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats

    DEFF Research Database (Denmark)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne

    2014-01-01

    of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine......Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains...... but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia....

  16. Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

    Directory of Open Access Journals (Sweden)

    Alessandro De Simone

    2016-06-01

    Full Text Available Genetic encoding of noncanonical amino acids (ncAAs through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS charges two tRNAs with Met: tRNAfMet initiates protein synthesis (starting AUG codon, whereas elongator tRNAMet participates in protein elongation (internal AUG codon(s. Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha and ethionine (Eth by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNAMet pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNAMet from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS / tRNAPyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution. This work is licensed under a Creative Commons Attribution 4.0 International

  17. 11C-methionine translocation in barley

    International Nuclear Information System (INIS)

    Nakanishi, Hiromi; Bughio, Naimatullah; Shigeta Ishioka, Noriko

    2000-01-01

    11 C-methionine was supplied to barley plants through a single leaf or via the roots and real time 11 C movement was monitored using a PETIS (positron emitting tracer imaging system). In Fe-deficient plants, 11 C-methionine was translocated from the tip of the absorbing leaf to the discrimination center' at the basal part of the shoot and then retranslocated to all the chlorotic leaves, while a negligible amount was retranslocated to the roots. In Fe-sufficient plants, methionine was translocated from the absorbing leaf to the discrimination center and then only to the newest leaf on the main shoot. A negligible amount was also retranslocated to the roots. Although, in Fe-sufficient plants, methionine translocation was observed from absorbing roots to shoots, in Fe-deficient plants, only a little amount was translocated from roots to shoots. In conclusion, methionine from the upper portion of a plant is not used as a precursor of mugineic acid under Fe-deficiency conditions. (author)

  18. Nucleotide sequence and developmental expression of Acanthamoeba S-adenosylmethionine synthetase gene.

    Science.gov (United States)

    Ahn, K S; Henney, H R

    1997-03-20

    We have isolated and characterized a cDNA (cDNA1) from an Acanthamoeba cDNA library encoding the enzyme S-adenosylmethionine (SAM) synthetase (ATP: L-methionine S-adenosyltransferase; EC 2.5.1.6). The nucleotide sequence exhibits about 61-73% overall similarity to the corresponding gene of other organisms. The cDNA displays extreme codon bias with a preference for C or G in the third position. A putative initiation site and an ATP-binding site are identified. An amino acid content of 388 and a molecular mass of about 44,000 Daltons are deduced for the enzyme. Putative phosphorylation sites which might be involved in regulation of the enzyme are revealed. The cDNA was expressed in Escherichia coli BL21(DE3), and the identity of the protein product confirmed by Western blotting analysis. Northern analyses of the expression of the Acanthamoeba SAM synthetase gene during development revealed a pronounced reduction in the level of transcripts as amoebae converted to cysts.

  19. The glycyl-tRNA synthetase of Chlamydia trachomatis.

    Science.gov (United States)

    Wagar, E A; Giese, M J; Yasin, B; Pang, M

    1995-01-01

    Aminoacyl-tRNA synthetases specifically charge tRNAs with their cognate amino acids. A prototype for the most complex aminoacyl-tRNA synthetases is the four-subunit glycyl-tRNA synthetase from Escherichia coli, encoded by two open reading frames. We examined the glycyl-tRNA synthetase gene from Chlamydia trachomatis, a genetically isolated bacterium, and identified only a single open reading frame for the chlamydial homolog (glyQS). This is the first report of a prokaryotic glycyl-tRNA synthetase encoded by a single gene. PMID:7665503

  20. Growth factors regulate glutamine synthetase activity in ...

    African Journals Online (AJOL)

    Khaled

    2012-07-10

    Jul 10, 2012 ... affected by growth medium, carbon source, nitrogen source and sodium chloride. LB supplemented with 7% glycerol ... Abbreviations: GS, Glutamine synthetase; MSM, minimal salt medium; NB, nutrient broth medium; NF, ... glutamate and ammonia, which in turn, cells are supplied with ammonia, and their ...

  1. Dexamethasone enhances glutamine synthetase activity and reduces N-methyl-D-aspartate neurotoxicity in mixed cultures of neurons and astrocytes

    Directory of Open Access Journals (Sweden)

    Edith Debroas

    2015-05-01

    Full Text Available Astrocytes are claimed to protect neurons against excitotoxicity by clearing glutamate from the extracellular space and rapidly converting it into glutamine. Glutamine, is then released into the extracellular medium, taken up by neurons and transformed back into glutamate which is then stored into synaptic vesicles. Glutamine synthetase (GS, the key enzyme that governs this glutamate/glutamine cycle, is known to be upregulated by glucocorticoids. In the present work we have thus studied in parallel the effects of dexamethasone on glutamine synthetase activity and NMDA-induced neuronal death in cultures derived from the brain cortex of murine embryos. We showed that dexamethasone was able to markedly enhance GS activity in cultures of astrocytes but not in near pure neuronal cultures. The pharmacological characteristics of the dexamethasone action strongly suggest that it corresponds to a typical receptor-mediated effect. We also observed that long lasting incubation (72 h of mixed astrocyte-neuron cultures in the presence of 100 nM dexamethasone significantly reduced the toxicity of NMDA treatment. Furthermore we demonstrated that methionine sulfoximine, a selective inhibitor of GS, abolished the dexamethasone-induced increase in GS activity and also markedly potentiated NMDA toxicity. Altogether these results suggest that dexamethasone may promote neuroprotection through a stimulation of astrocyte glutamine synthetase.

  2. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats.

    Science.gov (United States)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne; Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S; Simonsen, Mette; Ott, Peter; Vilstrup, Hendrik; Sørensen, Michael

    2014-03-01

    Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of (15)NH(4)(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of (15)NH(4)(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH(4)(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

  3. Effect of two types of methionine supplement on performance of ...

    African Journals Online (AJOL)

    An experiment was carried out to determine the effect of two types of synthetic methionine supplement (Dl methionine and MHA FA; Methionine Hydroxyl Analogue Free Acid, also known as Alimet) on performance of finisher broiler. Two hundred and thirty four (234) day old Hubbard Flex broilerschicks were allotted to six ...

  4. Abnormal maternal biomarkers of homocysteine and methionine ...

    African Journals Online (AJOL)

    Rabah M. Shawky

    2017-09-15

    Sep 15, 2017 ... heart defects. In both groups women will be excluded: If they were pregnant or taking folate antagonist medications (antiepileptic drugs) or vitamin supplementations at the time of the study. Measurement of plasma concentration of: Vitamin B-12, folic acid, Homocysteine, Methionine, S-adenosylmethionine.

  5. Influence of Methionine Supplementation on Nicotine Teratogenicity ...

    African Journals Online (AJOL)

    Human and animal studies have shown that maternal tobacco smoking during pregnancy adversely affects pre and postnatal growth and increases the risk of fetal mortality. The aim of the present study was to determine the toxicity of nicotine and protective effect of methionine on the toxic effects of nicotine. Pregnant ...

  6. Methionine supplementation in the productive efficiency, carcass ...

    African Journals Online (AJOL)

    The effect of dietary methionine supplementation at varying levels on the productive efficiency, carcass characteristics and economics of growing indigenous turkey was investigated. Four Isocaloric and Isonitrogenous diets were formulated. The diets were supplemented with 0.00%, 0.05%, 0.10%, and 0.15% respectively.

  7. Secretion Of Methionine By Microorganisms Associated With ...

    African Journals Online (AJOL)

    methionine were secreted after 96 hours and 72 hours respectively by the lactobacilli and Leuconostoc sp. Since lactic acid bacteria are micro-aerophilic, it is suggested that lactic acid bacteria (the two lactobacilli and Leuconostoc sp.), which are the major organisms involved in cassava fermentation for garri production, ...

  8. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  9. Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis

    DEFF Research Database (Denmark)

    Andersen, Jens Velde; McNair, Laura Frendrup; Schousboe, Arne

    2017-01-01

    cortical slices from female NMRI mice were incubated in media containing [1,2-(13) C]acetate or [U-(13) C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority...... of glutamine (13) C-labeling from [1,2-(13) C]acetate as intended. However, (13) C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-(13) C]acetate in the presence of MSO exceeded the level probable from exclusive...

  10. Physiological and biochemical aspects of methionine isomers and a methionine analogue in broilers.

    Science.gov (United States)

    Zhang, Shuai; Saremi, Behnam; Gilbert, Elizabeth R; Wong, Eric A

    2017-02-01

    Methionine is the first limiting amino acid in all poultry corn-soybean based diets. The objective of this study was to determine the effect of supplementation of L-methionine (L-Met), DL-methionine (DL-Met), and the methionine analogue, DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA), on biochemical and physiological parameters of broiler chickens. Male Cobb-500 broilers were fed from day of hatch (d 0) to d 35 posthatch using a basal diet deficient in methionine plus cysteine (Met + Cys) (control), or the basal diet supplemented with 0.22% DL-Met, 0.22% L-Met, or 0.31% DL-HMTBA to meet the Met + Cys requirements. Tissue (liver, duodenum, jejunum, and ileum) and blood samples were collected at various ages, from d 0 to d 35. Performance of the birds, blood parameters (e.g., acute phase proteins, white blood cell counts), mRNA expression of intestinal nutrient transporters and DNA methylation properties of liver tissues were examined. Both body weight and feed efficiency were improved in methionine supplemented groups compared to the control group. No significant differences were observed among DL-Met, L-Met, and DL-HMTBA for growth performance parameters. L-Met and DL-Met supplementation decreased the acute phase protein, serum amyloid A, while DL-HMTBA had no effect. Methionine supplementation had no effect on white blood cell differentiation count, hepatic total DNA methylation, or DNA methyltransferase activity. L-Met and DL-Met, but not DL-HMTBA, supplementation, resulted in enhanced expression of the ATB 0,+ and B 0 AT transporters in various small intestinal segments. All methionine sources increased expression of MCT1 in the jejunum. In conclusion, methionine supplementation improved growth performance of male broilers. Methionine supplementation was also associated with changes in intestinal nutrient transporter gene expression in certain segments and ages, suggesting that intestinal amino acid absorptive function can be regulated by the source of

  11. High Throughput Virtual Screening to Identify Novel natural product Inhibitors for MethionyltRNA-Synthetase of Brucella melitensis.

    Science.gov (United States)

    Kumari, Madhulata; Chandra, Subhash; Tiwari, Neeraj; Subbarao, Naidu

    2017-01-01

    The Brucella melitensis methionyl-tRNA-synthetase (MetRSBm) is a promising target for brucellosis drug development. The virtual screening of large libraries of a drug like molecules against a protein target is a common strategy used to identify novel inhibitors. A High throughput virtual screening was performed to identify hits to the potential antibrucellosis drug target, MetRSBm. The best inhibitor identified from the literature survey was 1312, 1415, and 1430. In the virtual screening 56,400 compounds of ChEMBL antimycobacterial library, 1596 approved drugs, 419 Natural product IV library, and 2396 methionine analogous were docked and rescoring, identified top 10 ranked compounds as anti-mycobacterial leads showing G-scores -10.27 to -8.42 (in kcal/mol), approved drugs G-scores -9.08 to -6.60 (in kcal/mol), Natural product IV library G-scores -10.55 to -6.02 (in kcal/mol), methionine analogous Gscores -11.20 to -8.51 (in kcal/mol), and compared with all three known inhibitors (as control) G-scores -3.88 to -3.17 (in kcal/mol). This result indicates these novel compounds have the best binding affinity for MetRSBm. In this study, we extrapolate that the analogous of methionine for find novel drug likeness has been identified [4-(L-histidyl)-2-phenylbenzoyl] methionine hydrochloride, might show the inhibitor of Brucella melitensis effect by interacting with MetRS enzyme. We suggests that Prumycin as a natural product is the novel drugs for brucellosis.

  12. Factors influencing methionine toxicity in young bobwhite quail

    Science.gov (United States)

    Serafin, J.A.

    1981-01-01

    Young Bobwhite quail (Colinus virginianus) were fed low and adequate protein purified diets with and without excess methionine to evaluate factors affecting methionine toxicity. Growth of quail fed an adequate protein (27%) diet, without supplemental glycine, was depressed by 1.75% and 2.25% excess methionine. Supplemental glycine (.3%) alleviated growth depression caused by 2.25% excess methionine. Quail fed 1.75% and 2.25% excess methionine developed signs of toxicity characterized by weakness, a lowered, outstretched neck when moving, and ataxia. In addition, quail would fall on their sides when disturbed and spin with their heads retracted. These conditions were transient in nature. Growth of quail fed a low protein (18.9%) diet was depressed by 1% and 1.5% excess methionine and DL-homocystine. Quail fed 1% and 1.5% excess methionine in this diet also developed signs of toxicity, the incidence of which was greater and the duration longer than occurred with quail fed adequate protein. Supplementing a low protein (20.15%) diet with .3% or .6% glycine or threonine or a combination of these amino acids did not alleviate growth depression caused by 1.5% excess methionine; however, 2% and 3% supplemental glycine were somewhat effective. Supplements of glycine (2%, 3%) and threonine (1%) completely reversed growth depression from 1% excess methionine but did not influence growth of controls, indicating that both amino acids counteract methionine toxicity. Both glycine and threonine alone improved growth by about the same extent in diets with 1% or 1.5% excess methionine; however, these amino acids alleviated less than 30% of the growth depression resulting from 1.5% excess methionine. The effectiveness of glycine in alleviating methionine toxicity in a low protein diet was decreased, and hemoglobin levels were depressed with 1.5% excess methionine compared to less amounts.

  13. Structure and Activity of an Aminoacyl-tRNA Synthetase that Charges tRNA with Nitro-Tryptophan

    Energy Technology Data Exchange (ETDEWEB)

    Buddha,M.; Crane, B.

    2005-01-01

    The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNATrp with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.

  14. Methionine + cystine levels and vitamin B6 supplementation on performance and enzyme expression of methionine metabolism of gilts from 75 to 100 kg

    Directory of Open Access Journals (Sweden)

    Cleiton Pagliari Sangali

    Full Text Available ABSTRACT This study was carried out to evaluate the effect of different levels of standardized ileal digestible (SID methionine + cystine (Met+Cys and vitamin B6 supplementation on the performance, blood variables, and gene expression of enzymes involved in methionine metabolism in female pigs between 75 and 100 kg. Fifty six female pigs were used (Talent × Topigs 20, averaging 75.06±1.68 kg in initial weight, allotted in a completely randomized block design arranged in a 2 × 4 factorial scheme, composed of two vitamin B6 supplementation levels (1.58 and 3.58 mg/kg and four levels of SID Met+Cys (0.370, 0.470, 0.570, and 0.670%, with seven replicates and one animal per experimental unit. No interactions between vitamin B6 supplementation and SID Met+Cys levels were observed. The levels of SID Met+Cys and vitamin B6 supplementation did not affect animal performance. Triacylglycerols showed a quadratic response to the SID Met+Cys levels, in which the lowest plasma concentration was estimated as 0.575%. Treatments did not affect the expression of the methionine synthase and cystathionine-γ-lyase enzymes or serum homocysteine levels. The SID Met+Cys requirement for female pigs from 75 to 100 kg is equal to or lower than 10.60 g/day, which corresponds to the level of 0.370% Met+Cys in the diet and a relationship 0.48% with the SID lysine.

  15. Sex-specific dysregulation of cysteine oxidation and the methionine and folate cycles in female cystathionine gamma-lyase null mice: a serendipitous model of the methylfolate trap

    Directory of Open Access Journals (Sweden)

    Hua Jiang

    2015-09-01

    Full Text Available In addition to its role in the endogenous synthesis of cysteine, cystathionine gamma-lyase (CGL is a major physiological source of the vasorelaxant hydrogen sulfide. Cgl null mice are potentially useful for studying the influence of this compound upon vascular tone and endothelial function. Here, we confirm a previous report that female Cgl null mice exhibit an approximate 45-fold increase in plasma total homocysteine compared to wild type controls. This level of homocysteine is approximately 3.5-fold higher than that observed in male Cgl null mice and is essentially equivalent to that observed in mouse models of cystathionine beta synthase deficient homocystinuria. Cgl null mice of both sexes exhibited decreased expression of methylenetetrahydrofolate reductase and cysteinesulfinate decarboxylase compared to WT controls. Female Cgl null mice exhibited a sex-specific induction of betaine homocysteine S-methyltransferase and methionine adenosyltransferase 1, alpha and a 70% decrease in methionine synthase expression accompanied by significantly decreased plasma methionine. Decreased plasma cysteine levels in female Cgl null mice were associated with sex-specific dysregulation of cysteine dioxygenase expression. Comparative histological assessment between cystathionine beta-synthase and Cgl null mice indicated that the therapeutic potential of cystathionine against liver injury merits possible further investigation. Collectively, our data demonstrates the importance of considering sex when investigating mouse models of inborn errors of metabolism and indicate that while female Cgl null mice are of questionable utility for studying the physiological role of hydrogen sulfide, they could serve as a useful model for studying the consequences of methionine synthase deficiency and the methylfolate trap.

  16. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling...

  17. Phosphinothricin-tripeptide synthetases from Streptomyces viridochromogenes.

    Science.gov (United States)

    Grammel, N; Schwartz, D; Wohlleben, W; Keller, U

    1998-02-10

    Phosphinothricyl-alanyl-alanine (Pt tripeptide (Ptt), bialaphos) is a metabolite produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus. It contains the unique phosphinoamino acid phosphinothricin (Pt), which after cleavage from Ptt is active as an inhibitor of glutamine synthetase. We have isolated three enzymes that assemble the building block of the Ptt peptide backbone in a nonribosomal mechanism. The first enzyme, named Ptt-synthetase I (PTTS I), activates N-acetyldemethylphosphinothricin (AcDMPt) as adenylate and thioester. Pt is not activated. PTTS I can also activate N-acetylphosphinothricin (AcPt) or N-acetylglutamate as structural analogues of AcDMPT. Native PTTS I has an estimated size of 62 kDa whereas the denatured form displays a size of 76 kDa. Immunoblot analysis and determination of its N-terminal protein sequence revealed that PTTS I is identical with the gene product of phsA. The phsA gene was previously identified near the Pt-resistance gene pat in the Ptt biosynthesis gene cluster in S. viridochromogenes. Besides PTTS I, two alanine-activating enzymes (PTTS II/III) were partially purified from S. viridochromogenes with estimated native sizes of ca. 120 kDa (enzyme 1) and ca. 140 kDa (enzyme 2). Both enzymes bind alanine as a thioester via the corresponding adenylate. Level of PTTS II/III and product formation were correlated with each other in several different strains of S. viridochromogenes. These results indicate that Ptt is synthesized by three peptide synthetases, each activating one single amino acid. The data also confirm previous genetic data, which suggest that AcDMPt-Ala-Ala is the precursor of Ptt.

  18. Bacterial variations on the methionine salvage pathway

    Directory of Open Access Journals (Sweden)

    Haas Dieter

    2004-03-01

    Full Text Available Abstract Background The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism. Results This work uses genome in silico analysis to propose methionine salvage pathways for Klebsiella pneumoniae, Leptospira interrogans, Thermoanaerobacter tengcongensis and Xylella fastidiosa. Experiments performed with mutants of B. subtilis and Pseudomonas aeruginosa substantiate the hypotheses proposed. The enzymes that catalyze the reactions are recruited from a variety of origins. The first, ubiquitous, enzyme of the pathway, MtnA (methylthioribose-1-phosphate isomerase, belongs to a family of proteins related to eukaryotic intiation factor 2B alpha. mtnB codes for a methylthioribulose-1-phosphate dehydratase. Two reactions follow, that of an enolase and that of a phosphatase. While in B. subtilis this is performed by two distinct polypeptides, in the other organisms analyzed here an enolase-phosphatase yields 1,2-dihydroxy-3-keto-5-methylthiopentene. In the presence of dioxygen an aci-reductone dioxygenase yields the immediate precursor of methionine, ketomethylthiobutyrate. Under some conditions this enzyme produces carbon monoxide in B. subtilis, suggesting a route for a new gaseous mediator in bacteria. Ketomethylthiobutyrate is finally transaminated by an aminotransferase that exists usually as a broad specificity enzyme (often able to transaminate aromatic aminoacid keto-acid precursors or histidinol-phosphate. Conclusion A functional methionine salvage pathway was experimentally demonstrated, for the first time, in P. aeruginosa. Apparently, methionine salvage pathways are frequent in Bacteria (and in Eukarya, with recruitment of different polypeptides to perform the needed reactions (an ancestor of a translation initiation factor and Ru

  19. Absence of the glutamine-synthetase-linked methylammonium (ammonium)-transport system in the cyanobiont of Cycas-cyanobacterial symbiosis.

    Science.gov (United States)

    Rai, A N; Lindblad, P; Bergman, B

    1986-11-01

    Using the ammonium analogue (14)CH3NH 3 (+) , ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-DL-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (ΔΨ), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous (14)CH3NH 3 (+) or NH 4 (+) because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.

  20. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

    DEFF Research Database (Denmark)

    Noh, Soo Min; Shin, Seunghyeon; Min Lee, Gyun

    2018-01-01

    and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated......To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1...... in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation...

  1. Comparison of methionine sources around requirement levels using a methionine efficacy method in 0 to 28 day old broilers.

    Science.gov (United States)

    Agostini, P S; Dalibard, P; Mercier, Y; Van der Aar, P; Van der Klis, J D

    2016-03-01

    The addition of methionine in the poultry feed industry is still facing the relative efficacy dilemma between DL-methionine (DLM) and hydroxy-methionine (HMTBA). The aim of this study was to compare the effect of dietary DLM and HMTBA on broiler performance at different levels of total sulfur amino acids (TSAA). The treatments consisted of a basal diet without methionine addition, and 4 increasing methionine doses for both sources resulting in TSAA/Lysine ratios from 0.62 to 0.73 in the starter phase and 0.59 to 0.82 in the grower phase. The comparison of product performance was performed by three-way ANOVA analysis and by methionine efficacy calculation as an alternative method of comparison. Growth results obtained during the starter phase with the different methionine supplementations did not show significant growth responses to TSAA levels, indicating a lower methionine requirement in the starter phase than currently assumed. However, a significant methionine dose effect was obtained for the period 10 to 28 day of age and for the entire growth period of 0 to 28 day of age. Excepting a significant gender effect, the statistical analysis did not allow for the discrimination of methionine sources, and no interaction between source and dose level was observed up to 28 days of age. A significant interaction between source and dose level was observed for methionine efficacy for the grower phase, and the total growth period showed better HMTBA efficacy at higher TSAA value. The exponential model fitted to each methionine source for body weight response depending on methionine intake or for feed conversion ratio (FCR) depending on methionine doses did not allow the methionine sources to be distinguished. Altogether, these results conclude that methionine sources lead to similar performances response when compared at TSAA values around the broiler requirement level. These results also showed that at TSAA values above requirement, HMTBA had a better methionine efficacy

  2. Regulation of thrombosis and vascular function by protein methionine oxidation

    Science.gov (United States)

    Gu, Sean X.; Stevens, Jeff W.

    2015-01-01

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

  3. Dietary Methionine Restriction: Novel Treatment for Hormone Independent Prostate Cancer

    National Research Council Canada - National Science Library

    Epner, Daniel

    2003-01-01

    .... We used Southern blot analysis with methylation-sensitive restriction enzymes, western blot analysis, and RT-PCR to determine whether methionine restriction restored expression of growth inhibitory...

  4. Methionine Metabolites in Patients With Sepsis.

    Science.gov (United States)

    Wexler, Orren; Gough, Michael S; Morgan, Mary Anne M; Mack, Cynthia M; Apostolakos, Michael J; Doolin, Kathleen P; Mooney, Robert A; Arning, Erland; Bottiglieri, Teodoro; Pietropaoli, Anthony P

    2018-01-01

    Sepsis is characterized by microvascular dysfunction and thrombophilia. Several methionine metabolites may be relevant to this sepsis pathophysiology. S-adenosylmethionine (SAM) serves as the methyl donor for trans-methylation reactions. S-adenosylhomocysteine (SAH) is the by-product of these reactions and serves as the precursor to homocysteine. Relationships between plasma total homocysteine concentrations (tHcy) and vascular disease and thrombosis are firmly established. We hypothesized that SAM, SAH, and tHcy levels are elevated in patients with sepsis and associated with mortality. This was a combined case-control and prospective cohort study consisting of 109 patients with sepsis and 50 control participants without acute illness. The study was conducted in the medical and surgical intensive care units of the University of Rochester Medical Center. Methionine, SAM, SAH, and tHcy concentrations were compared in patients with sepsis versus control participants and in sepsis survivors versus nonsurvivors. Patients with sepsis had significantly higher plasma SAM and SAH concentrations than control participants (SAM: 164 [107-227] vs73 [59-87 nM], P sepsis patients compared to healthy control participants (4 [2-6]) vs 7 [5-9] μM; P = .04). In multivariable analysis, quartiles of SAM, SAH, and tHcy were independently associated with sepsis ( P = .006, P = .05, and P Sepsis nonsurvivors had significantly higher plasma SAM and SAH concentrations than survivors (SAM: 223 [125-260] vs 136 [96-187] nM; P = .01; SAH: 139 [81-197] vs 86 [55-130] nM, P = .006). Plasma tHcy levels were similar in survivors vs nonsurvivors. The associations between SAM or SAH and hospital mortality were no longer significant after adjusting for renal dysfunction. Methionine metabolite concentrations are abnormal in sepsis and linked with clinical outcomes. Further study is required to determine whether these abnormalities have pathophysiologic significance.

  5. Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

    Science.gov (United States)

    Hussain, Tahir; Yogavel, Manickam; Sharma, Amit

    2015-04-01

    Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

    Science.gov (United States)

    Montanini, Barbara; Betti, Marco; Márquez, Antonio J; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

    2003-01-01

    The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells. PMID:12683951

  7. Genetic Polymorphism of Folate and Methionine Metabolizing Enzymes and their Susceptibility to Malignant Lymphoma

    International Nuclear Information System (INIS)

    Habib, E.E.; Aziz, M.; Kotb, M.

    2005-01-01

    Folate and methionine metabolism is involved in DNA synthesis and methylation. Polymorphisms in the genes of folate metabolism enzymes have been associated with some forms of cancer. In the present study, 2 polymorphisms were evaluated for a folate metabolic enzyme, methylene-tetrahydrofolate reductase (MTHFR), and one was evaluated for methionine synthase (MS). The 2 polymorphisms MTHFR 677 C-7T and MTHFR 1298 A-7C, are reported to reduce the enzyme activity, which causes intracellular accumulation of 5, 10 vm ethylene-tetrahydrofolate and results in a reduced incidence of DNA double strand breakage. The MS 2756 A-7G polymorphism also reduces the enzyme activity and results in the hypo methylation of DNA. Patients and Methods: To test this hypothesis, genetic polymorphisms in the folate metabolic pathway were investigated using the DNA from a case-control study on 31 patients having malignant lymphoma from the Oncology Outpatient Clinic of the New Children's Hospital, Cairo University and 30 controls who were actually normal children attending for vaccination to the same hospital. We found that there is a higher susceptibility with the MTHFR 677CC and MTHFR 1298 AA genotypes (OR=4.3, 95% CI 1.12-16). When those harbor at least one variant allele in either polymorphism of MTHFR they were defined as reference. For the MS 2756 AG genotype polymorphism there was also a higher susceptibility to developing malignant lymphoma (OR=2.6; 95% CI 1.16.4). Results suggest that folate and methionine metabolism may play an important role in the pathogenesis of malignant lymphoma. Further studies to confirm this association and detailed biologic mechanisms are now required

  8. Lean Body Mass Harbors Sensing Mechanisms that Allow Safeguarding of Methionine Homeostasis

    Directory of Open Access Journals (Sweden)

    Yves Ingenbleek

    2017-09-01

    Full Text Available Protein-depleted states generate allosteric inhibition of liver cystathionine β-synthase (CBS, which governs the first enzymatic step of the transsulfuration cascade, resulting in upstream accretion of homocysteine (Hcy in body fluids. A similar Hcy increase may arise from normal hepatocytes undergoing experimentally-induced impairment of betaine-homocysteine methyltransferase (BHTM activity or from components of lean body mass (LBM submitted to any inflammatory disorder. LBM comprises a composite agglomeration of extrarenal tissues characterized by naturally occurring BHTM inactivity. As a result of cellular injury, LBM releases high concentrations of Hcy into the extracellular space, contrasting with the disruption of normal remethylation pathways. Hyperhomocysteinemia acts as a biomarker, reflecting the severity of insult and operating as an alarm signal. Elevated Hcy levels constitute a precursor pool recognized by a CBS coding region that reacts to meet increased methionine requirements in LBM tissues, using its enhanced production in hepatocytes. Preservation of methionine homeostasis benefits from its high metabolic priority and survival value.

  9. Molecular flexibility and structural instabilities in crystalline L-methionine

    NARCIS (Netherlands)

    Fischer, Jennifer; Lima, Jose A.; Freire, Paulo T. C.; Melo, Francisco E. A.; Havenith, Remco W. A.; Mendes Filho, Josue; Broer, Ria; Eckert, Juergen; Bordallo, Heloisa N.

    2013-01-01

    We have investigated the dynamics in polycrystalline samples of L-methionine related to the structural transition at about 307 K by incoherent inelastic and quasielastic neutron scattering, X-ray powder diffraction as well as ab-initio calculations. L-Methionine is a sulfur amino acid which can be

  10. Comparative efficacy of herbal and synthetic methionine on ...

    African Journals Online (AJOL)

    HM) compared to synthetic methionine (SM) in the diets of domestic laying hens. The herbal methionine (Meth-o-Tas®) was supplied by Intas Pharmaceutical Limited, India. The HM and SM were added to a standard diet at 0.5 and 1.0 kg per ...

  11. Influence of Supplemental Methionine on Growth Performance of ...

    African Journals Online (AJOL)

    The animals were fed diets containing 0 (control), 2 and 4g/kg methionine. Results indicated significant differences (P<0.05) between treatment means in terms of feed intake and live weight changes. Animals fed diets containing 2g/ kg methionine were better (P <0.05) compared to other treatments in terms of feed intake ...

  12. Molecular mechanism of methionine differentiation in high and low ...

    African Journals Online (AJOL)

    Since maize is a primary food stuff for humans and livestock, its amino acid balance is important for proper nutrition. Methionine, an essential amino acid and a primary source of sulfur, is lacking in maize endosperm. Several maize populations were developed through breeding with enhanced methionine content in ...

  13. Methionine as a Precursor of Ethylene—Commentary

    Science.gov (United States)

    Lieberman et al. showed in a 1966 publication of Plant Physiology that methionine is a precursor of ethylene. It was the first paper that showed ethylene carbons are derived from carbons 3 and 4 of methionine. This paper catalyzed remarkable interest among plant biologists to elucidate the biosynth...

  14. The effects of an L-methionine combination supplement on ...

    African Journals Online (AJOL)

    Twenty-one ultramarathon runners (17 males, 4 females) preparing for participation in an 87.3 km ultramarathon. Interventions. L-methionine combination supplement (L-methionine, vitamin B6, vitamin B12, folic acid and magnesium) or placebo containing potato starch. Main outcome measures. Incidence of URTS was ...

  15. Acute Administration of Methionine Affects Performance of Swiss ...

    African Journals Online (AJOL)

    Acetylcholinesterase activities in all groups were not statistically significant. It can be concluded that acute methionine administration has some benefits in memory enhancement. However, a short course folate supplementation impairslearning and working memory especially when combined with methionine which may be ...

  16. S-adenosyl-L-methionine for alcoholic liver diseases

    DEFF Research Database (Denmark)

    Rambaldi, A; Gluud, C

    2006-01-01

    Alcohol is a major cause of liver disease and disrupts methionine and oxidative balances. S-adenosyl-L-methionine (SAMe) acts as a methyl donor for methylation reactions and participates in the synthesis of glutathione, the main cellular antioxidant. Randomised clinical trials have addressed...... the question whether SAMe may benefit patients with alcoholic liver diseases....

  17. Oxidation Resistance of the Sulfur Amino Acids: Methionine and Cysteine

    Directory of Open Access Journals (Sweden)

    Peng Bin

    2017-01-01

    Full Text Available Sulfur amino acids are a kind of amino acids which contain sulfhydryl, and they play a crucial role in protein structure, metabolism, immunity, and oxidation. Our review demonstrates the oxidation resistance effect of methionine and cysteine, two of the most representative sulfur amino acids, and their metabolites. Methionine and cysteine are extremely sensitive to almost all forms of reactive oxygen species, which makes them antioxidative. Moreover, methionine and cysteine are precursors of S-adenosylmethionine, hydrogen sulfide, taurine, and glutathione. These products are reported to alleviate oxidant stress induced by various oxidants and protect the tissue from the damage. However, the deficiency and excess of methionine and cysteine in diet affect the normal growth of animals; thereby a new study about defining adequate levels of methionine and cysteine intake is important.

  18. Detection and Measurement of Methionine Oxidation in Proteins.

    Science.gov (United States)

    Sen, K Ilker; Hepler, Robert; Nanda, Hirsh

    2017-02-02

    Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product. Thus, methionine oxidation is a critical quality attribute that needs to be monitored throughout development. Here we describe a method using LC/MS/MS to identify site-specific methionine modifications in proteins. Antibodies are stressed with hydrogen peroxide, and the level of Met oxidation is compared to that of reference molecules. The protocols presented here are not specific to methionine and can be used more generally to identify other PTM risk sites in molecules after various types of treatments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  19. Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver.

    OpenAIRE

    Johnson, D L; Yang, D C

    1981-01-01

    The particulate aminoacyl-tRNA synthetases of rat liver were copurified about 1000-fold with more than 20% yields for individual synthetase activities. Measurements of aminoacylation activities showed that lysyl-, arginyl-, leucyl-, isoleucyl-, and methionyl-tRNA synthetases in the purified complex cosedimented at 18 S. The molecular weight of the synthetase complex is about one million, as estimated by gel filtration. The stoichiometry of the synthetase in the complex was determined by activ...

  20. Methionine synthase A2756G and reduced folate carrier1 A80G ...

    African Journals Online (AJOL)

    Maha Moustafa

    2015-10-09

    Oct 9, 2015 ... the RFC1 80G>A polymorphism has been associated with reduced red cell folate concentrations among healthy women. [21], and with reduced serum folate concentrations in mothers of down syndrome children (MDS) [22]. However, less than. 1.000 MDS were available for those meta-analyses, and sub-.

  1. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    Science.gov (United States)

    ... hyperammonemia, type I Genetics Education Materials for School Success (GEMSS) Orphanet: Carbamoyl-phosphate synthetase 1 deficiency Patient ... for Links Data Files & API Site Map Subscribe Customer Support USA.gov Copyright Privacy Accessibility FOIA Viewers & ...

  2. Role of Methionine Adenosyltransferase Genes in Hepatocarcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ramani, Komal [Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, Southern California Research Center for Alcoholic Liver and Pancreatic Diseases & Cirrhosis, Keck School of Medicine USC, Los Angeles, California 90033 (United States); Mato, José M. [CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Technology, Park of Bizkaia, 48160 Derio, Bizkaia (Spain); Lu, Shelly C., E-mail: shellylu@usc.edu [Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, Southern California Research Center for Alcoholic Liver and Pancreatic Diseases & Cirrhosis, Keck School of Medicine USC, Los Angeles, California 90033 (United States)

    2011-03-24

    Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. Detection of HCC can be difficult, as most of the patients who develop this tumor have no symptoms other than those related to their longstanding liver disease. There is an urgent need to understand the molecular mechanisms that are responsible for the development of this disease so that appropriate therapies can be designed. Methionine adenosyltransferase (MAT) is an essential enzyme required for the biosynthesis of S-adenosylmethionine (AdoMet), an important methyl donor in the cell. Alterations in the expression of MAT genes and a decline in AdoMet biosynthesis are known to be associated with liver injury, cirrhosis and HCC. This review focuses on the role of MAT genes in HCC development and the scope for therapeutic strategies using these genes.

  3. Dietary Methionine Restriction Alleviates Hyperglycemia in Pigs with Intrauterine Growth Restriction by Enhancing Hepatic Protein Kinase B Signaling and Glycogen Synthesis.

    Science.gov (United States)

    Ying, Zhixiong; Zhang, Hao; Su, Weipeng; Zhou, Le; Wang, Fei; Li, Yue; Zhang, Lili; Wang, Tian

    2017-10-01

    Background: Individuals with intrauterine growth restriction (IUGR) are prone to developing type 2 diabetes mellitus (T2DM). Dietary methionine restriction (MR) improves insulin sensitivity and glucose homeostasis in individuals with normal birth weight (NBW). Objective: This study investigated the effects of MR on plasma glucose concentration and hepatic and muscle glucose metabolism in pigs with IUGR. Methods: Thirty female NBW and 60 same-sex spontaneous IUGR piglets (Landrace × Yorkshire) were selected. After weaning (day 21), the piglets were fed diets with adequate methionine (NBW-CON and IUGR-CON) or 30% less methionine (IUGR-MR) ( n = 6). At day 180, 1 pig with a body weight near the mean of each replication was selected for biochemical analysis. Results: The IUGR-CON group showed 41.6%, 68.6%, and 67.1% higher plasma glucose concentration, hepatic phosphoenolpyruvate carboxykinase activity, and glucose-6-phosphatase activity, respectively, than the NBW-CON group ( P glycogen content and glycogen synthase activity were 36.9% and 38.8% lower, respectively, in the IUGR-CON than the NBW-CON group ( P glycogen content and glycogen synthase activity of the IUGR-MR pigs were 62.9% and 50.8% higher than those of the IUGR-CON pigs ( P glycogen synthesis, implying a potential nutritional strategy to prevent type 2 diabetes mellitus in IUGR offspring. © 2017 American Society for Nutrition.

  4. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    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.

  6. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been...

  7. Methionine Sulfoxide Reductase A Deficiency Exacerbates Cisplatin-Induced Nephrotoxicity via Increased Mitochondrial Damage and Renal Cell Death.

    Science.gov (United States)

    Noh, Mi Ra; Kim, Ki Young; Han, Sang Jun; Kim, Jee In; Kim, Hwa-Young; Park, Kwon Moo

    2017-10-10

    Methionine sulfoxide reductase A (MsrA), which is abundantly localized in the mitochondria, reduces methionine-S-sulfoxide, scavenging reactive oxygen species (ROS). Cisplatin, an anticancer drug, accumulates at high levels in the mitochondria of renal cells, causing mitochondrial impairment that ultimately leads to nephrotoxicity. Here, we investigated the role of MsrA in cisplatin-induced mitochondrial damage and kidney cell death using MsrA gene-deleted (MsrA -/- ) mice. Cisplatin injection resulted in increases of ROS production, methionine oxidation, and oxidative damage in the kidneys. This oxidative stress was greater in MsrA -/- mouse kidneys than in wild-type (MsrA +/+ ) mouse kidneys. MsrA gene deletion exacerbated cisplatin-induced reductions in the expression and activity of MsrA and MsrBs, and the expression of thioredoxin 1, glutathione peroxidase 1 and 4, mitochondrial superoxide dismutase, cystathionine-β-synthase, and cystathionine-γ-lyase. Cisplatin induced swelling, cristae loss, and fragmentation of mitochondria with increased lipid peroxidation, more so in MsrA -/- than in MsrA +/+ kidneys. The ratio of mitochondrial fission regulator (Fis1) to fusion regulator (Opa1) was higher in MsrA -/- than MsrA +/+ mice. MsrA deletion exacerbated cisplatin-induced increases in Bax to Bcl-2 ratio, cleaved caspase-3 level, and apoptosis, whereas MsrA overexpression attenuated cisplatin-induced oxidative stress and apoptosis. MsrA gene deletion in mice exacerbates cisplatin-induced renal injury through increases of mitochondrial susceptibility, whereas MsrA overexpression protects cells against cisplatin. This study demonstrates that MsrA protects kidney cells against cisplatin-induced methionine oxidation, oxidative stress, mitochondrial damage, and apoptosis, suggesting that MsrA could be a useful target protein for the treatment of cisplatin-induced nephrotoxicity. Antioxid. Redox Signal. 27, 727-741.

  8. A machine learning approach for predicting methionine oxidation sites.

    Science.gov (United States)

    Aledo, Juan C; Cantón, Francisco R; Veredas, Francisco J

    2017-09-29

    The oxidation of protein-bound methionine to form methionine sulfoxide, has traditionally been regarded as an oxidative damage. However, recent evidences support the view of this reversible reaction as a regulatory post-translational modification. The perception that methionine sulfoxidation may provide a mechanism to the redox regulation of a wide range of cellular processes, has stimulated some proteomic studies. However, these experimental approaches are expensive and time-consuming. Therefore, computational methods designed to predict methionine oxidation sites are an attractive alternative. As a first approach to this matter, we have developed models based on random forests, support vector machines and neural networks, aimed at accurate prediction of sites of methionine oxidation. Starting from published proteomic data regarding oxidized methionines, we created a hand-curated dataset formed by 113 unique polypeptides of known structure, containing 975 methionyl residues, 122 of which were oxidation-prone (positive dataset) and 853 were oxidation-resistant (negative dataset). We use a machine learning approach to generate predictive models from these datasets. Among the multiple features used in the classification task, some of them contributed substantially to the performance of the predictive models. Thus, (i) the solvent accessible area of the methionine residue, (ii) the number of residues between the analyzed methionine and the next methionine found towards the N-terminus and (iii) the spatial distance between the atom of sulfur from the analyzed methionine and the closest aromatic residue, were among the most relevant features. Compared to the other classifiers we also evaluated, random forests provided the best performance, with accuracy, sensitivity and specificity of 0.7468±0.0567, 0.6817±0.0982 and 0.7557±0.0721, respectively (mean ± standard deviation). We present the first predictive models aimed to computationally detect methionine sites that

  9. effect of supplementation of soyabea~ diet with l-asd d,l-methionine ...

    African Journals Online (AJOL)

    BSN

    metliionine degradation tn its derivative such as taurine and methionine sulfoxidc as reported by Mmai. '/al., (1989) for young carp fed crystalline methionine. This would render methionine unavailable for protein syntliesis. The growth rat..: of mudfish fed soyahean diets supplemented with crystalline L-methionine was.

  10. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  11. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  12. New perspectives on glutamine synthetase in grasses.

    Science.gov (United States)

    Swarbreck, Stéphanie M; Defoin-Platel, M; Hindle, M; Saqi, M; Habash, Dimah Z

    2011-02-01

    Members of the glutamine synthetase (GS) gene family have now been characterized in many crop species such as wheat, rice, and maize. Studies have shown that cytosolic GS isoforms are involved in nitrogen remobilization during leaf senescence and emphasized a role in seed production particularly in small grain crop species. Data from the sequencing of genomes for model crops and expressed sequence tag (EST) libraries from non-model species have strengthened the idea that the cytosolic GS genes are organized in three functionally and phylogenetically conserved subfamilies. Using a bioinformatic approach, the considerable publicly available information on high throughput gene expression was mined to search for genes having patterns of expression similar to GS. Interesting new hypotheses have emerged from searching for co-expressed genes across multiple unfiltered experimental data sets in rice. This approach should inform new experimental designs and studies to explore the regulation of the GS gene family further. It is expected that understanding the regulation of GS under varied climatic conditions will emerge as an important new area considering the results from recent studies that have shown nitrogen assimilation to be critical to plant acclimation to high CO(2) concentrations.

  13. S-Adenosyl-L-Methionine (SAMe): An Introduction

    Science.gov (United States)

    ... dietary supplement. SAMe was discovered in the early 1950s. It’s made in the body from methionine, an ... of Health & Human Services , National Institutes of Health , USA.gov National Center for Complementary and Integrative Health ( ...

  14. Gleaning unexpected fruits from hard-won synthetases: probing principles of permissivity in non-canonical amino acid-tRNA synthetases.

    Science.gov (United States)

    Cooley, Richard B; Karplus, P Andrew; Mehl, Ryan A

    2014-08-18

    The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins is an important tool for understanding biological function. Traditionally, each new ncAA targeted for incorporation requires a resource-consuming process of generating new ncAA aminoacyl tRNA synthetase/tRNACUA pairs. However, the discovery that some tRNA synthetases are "permissive", in that they can incorporate multiple ncAAs, means that it is no longer always necessary to develop a new synthetase for each newly desired ncAA. Developing a better understanding of what factors make ncAA synthetases more permissive would increase the utility of this new approach. Here, we characterized two synthetases selected for the same ncAA that have markedly different "permissivity profiles." Remarkably, the more permissive synthetase incorporated an ncAA for which we had not been able to generate a synthetase through de novo selection methods. Crystal structures revealed that the two synthetases recognize their parent ncAA through a conserved core of interactions, with the more permissive synthetase displaying a greater degree of flexibility in its interaction geometries. We also observed that intraprotein interactions not directly involved in ncAA binding can play a crucial role in synthetase permissivity and suggest that optimization of such interactions might provide an avenue to engineering synthetases with enhanced permissivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-12-13

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

  16. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  17. Cleaning up polyketide synthases.

    Science.gov (United States)

    Kwan, Jason C; Schmidt, Eric W

    2012-03-23

    Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. High-coverage proteomics reveals methionine auxotrophy in Deinococcus radiodurans.

    Science.gov (United States)

    Zhou, Yanxia; Shen, Pan; Lan, Qiuyan; Deng, Chen; Zhang, Yao; Li, Yanchang; Wei, Wei; Wang, Yihao; Su, Na; He, Fuchu; Xie, Qiong; Lyu, Zhitang; Yang, Dong; Xu, Ping

    2017-07-01

    Deinococcus radiodurans is a robust bacterium best known for its capacity to resist to radiation. In this study, the SDS-PAGE coupled with high-precision LC-MS/MS was used to study the D. radiodurans proteome. A total of 1951 proteins were identified which covers 63.18% protein-coding genes. Comparison of the identified proteins to the key enzymes in amino acid biosyntheses from KEGG database showed the methionine biosynthesis module is incomplete while other amino acid biosynthesis modules are complete, which indicated methionine auxotrophy in D. radiodurans. The subsequent amino acid-auxotrophic screening has verified methionine instead of other amino acids is essential for the growth of D. radiodurans. With molecular evolutionary genetic analysis, we found the divergence in methionine biosynthesis during the evolution of the common ancestor of bacteria. We also found D. radiodurans lost the power of synthesizing methionine because of the missing metA and metX in two types of methionine biosyntheses. For the first time, this study used high-coverage proteome analysis to identify D. radiodurans amino acid auxotrophy, which provides the important reference for the development of quantitative proteomics analysis using stable isotope labeling in metabolomics of D. radiodurans and in-depth analysis of the molecular mechanism of radiation resistance. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Prediction of Methionine and Homocysteine levels in Zucker diabetic fatty (ZDF) rats as a T2DM animal model after consumption of a Methionine-rich diet

    OpenAIRE

    Han, Nayoung; Chae, Jung-woo; Jeon, Jihyun; Lee, Jaeyeon; Back, Hyun-moon; Song, Byungjeong; Kwon, Kwang-il; Kim, Sang Kyum; Yun, Hwi-yeol

    2018-01-01

    Background Although alterations in the methionine metabolism cycle (MMC) have been associated with vascular complications of diabetes, there have not been consistent results about the levels of methionine and homocysteine in type 2 diabetes mellitus (T2DM). The aim of the current study was to predict changes in plasma methionine and homocysteine concentrations after simulated consumption of methionine-rich foods, following the development of a mathematical model for MMC in Zucker Diabetic Fat...

  1. The glutamine synthetase gene family in Populus

    Directory of Open Access Journals (Sweden)

    Cánovas Francisco M

    2011-08-01

    Full Text Available Abstract Background Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. Results The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1 and 1 which codes for the choroplastic GS isoform (GS2. Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. Conclusions Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.

  2. Evolutionary divergence of chloroplast FAD synthetase proteins

    Directory of Open Access Journals (Sweden)

    Arilla-Luna Sonia

    2010-10-01

    Full Text Available Abstract Background Flavin adenine dinucleotide synthetases (FADSs - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity.

  3. The low-methionine content of vegan diets may make methionine restriction feasible as a life extension strategy.

    Science.gov (United States)

    McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco

    2009-02-01

    Recent studies confirm that dietary methionine restriction increases both mean and maximal lifespan in rats and mice, achieving "aging retardant" effects very similar to those of caloric restriction, including a suppression of mitochondrial superoxide generation. Although voluntary caloric restriction is never likely to gain much popularity as a pro-longevity strategy for humans, it may be more feasible to achieve moderate methionine restriction, in light of the fact that vegan diets tend to be relatively low in this amino acid. Plant proteins - especially those derived from legumes or nuts - tend to be lower in methionine than animal proteins. Furthermore, the total protein content of vegan diets, as a function of calorie content, tends to be lower than that of omnivore diets, and plant protein has somewhat lower bioavailability than animal protein. Whole-food vegan diets that moderate bean and soy intake, while including ample amounts of fruit and wine or beer, can be quite low in methionine, while supplying abundant nutrition for health (assuming concurrent B12 supplementation). Furthermore, low-fat vegan diets, coupled with exercise training, can be expected to promote longevity by decreasing systemic levels of insulin and free IGF-I; the latter effect would be amplified by methionine restriction - though it is not clear whether IGF-I down-regulation is the sole basis for the impact of low-methionine diets on longevity in rodents.

  4. Detection of oxidized methionine in selected proteins, cellular extracts, and blood serums by novel anti-methionine sulfoxide antibodies

    Science.gov (United States)

    Oien, Derek B.; Canello, Tamar; Gabizon, Ruth; Gasset, Maria; Lundquist, Brandi L.; Burns, Jeff M; Moskovitz, Jackob

    2009-01-01

    Methionine sulfoxide (MetO) is a common posttranslational modification to proteins occurring in vivo. These modifications are prevalent when reactive oxygen species levels are increased. To enable the detection of MetO in pure and extracted proteins from various sources, we have developed novel antibodies that can recognize MetO-proteins. These antibodies are polyclonal antibodies raised against an oxidized methionine-rich zein protein (MetO-DZS18) that are shown to recognize methionine oxidation in pure proteins and mouse and yeast extracts. Furthermore, mouse serum albumin and immunoglobulin (IgG) were shown to accumulate MetO as function of age especially in serums of methionine sulfoxide reductase A knockout mice. Interestingly, high levels of methionine-oxidized IgG in serums of subjects diagnosed with Alzheimer’s disease were detected by western blot analysis using these antibodies. It is suggested that anti-MetO-DZS18 antibodies can be applied in the identification of proteins that undergo methionine oxidation under oxidative stress, aging, or disease state conditions. PMID:19388147

  5. Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

    Science.gov (United States)

    Vats, P.; Mukherjee, A. K.; Kumria, M. M. L.; Singh, S. N.; Patil, S. K. B.; Rangnathan, S.; Sridharan, K.

    Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76+/-0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82+/-2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure

  6. [Advances in isoprene synthase research].

    Science.gov (United States)

    Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

    2017-11-25

    Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

  7. S-Adenosylmethionine and S-adenosylhomocystein metabolism in isolated rat liver. Effects of L-methionine, L-homocystein, and adenosine.

    Science.gov (United States)

    Hoffman, D R; Marion, D W; Cornatzer, W E; Duerre, J A

    1980-11-25

    The effects of varying concentrations of L-methionine, L-homocysteine, and adenosine on the tissue levels of S-adenosylmethionine (AdoMet) and S-adenosyl-homocystein (AdoHcy) were investigated in perfused liver. In the normal liver, the intracellular concentration of AdoMet was dependent upon the availability of methionine. In the presence of high concentrations of methionine the maximum level of AdoMet attainable was 300 nmol/g of liver. The exogenous concentration of methionine did not alter the hepatic concentration of AdoHcy (8 to 20 nmol/g) while adenosine or homocysteine blocked hydrolysis of AdoHcy resulting in elevated levels of AdoHcy (400 to 600 nmol/g) and AdoMet (300 to 600 nmol/g). The addition of both adenosine (4mM) and homocysteine (3.4 mM) to the perfusate further increased the levels of AdoHcy (4 mumol/g) and AdoMet (1.2 mumol/g). As the concentration of AdoHcy increased, significant amounts of this compound were released into the perfusate, while AdoMet was not detected. Under all conditions where AdoHcy accumulated in the cell, a concomitant increase in the AdoMet level occurred. Apparently AdoHcy acts as a positive effector of the S-adenosylmethionine synthase. The hepatocytes did not take up significant amounts of [methyl-14C]AdoMet from the perfusate nor were any [14C]methyl groups from this compound incorporated into histones, DNA, or phospholipids. In contrast, [14C]methyl groups were readily incorporated into these macromolecules from exogenous [methyl-14C]methionine. The addition of adenosine (4 mM) and homocystein (3.4 mM) shifted the AdoMet:AdoHcy ratio from 8.2 to 0.3. Under these conditions, transmethylation was inhibited markedly.

  8. Regions involved in fengycin synthetases enzyme complex formation

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Cheng

    2017-12-01

    Full Text Available Background: Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order to form the complex. This study investigates how these enzymes interact in the complex and analyzes the regions in the enzymes that are critical to the interactions. Methods: Deletions were generated in the fengycin synthetases. The interaction of these mutant proteins with their partner enzymes in the complex was analyzed in vitro by a glutathione S-transferase (GST or nickel pulldown assay. Results: The communication-mediating donor (COM-D domains of the fengycin synthetases, when fused to GST, specifically pulled down their downstream partner enzymes in the GST-pulldown assays. The communication-mediating acceptor (COM-A domains were required for binding between two partner enzymes, although the domains alone did not confer specificity of the binding to their upstream partner enzymes. This study found that the COM-A domain, the condensation domain, and a portion of the adenylation domain in fengycin synthetase B (FenB were required for specific binding to fengycin synthetase A (FenA. Conclusion: The interaction between the COM-D and COM-A domains in two partner enzymes is critical for nonribosomal peptide synthesis. The COM-A domain alone is insufficient for interacting with its upstream partner enzyme in the enzyme complex with specificity; a region that contains COM-A, condensation, and a portion of adenylation domains in the downstream partner enzyme is required. Keywords: communication-mediating donor and acceptor domain, fengycin synthetase, protein-protein interaction

  9. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  10. Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli

    DEFF Research Database (Denmark)

    Mirza, Nadia Muhammad Akram; Crocoll, Christoph; Olsen, Carl Erik

    2016-01-01

    in Escherichia coli cytosol. Introduction of two plasmids encoding the methionine chain elongation pathway into E. coli resulted in production of 25mgL(-1) of dihomo-methionine. In addition to chain-elongated methionine products, side-products from chain elongation of leucine were produced. Methionine...... supplementation enhanced dihomo-methionine production to 57mgL(-1), while keeping a steady level of the chain-elongated leucine products. Engineering of the de-compartmentalized pathway of dihomo-methionine in E. coli cytosol provides an important first step for microbial production of the health...

  11. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    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.

  12. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

    Science.gov (United States)

    Quadri, L E; Weinreb, P H; Lei, M; Nakano, M M; Zuber, P; Walsh, C T

    1998-02-10

    The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

  13. Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel).

    Science.gov (United States)

    Zhang, Meng-Yi; Wei, Dong; Li, Ran; Jia, Hong-Ting; Liu, Yu-Wei; Taning, Clauvis Nji Tizi; Wang, Jin-Jun; Smagghe, Guy

    2018-04-01

    In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS-specific inhibitor (L-methionine S-sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS-c and BdGS-m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS-c) and mitochondria (BdGS-m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS-c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS-c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS-c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS-c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management. © 2018 Wiley Periodicals, Inc.

  14. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  15. Heterogeneous distribution of glutamine synthetase during rat liver development

    NARCIS (Netherlands)

    Gaasbeek Janzen, J. W.; Gebhardt, R.; ten Voorde, G. H.; Lamers, W. H.; Charles, R.; Moorman, A. F.

    1987-01-01

    Two days before birth, immunohistochemical detection of glutamine synthetase already reveals a heterogeneous distribution pattern related to the vascular architecture of the liver. Only a small number of hepatocytes in the vicinity of the efferent venules show relatively high staining intensity.

  16. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

  17. Glutamine Synthetase Deficiency in Murine Astrocytes Results in Neonatal Death

    NARCIS (Netherlands)

    He, Youji; Hakvoort, Theodorus B. M.; Vermeulen, Jacqueline L. M.; Labruyère, Wilhelmina T.; de Waart, D. Rudi; van der Hel, W. Saskia; Ruijter, Jan M.; Uylings, Harry B. M.; Lamers, Wouter H.

    2010-01-01

    Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/f)l mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in

  18. Restoration Of Glutamine Synthetase Activity, Nitric Oxide Levels ...

    African Journals Online (AJOL)

    Background: Propolis has been proposed to be protective on neurodegenerative disorders. To understand the neuroprotective effects of honeybee propolis, glutamine synthetase (GS) activity, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and total antioxidant status (TAS) were studied in different brain ...

  19. Radiation stability of methionine-35S and selenomethionine-75Se

    International Nuclear Information System (INIS)

    Galateanu, I.; Lungu, V.V.; Viorel, D.

    1976-01-01

    The radiation stability of methionine- 35 S and selenomethionine 75 Se was investigated using the methods of thin-layer chromatography, gas chromatography and ESR. Radiation decomposition of methionine- 35 S mainly consists in an oxidation process and in the release of volatile products. The ESR-spectra of irradiated DL-methionine indicated a strong localization of the unpaired electrons on sulfur atoms. Radiation damage to selenomethionine- 75 Se as a function of radiation dose proved an increased stability of this compound and its radiation decomposition consists in the formation of oxidized products and by direct rupture of the selenium bounds accompanied by the formation of volatile compounds like CH 3 SEH and SeH 2 . The self-radiolysis of the aqueous solution of selenomethionine- 75 Se during its storage in air leads, however, to a lower decomposition rate which consists in the release of inorganic selenium and in an oxidation process. (author)

  20. The Synthesis of Polyamines from Methionine in Intact and Disrupted Leaf Protoplasts of Virus-Infected Chinese Cabbage 1

    Science.gov (United States)

    Cohen, Seymour S.; Balint, Robert; Sindhu, Ram K.

    1981-01-01

    In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-14C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine. Protein synthesis is similar for 4 hours in both healthy and infected protoplasts. Accumulation of labeled spermidine stops after 2 hours in healthy protoplasts but continues in the infected protoplasts. Much of the newly synthesized protein and spermidine is present in the easily sedimentable fraction of the readily disrupted protoplasts. Disrupted and diluted protoplasts have a decreased ability to metabolize methionine to protein and spermidine. The residual synthetic activity is essentially entirely in the easily sedimentable fraction. However, this fraction is unable to synthesize spermine, an activity found in protoplasts and disrupted protoplasts. Disrupted protoplasts contain spermidine synthase (EC 2.5.1.16) and about a quarter of this activity is present in a low-speed sedimentable fraction containing the chloroplasts. The protoplast system is suitable for an analysis of polyamine synthesis in turnip yellow mosaic virus infection and appears particularly suitable for study of the distribution of the enzymes involved. Images PMID:16662066

  1. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  2. Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis

    Directory of Open Access Journals (Sweden)

    Knodel Marvin H

    2003-06-01

    Full Text Available Abstract Background Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. Results The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 μmol/min/mg protein and the Km for methionine and ATP was 288 μM and 76 μM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC of 500 μM, while the MIC for 8-azaguanine was >1.0 mM. Conclusion The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting

  3. Different Levels of Digestible Methionine on Performance of Broiler Starter

    Directory of Open Access Journals (Sweden)

    WL Bryden

    2010-01-01

    Full Text Available Dietary protein and amino acid supply is the most expensive component of poultry diets. Therefore several efforts made by the industry to minimize the cost of the protein portion of the diet. Accordingly, there has been a recent move to use digestible amino acid values in the formulation of poultry diets. The efficiency of protein utilization depends to a large extent on the amino acid composition of the diet. The study was conducted to determine the digestible methionine requirement of broilers during the starter periods. One hundred and seventy five (175 chicks were allocated to 5 treatments with five replicates of seven chicks per replicate in a completely randomized design. Chicks were fed experimental diets from one day old to 21 days of age. Dietary treatments included 5 titrated levels each of digestible methionine (3.0, 4.5, 6.0, 7.5, and 9.0 g/kg diet added to a basal diet. The allowance of digestible methionine, rather than digestible sulphur amino acids was used in formulating the diets. Supplemental synthetic DL-Methionine which were considered to be 100% digestible were added to diets to obtain the concentration of the digestible amino acid. Each week until the conclusion of the trial, birds were individually weighed, feed intake per pen was measured, and feed conversion ratio (FCR was computed. This study suggested that the digestible methionine requirement for broiler starter is 4.7 g/kg for optimal body weight gain and 4.6 g/kg for optimal feed conversion ratio. (Animal Production 12(1: 6-11 (2010Key Words: amino acid, broiler, digestible, methionine, starter

  4. Stereochemical course of enzyme-catalyzed aminopropyl transfer: spermidine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Kullberg, D.W.; Orr, G.R.; Coward, J.K.

    1986-05-01

    The R and S enantionmers of S-adenosyl-3-(/sup 2/H)3-(methylthio)-1-propylamine (decarboxylated S-adenosylmethionine), previously synthesized in this laboratory, were incubated with (1,4-/sup 2/H/sub 4/)-putrescine in the presence of spermidine synthase from E. coli. The resulting chiral (/sup 2/H/sub 5/)spermidines were isolated and converted to their N/sub 1/,N/sub 7/-dibocspermidine-N/sub 4/-(1S,4R)-camphanamides. The derivatives were analyzed by 500 MHz /sup 1/H-NMR and the configuration of the chiral center assigned by correlation with the spectra of synthetic chiral (/sup 2/H/sub 3/)dibocspermidine camphanamide standards. The enzyme-catalyzed aminopropyl transfer was shown to occur with net retention of configuration, indicative of a double-displacement mechanism. This result concurs with that of a previous steady-state kinetics study of spermidine synthase isolated from E. coli, but contradicts the single-displacement mechanism suggested by a stereochemical analysis of chiral spermidines biosynthesized in E. coli treated with chirally deuterated methionines. It also indicates that this aminopropyltransferase is mechanistically distinct from the methyltransferases, which have been shown to act via a single-displacement mechanism (net inversion at -CH/sub 3/) in all cases studied to date.

  5. Absorption of l-methionine from the human small intestine

    Science.gov (United States)

    Schedl, Harold P.; Pierce, Charles E.; Rider, Alan; Clifton, James A.

    1968-01-01

    Absorption of L-methionine was measured in all parts of the human small intestine using transintestinal intubation and perfusion. In four normal subjects, adsorption was higher in the proximal than in the distal intestine. In two patients with nontropical sprue in relapse, there was a proximal zone of low absorption with higher absorption distally. In all parts of the small intestine, absorption showed rate-limiting kinetics as methionine concentration was increased. In normal subjects, the proximal Km (Michaelis constant) was more than 3 times higher than the distal, which suggests a difference in transport mechanisms between the two segments. PMID:12066784

  6. Traumatic brain injury alters methionine metabolism: implications for pathophysiology

    Directory of Open Access Journals (Sweden)

    Pramod K Dash

    2016-04-01

    Full Text Available Methionine is an essential proteinogenic amino acid that is obtained from the diet. In addition to its requirement for protein biosynthesis, methionine is metabolized to generate metabolites that play key roles in a number of cellular functions. Metabolism of methionine via the transmethylation pathway generates S-adenosylmethionine (SAM that serves as the principal methyl (-CH3 donor for DNA and histone methyltransferases to regulate epigenetic changes in gene expression. SAM is also required for methylation of other cellular proteins that serve various functions and phosphatidylcholine synthesis that participate in cellular signaling.. Under conditions of oxidative stress, homocysteine (which is derived from SAM enters the transsulfuration pathway to generate glutathione, an important cytoprotective molecule against oxidative damage. As both experimental and clinical studies have shown that traumatic brain injury (TBI alters DNA and histone methylation and causes oxidative stress, we examined if TBI alters the plasma levels of methionine and its metabolites in human patients. Blood samples were collected from healthy volunteers (n = 20 and patients with mild TBI (GCS > 12; n = 20 or severe TBI (GCS < 8; n = 20 within the first 24 hours of injury. The levels of methionine and its metabolites in the plasma samples were analyzed by either liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry (LC-MS or GC-MS. Severe TBI decreased the levels of methionine, SAM, betaine and 2-methylglycine as compared to healthy volunteers, indicating a decrease in metabolism through the transmethylation cycle. In addition, precursors for the generation of glutathione, cysteine and glycine were also found to be decreased as were intermediate metabolites of the gamma-glutamyl cycle (gamma-glutamyl amino acids and 5-oxoproline. Mild TBI also decreased the levels of methionine, α-ketobutyrate, 2 hydroxybutyrate and glycine, albeit to lesser

  7. Seleno methionine-75 as a scanning agent for neuroblastoma

    International Nuclear Information System (INIS)

    Covington, E.E.; D'Angio, G.J.; Helson, L.; Romano, R.W.

    1974-01-01

    Neuroblastoma is a functioning tumor and patients with this tumor are known to excrete vanilmandelic acid and other degradation products of norepinephrine. It also accumulates and produces excess cystathionine for which methionine is a precursor in the normal anabolic pathway. This was the rationale for testing 75 Se-methionine as a possible scanning agent in patients with neuroblastoma. D'Angio et al reported the results of a preliminary investigation in which 3 of 4 patients with neuroblastoma, all with known metastases of the skull, had positive scans correctly localizing the disease. These preliminary data seemed encouraging, and further investigation was undertaken. The results are reported

  8. The use of low protein liquid diets to determine the methionine requirement and the efficacy of methionine hydroxy analogue for the three-week-old pig.

    Science.gov (United States)

    Reifsnyder, D H; Young, C T; Jones, E E

    1984-09-01

    A mechanical feeding device that dispenses liquid diets hourly was developed to feed 3-week-old pigs under carefully controlled and sanitary conditions. Pigs were weaned at 19-21 days of age, placed in individual cages of the automatic feeder, and trained to eat low protein (9%) milk diets, which were supplemented with essential amino acids, glutamic acid and monosodium glutamate so as to be equivalent to 14% protein nitrogen. The basal 9% protein diet contained 0.25% L-methionine and 0.08% L-cysteine and was supplemented with L- or DL-methionine or DL-methionine hydroxy analogue (MHA) at various levels for evaluation of the methionine requirement. Pigs fed the basal diet showed a significant decrease in gain, feed efficiency and plasma urea (P less than 0.05) relative to animals that received supplemental methionine or MHA. The plasma methionine concentration remained below 0.2 mumol/ml plasma when pigs were fed diets containing 0.25-0.51% methionine; however, a significant increase in plasma methionine (P less than 0.05) was seen when pigs were fed diets that contained greater than 0.51% methionine activity in the form of L- or DL-methionine or DL-MHA. The highest average daily gain (470 g) obtained with a diet containing 0.51% methionine was significantly better (P less than 0.05) than diets containing more or less L- or DL-methionine, and the feed efficiency of this diet (1.58 kg feed per kilogram gain) was also significantly better (P less than 0.05) than the feed efficiency obtained with other dietary methionine levels. MHA (0.17%) added to the basal diet significantly improved the average daily gain (P less than 0.05) and lead to a significant decrease in plasma urea (P less than 0.05) relative to pigs that received the basal diet. Supplemental MHA (greater than 0.51% methionine level) produced significant increases (P less than 0.05) in plasma methionine. These data show that the methionine requirement of the 3-week-old pig can be satisfied with L- or DL-methionine

  9. Genomic, Biochemical, and Modeling Analyses of Asparagine Synthetases from Wheat

    Directory of Open Access Journals (Sweden)

    Hongwei Xu

    2018-01-01

    Full Text Available Asparagine synthetase activity in cereals has become an important issue with the discovery that free asparagine concentration determines the potential for formation of acrylamide, a probably carcinogenic processing contaminant, in baked cereal products. Asparagine synthetase catalyses the ATP-dependent transfer of the amino group of glutamine to a molecule of aspartate to generate glutamate and asparagine. Here, asparagine synthetase-encoding polymerase chain reaction (PCR products were amplified from wheat (Triticum aestivum cv. Spark cDNA. The encoded proteins were assigned the names TaASN1, TaASN2, and TaASN3 on the basis of comparisons with other wheat and cereal asparagine synthetases. Although very similar to each other they differed slightly in size, with molecular masses of 65.49, 65.06, and 66.24 kDa, respectively. Chromosomal positions and scaffold references were established for TaASN1, TaASN2, and TaASN3, and a fourth, more recently identified gene, TaASN4. TaASN1, TaASN2, and TaASN4 were all found to be single copy genes, located on chromosomes 5, 3, and 4, respectively, of each genome (A, B, and D, although variety Chinese Spring lacked a TaASN2 gene in the B genome. Two copies of TaASN3 were found on chromosome 1 of each genome, and these were given the names TaASN3.1 and TaASN3.2. The TaASN1, TaASN2, and TaASN3 PCR products were heterologously expressed in Escherichia coli (TaASN4 was not investigated in this part of the study. Western blot analysis identified two monoclonal antibodies that recognized the three proteins, but did not distinguish between them, despite being raised to epitopes SKKPRMIEVAAP and GGSNKPGVMNTV in the variable C-terminal regions of the proteins. The heterologously expressed TaASN1 and TaASN2 proteins were found to be active asparagine synthetases, producing asparagine and glutamate from glutamine and aspartate. The asparagine synthetase reaction was modeled using SNOOPY® software and information from

  10. Evaluation of Four Commercial Sources of Synthetic Methionine on ...

    African Journals Online (AJOL)

    A total of 252 day old Ross broiler chicks was used in a feeding trial to evaluate the effect of four different commercial brands of synthetic methionine supplement on the performance of broiler chickens. The chicks were distributed into four dietary treatments each with three replicates and 21 chicks/replicate; in a Completely ...

  11. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Science.gov (United States)

    2010-04-01

    ... HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L... in paragraph (d) of this section. The minimum amount of the additive to achieve the desired effect...

  12. Methionine in Velvet Bean ( Mucuna pruriens ) Based Broiler Starter ...

    African Journals Online (AJOL)

    The performance of broiler chicks fed starter diets containing 30% raw or heat treated, and 20% heat treated velvet beans with varying levels of methionine was determined. The influence of varying levels of heat treated velvet beans on growth and carcass characteristics of finishing broilers was also investigated. There was ...

  13. 10597 influence of hypo and hyper methionine supplementation

    African Journals Online (AJOL)

    dell

    It is therefore important to ensure that poultry diets contain enough methionine to ensure the desired growth, feed conversion and immune response, but not too much so as to be toxic to the birds. Newcastle disease is one of the most rampant viral diseases of poultry with a prevalence rate of 28.9% in Nigeria [10]. Two of the ...

  14. Biological activity of maleic methionine and methyl maleic ...

    African Journals Online (AJOL)

    nificantly increased wool growth. On the other hand,. Landman (1981) was unable to detect any beneficial effect of this product on hair growth of Angora goats. As far as could be established from the literature,. C5MM has not been tested as a methionine source for ruminants. Due to the fact that experiments of this nature in.

  15. Effects of infused methionine, lysine and rumen-protected ...

    African Journals Online (AJOL)

    Keratin contains about l}Vo arginine, thus a lysine-induced arginine deficiency may depress fibre production as in the study of Sahlu & Fernandez (1992) with. Angora goats. Supply of both methionine and lysine appeared to limit wool growth of sheep limit fed high roughage diets contain- ing non-protein nitrogen as the ...

  16. Effect of methionine and glucosamine conjugation on the anticancer ...

    Indian Academy of Sciences (India)

    ... methionine conjugated aromatic dinitrobenzamide mustard of 2-chlorobenzoic acid is the most effective one. It acts by inducing apoptosis through G2/M phase arrest and encouragingly, is much less toxic to nontumorigenic human embryonic kidney (HEK-293T) and mouse embryonic fibroblast (NIH 3T3) cell lines in vitro.

  17. The effects of an L-methionine combination supplement on ...

    African Journals Online (AJOL)

    Enrique

    -factors for the remethylation of L-homo- cysteine to L-methionine. SAH can be converted by 3 enzy- ... tion with vitamins E and C decreases exercise-induced oxidative ..... Rowbottom D, Green K. Acute exercise effects on the immune system.

  18. Utilization of 35S methionine by the goat

    International Nuclear Information System (INIS)

    Champredon, C.; Pion, R.

    1977-01-01

    A mixture of 2.5 g of D,L-methionine and 2.1 mCi of L- 35 S methionine is injected into the rumen of two young dry goats. Abomasal contents and blood are sampled for 4 days after intraruminal injection of the tracer. Total radioactivity and specific activity of sulfur amino acids are measured in free- and protein-bound fractions of abomasal contents and blood. The radioactivity of the abomasal content soluble fraction (TCA) increases very rapidly. The main labelled compound in the TCA extract during the first hour is methionine. Total plasma radioactivity increases during the 9 hours following the injection, then decreases slowly. It is mainly found in the extract during the first hours of the experiment, but is almost totally recovered in the protein-bound fraction 48 hours after the injection. It is concluded that a part of the 35 S is absorbed directly from the rumen as unidentified sulfur-labelled compounds and is carried by the bloodstream, but a significant proportion of the 35 S introduced into the rumen as methionine is incorporated into microbial protein or enters the intestine directly [fr

  19. Methionine metabolism and phenotypic variability in X-linked adrenoleukodystrophy

    NARCIS (Netherlands)

    Linnebank, M.; Kemp, S.; Wanders, R. J. A.; Kleijer, W. J.; van der Sterre, M. L. T.; Gärtner, J.; Fliessbach, K.; Semmler, A.; Sokolowski, P.; Köhler, W.; Schlegel, U.; Schmidt, S.; Klockgether, T.; Wüllner, U.

    2006-01-01

    A combined genotype of polymorphisms of methionine metabolism has been associated with CNS demyelination in methotrexate-treated patients. Within a sample of 86 patients with X-linked adrenoleukodystrophy, this genotype was overrepresented in a subgroup of 15 patients with adrenomyeloneuropathy

  20. Effect of Low Protein-Methionine-and-Lysine-Supplemented Diets ...

    African Journals Online (AJOL)

    Two experiments were conducted to investigate the effect of supplementing low CP diets with methionine and lysine on broiler performance, carcass measure and their immune response against Infectious Bursa Disease (IBD) virus. In Experiment 1, ten diets were formulated. Diet 1 (control diet) contained 23.0% CP and ...

  1. Diorganotin(IV) Complexes with Methionine Methyl Ester. Equilibria ...

    African Journals Online (AJOL)

    IV) (DBT) and diphenyltin(IV) (DPT) was investigated at 25 °C and 0.1 mol dm–3 ionic strength in water for dimethyltin(IV) and in 50 % dioxane–water mixture for dibutyltin(IV) and diphenyltin(IV). Methionine methyl ester forms1:1 and 1:2 ...

  2. Diorganotin(IV) Complexes with Methionine Methyl Ester. Equilibria ...

    African Journals Online (AJOL)

    NICO

    Diorganotin(IV) Complexes with Methionine Methyl Ester. Equilibria and Displacement by DNA Constituents. M.M. Shoukry1,2,* Ayser Al-Alousi2 and Sameya M. Tarek. 1Deparment of Chemistry, Faculty of Science, Islamic University, Madina, Saudi Arabia. 2Department of Chemistry, Faculty of Science, Cairo University, ...

  3. A Rare Cause of Neonatal Hemolytic Anemia: Glutathione Synthetase Deficiency.

    Science.gov (United States)

    Soylu Ustkoyuncu, Pembe; Mutlu, Fatma Türkan; Kiraz, Aslihan; Tag Balkis, Zuhal; Yel, Sibel

    2018-01-01

    Isolated hemolysis or hemolytic anemia and 5-oxoprolinuria are 2 distinct medical conditions in the clinical spectrum associated with glutathione synthetase deficiency. A 1-day-old female baby presented with anemia and respiratory distress. Her hemoglobin level was 9.5 g/dL and the total serum bilirubin level was 5.6 mg/dL. Metabolic acidosis was detected in her blood gas analysis. Metabolic acidosis recurred despite treatment and further investigation was required. Her 5-oxoproline level was 3815 mmol/mol creatinine in urine organic acid analysis, and a homozygous mutation [p.R125H (c.374G>A)] was found in the glutathione synthetase gene. GSD has been observed in very few patients and is rarely considered in the differential diagnosis of hemolytic anemia in newborns.

  4. Glutamine synthetase gene evolution: A good molecular clock

    International Nuclear Information System (INIS)

    Pesole, G.; Lanvave, C.; Saccone, C.; Bozzetti, M.P.; Preparata, G.

    1991-01-01

    Glutamine synthetase gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. The calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. The data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves

  5. A stochastic modeling of isotope exchange reactions in glutamine synthetase

    Science.gov (United States)

    Kazmiruk, N. V.; Boronovskiy, S. E.; Nartsissov, Ya R.

    2017-11-01

    The model presented in this work allows simulation of isotopic exchange reactions at chemical equilibrium catalyzed by a glutamine synthetase. To simulate the functioning of the enzyme the algorithm based on the stochastic approach was applied. The dependence of exchange rates for 14C and 32P on metabolite concentration was estimated. The simulation results confirmed the hypothesis of the ascertained validity for preferred order random binding mechanism. Corresponding values of K0.5 were also obtained.

  6. Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Bonnefond, Luc; Frugier, Magali; Touzé, Elodie; Lorber, Bernard; Florentz, Catherine; Giegé, Richard, E-mail: r.giege@ibmc.u-strasbg.fr; Rudinger-Thirion, Joëlle; Sauter, Claude [Département ‘Machineries Traductionnelles’, Architecture et Réactivité de l’ARN, Université Louis Pasteur de Strasbourg, CNRS, IBMC, 15 Rue René Descartes, 67084 Strasbourg (France)

    2007-04-01

    Crystals of human mitochondrial tyrosyl-tRNA synthetase lacking the C-terminal S4-like domain diffract to 2.7 Å resolution and are suitable for structure determination. Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA{sup Tyr} charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4{sub 3}2{sub 1}2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 Å resolution. Complete data sets could be collected and led to structure solution by molecular replacement.

  7. The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad.

    Science.gov (United States)

    Bieganowski, Pawel; Brenner, Charles

    2003-08-29

    Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

  8. LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B Encode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis thaliana[C][W][OA

    Science.gov (United States)

    Kim, Sung Soo; Grienenberger, Etienne; Lallemand, Benjamin; Colpitts, Che C.; Kim, Sun Young; Souza, Clarice de Azevedo; Geoffroy, Pierrette; Heintz, Dimitri; Krahn, Daniel; Kaiser, Markus; Kombrink, Erich; Heitz, Thierry; Suh, Dae-Yeon; Legrand, Michel; Douglas, Carl J.

    2010-01-01

    Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors. PMID:21193570

  9. A kinetic and mechanistic study on the oxidation of l-methionine and N-acetyl l-methionine by cerium(IV in sulfuric acid medium

    Directory of Open Access Journals (Sweden)

    T. Sumathi

    2016-09-01

    Full Text Available The kinetics of oxidation of l-methionine and N-acetyl l-methionine by Ce(IV in sulfuric acid–sulfate media in the range of 288.1–298.1 K has been investigated. The major oxidation products of methionine and N-acetyl l-methionine have been identified as methionine sulfoxide and N-acetyl methionine sulfoxide. The major oxidation products have been confirmed by qualitative analysis and boiling point. The reaction was first order with respect to l-methionine, N-acetyl l-methionine and Ce(IV. Increase in [H+], ionic strength and HSO4- did not affect the reaction rate. Under the experimental conditions, Ce4+ was the effective oxidizing species of cerium. Increase in dielectric constant of the medium decreased the reaction rate. Under nitrogen atmosphere, the reaction system can initiate polymerization of acrylonitrile, indicating the generation of free radicals. Activation parameters associated with the overall reaction have been calculated.

  10. Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Dharmendra; Subramanian, Kartik; Bisaria, Virendra S.; Sreekrishnan, T.R.; Gomes, James [Indian Inst. of Technology, Dept. of Biochemical Engineering and Biotechnology, New Delhi (India)

    2005-02-01

    The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l{sup -1} of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l{sup -1} h{sup -1}) every 2 h to the production medium increased the production of methionine to 3.39 g l{sup -1}. A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers. (Author)

  11. Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

    Directory of Open Access Journals (Sweden)

    Iyer Lakshminarayan M

    2010-08-01

    Full Text Available Abstract Background Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS in non-ribosomal peptide ligation. Results Here we describe novel examples of AAtRS related proteins that are likely to be involved in the synthesis of widely distributed peptide-derived metabolites. Using sensitive sequence profile methods we show that the cyclodipeptide synthases (CDPSs are members of the HUP class of Rossmannoid domains and are likely to be highly derived versions of the class-I AAtRS catalytic domains. We also identify the first eukaryotic CDPSs in fungi and in animals; they might be involved in immune response in the latter organisms. We also identify a paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptide- derived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS. We further identify an analogous system wherein the MtRS has been replaced by more typical peptide ligases with the ATP-grasp or modular condensation-domains. Conclusions The prevalence of these predicted biosynthetic pathways in phylogenetically distant, pathogenic or symbiotic bacteria suggests that metabolites synthesized by them might participate in interactions with the host. More generally, these findings point to a complete spectrum of recruitment of AAtRS to various non-ribosomal biosynthetic pathways, ranging from the

  12. Comparison of L-(1-/sup 11/C)methionine and L-methyl-(/sup 11/C)methionine for measuring in vivo protein synthesis rates with PET

    Energy Technology Data Exchange (ETDEWEB)

    Ishiwata, K.; Vaalburg, W.; Elsinga, P.H.; Paans, A.M.; Woldring, M.G.

    1988-08-01

    To evaluate the feasibility of using either L-(1-11C)-methionine or L-(methyl-11C)methionine for measuring protein synthesis rates by positron emission tomography (PET) in normal and neoplastic tissues, distribution and metabolic studies with 14C- and 11C-labeled methionines were carried out in rats bearing Walker 256 carcinosarcoma. The tissue distributions of the two 14C-labeled methionines were similar except for liver tissue. Similar distribution patterns were observed in vivo by PET using 11C-labeled methionines. The highest 14C incorporation rate into the protein-bound fraction was found in the liver followed by tumor, brain, and pancreas. The incorporation rates in liver and pancreas were different for the two methionines. By chloroform-methanol fractionation of these four tissues, in liver significantly different amounts of 14C were observed in macromolecules. Also in brain tissue slight differences were found. By HPLC analyses of the protein-free fractions of plasma, tumor, and brain tissue at 60 min after injection, for both methionines several 14C-labeled metabolites in different amounts, were detected. About half of the 14C-labeled material in the protein-free fraction was found to be methionine. In these three tissues the amount of nonprotein metabolites and (14C)bicarbonate amount ranged from 10% to 17% and 12% to 15% for L-(1-14C)methionine and L-(methyl-14C)methionine, respectively. From these results it can be concluded that the minor metabolic pathways have to be investigated in order to quantitatively model the protein synthesis by PET.

  13. Homocysteine and cysteine loads in patients with homocystinuria due to cystathionine synthase deficiency: effects of vitamin B-6.

    Science.gov (United States)

    Rassin, D K; Longhi, R C; Sternowsky, H J; Sturman, J A; Gaull, G E

    1977-08-15

    The metabolic response of patients with homocystinuria due to cystabhionine synthase deficiency to oral loads of homocysteine indicates: that even severely affected patients with homocystinuria have pools of cystine in their tissues; that control of sulfur amino acid metabolism favors increased concentrations of methionine rather than homocystine in the plasma; and that even patients who apparently are not B-6-responsive respond differently to the loads of homocysteine when challenged during B-6-treatment compared with their response before B-6 treatment. Loading tests with homocysteine indicate that B-6 treatment be of some benefit even in individuals who do not have an obvious biochemical response to such therapy.

  14. Glutamine versus ammonia utilization in the NAD synthetase family.

    Directory of Open Access Journals (Sweden)

    Jessica De Ingeniis

    Full Text Available NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS. Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS

  15. Phosphinothricin tripeptide synthetases in Streptomyces viridochromogenes Tü494.

    Science.gov (United States)

    Schwartz, Dirk; Grammel, Nicolas; Heinzelmann, Eva; Keller, Ullrich; Wohlleben, Wolfgang

    2005-11-01

    The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster of S. viridochromogenes. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from S. viridochromogenes confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (the1 and the2) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.

  16. Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Saugmann, P; Richter, Erik

    1979-01-01

    Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...... groups of rats. These changes may partly explain the demonstrated training-induced increase in glucose tolerance. None of the findings could be ascribed to differences in foold intake or body weight....

  17. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

    2011-01-01

    Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

  18. Methods and compositions for the production of orthogonal tRNA-aminoacyl-tRNA synthetase pairs

    Science.gov (United States)

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

    2011-09-06

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  19. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... of these enzymes for correct genetic code expression as well as early structural data and related enzymology will be reviewed. Despite structural diversity, all synthetases follow a two-step mechanism for tRNA aminoacylation. Specificity, however, is not absolute since synthetases were shown to catalyze ...

  20. [The anti-synthetase syndrome: muscle disease and multisystem disorder at the same time

    NARCIS (Netherlands)

    Hengstman, G.J.D.; Venrooij, W.J.W. van; Hoogen, F.H.J. van den; Engelen, B.G.M. van

    2003-01-01

    In three women, aged 60, 45 and 38 years, who presented with exertional dyspnoea (due to lung fibrosis) and Raynaud's phenomenon, dermatomyopathy and Raynaud's phenomenon, and symmetrical arthralgia and myalgia, respectively, the anti-synthetase syndrome was diagnosed. The anti-synthetase syndrome

  1. Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

    Science.gov (United States)

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

    2008-04-08

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  2. Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family.

    Science.gov (United States)

    Shiba, K; Motegi, H; Yoshida, M; Noda, T

    1998-11-15

    We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.

  3. Evidence for participation of the methionine sulfoxide reductase repair system in plant seed longevity

    OpenAIRE

    Châtelain, Emilie; Satour, Pascale; Laugier, Edith; Ly Vu, Benoit; Payet, Nicole; Rey, Pascal; Montrichard, Françoise

    2013-01-01

    Seeds are in a natural oxidative context leading to protein oxidation. Although inevitable for proper progression from maturation to germination, protein oxidation at high levels is detrimental and associated with seed aging. Oxidation of methionine to methionine sulfoxide is a common form of damage observed during aging in all organisms. This damage is reversible through the action of methionine sulfoxide reductases (MSRs), which play key roles in lifespan control in yeast and animal cells. ...

  4. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...

  5. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    OpenAIRE

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

    2009-01-01

    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 ...

  6. Ectopic expression of GsPPCK3 and SCMRP in Medicago sativa enhances plant alkaline stress tolerance and methionine content.

    Science.gov (United States)

    Sun, Mingzhe; Sun, Xiaoli; Zhao, Yang; Zhao, Chaoyue; Duanmu, Huizi; Yu, Yang; Ji, Wei; Zhu, Yanming

    2014-01-01

    So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), H(+)-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.

  7. Ectopic expression of GsPPCK3 and SCMRP in Medicago sativa enhances plant alkaline stress tolerance and methionine content.

    Directory of Open Access Journals (Sweden)

    Mingzhe Sun

    Full Text Available So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs and PEPC kinases (PPCKs fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3 as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase, H(+-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.

  8. Ectopic Expression of GsPPCK3 and SCMRP in Medicago sativa Enhances Plant Alkaline Stress Tolerance and Methionine Content

    Science.gov (United States)

    Zhao, Yang; Zhao, Chaoyue; DuanMu, Huizi; Yu, Yang; Ji, Wei; Zhu, Yanming

    2014-01-01

    So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), H+-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops. PMID:24586886

  9. Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp.

    Science.gov (United States)

    Mofid, Mohammad Reza; Finking, Robert; Marahiel, Mohamed A

    2002-05-10

    The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and

  10. Higher endogenous methionine in transgenic Arabidopsis seeds affects the composition of storage proteins and lipids.

    Science.gov (United States)

    Cohen, Hagai; Pajak, Agnieszka; Pandurangan, Sudhakar; Amir, Rachel; Marsolais, Frédéric

    2016-06-01

    Previous in vitro studies demonstrate that exogenous application of the sulfur-containing amino acid methionine into cultured soybean cotyledons and seedlings reduces the level of methionine-poor storage proteins and elevates those that are methionine-rich. However, the effect of higher endogenous methionine in seeds on the composition of storage products in vivo is not studied yet. We have recently produced transgenic Arabidopsis seeds having significantly higher levels of methionine. In the present work we used these seeds as a model system and profiled them for changes in the abundances of 12S-globulins and 2S-albumins, the two major groups of storage proteins, using 2D-gels and MALDI-MS detection. The findings suggest that higher methionine affects from a certain threshold the accumulation of several subunits of 12S-globulins and 2S-albumins, regardless of their methionine contents, resulting in higher total protein contents. The mRNA abundances of most of the genes encoding these proteins were either correlated or not correlated with the abundances of these proteins, implying that methionine may regulate storage proteins at both transcriptional and post-transcriptional levels. The elevations in total protein contents resulted in reduction of total lipids and altered the fatty acid composition. Altogether, the data provide new insights into the regulatory roles of elevated methionine levels on seed composition.

  11. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    Science.gov (United States)

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  12. The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection.

    Directory of Open Access Journals (Sweden)

    Marcell Crispim

    2018-01-01

    Full Text Available Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

  13. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

    Science.gov (United States)

    Pyne, C; Bognar, A L

    1992-03-01

    The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.

  14. Holocarboxylase synthetase deficiency pre and post newborn screening

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-06-01

    Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

  15. Purification and properties of the dihydrofolate synthetase from Serratia indica

    International Nuclear Information System (INIS)

    Ikeda, Masamichi; Iwai, Kazuo

    1976-01-01

    The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg 2+ and K + as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

  16. Prediction of Methionine and Homocysteine levels in Zucker diabetic fatty (ZDF) rats as a T2DM animal model after consumption of a Methionine-rich diet.

    Science.gov (United States)

    Han, Nayoung; Chae, Jung-Woo; Jeon, Jihyun; Lee, Jaeyeon; Back, Hyun-Moon; Song, Byungjeong; Kwon, Kwang-Il; Kim, Sang Kyum; Yun, Hwi-Yeol

    2018-01-01

    Although alterations in the methionine metabolism cycle (MMC) have been associated with vascular complications of diabetes, there have not been consistent results about the levels of methionine and homocysteine in type 2 diabetes mellitus (T2DM). The aim of the current study was to predict changes in plasma methionine and homocysteine concentrations after simulated consumption of methionine-rich foods, following the development of a mathematical model for MMC in Zucker Diabetic Fatty (ZDF) rats, as a representative T2DM animal model. The model building and simulation were performed using NONMEM® (ver. 7.3.0) assisted by Perl-Speaks-NONMEM (PsN, ver. 4.3.0). Model parameters were derived using first-order conditional estimation method with interactions permitted among the parameters (FOCE-INTER). NCA was conducted using Phoenix (ver. 6.4.0). For all tests, we considered a P -value < 0.05 to reflect statistical significance. Our model featured seven compartments that considered all parts of the cycle by applying non-linear mixed effects model. Conversion of S-adenosyl-L-homocysteine (SAH) to homocysteine increased and the metabolism of homocysteine was reduced under diabetic conditions, and consequently homocysteine accumulated in the elimination phase.Using our model, we performed simulations to compare the changes in plasma methionine and homocysteine concentrations between ZDF and normal rats, by multiple administrations of the methionine-rich diet of 1 mmol/kg, daily for 60 days. The levels of methionine and homocysteine were elevated approximately two- and three-fold, respectively, in ZDF rats, while there were no changes observed in the normal control rats. These results can be interpreted to mean that both methionine and homocysteine will accumulate in patients with T2DM, who regularly consume high-methionine foods.

  17. A sub-threshold dose of pilocarpine increases glutamine synthetase in reactive astrocytes and enhances the progression of amygdaloid-kindling epilepsy in rats.

    Science.gov (United States)

    Sun, Hong-Liu; Deng, Da-Ping; Pan, Xiao-Hong; Wang, Chao-Yun; Zhang, Xiu-Li; Chen, Xiang-Ming; Wang, Chun-Hua; Liu, Yu-Xia; Li, Shu-Cui; Bai, Xian-Yong; Zhu, Wei

    2016-03-02

    The prognosis of patients exposed to a sub-threshold dose of a proconvulsant is difficult to establish. In this study, we investigated the effect of a single sub-threshold dose of the proconvulsant pilocarpine (PILO) on the progression of seizures that were subsequently induced by daily electrical stimulation (kindling) of the amygdaloid formation. Male Sprague–Dawley rats were each implanted with an electrode in the right basolateral amygdala and an indwelling cannula in the right ventricle. The animals were randomized into groups and were administered one of the following treatments: saline, PILO, saline+L-α-aminoadipic acid (L-AAA; one dosage tested), PILO+L-AAA, or PILO+L-methionine sulfoximine (three dosages tested). Amygdaloid stimulation and electroencephalography were performed once daily. We performed immunohistochemistry and western blot for glial fibrillary acidic protein and glutamine synthetase (GS). We also assayed the enzymic activity of GS in discrete brain regions. An intraperitoneal injection of a sub-threshold PILO dose enhanced the progression of amygdaloid-kindling seizures and was accompanied by an increase in reactive-astrocyte and GS (content and activity) in the hippocampus and piriform cortex. L-AAA and L-methionine sulfoximine, inhibitors of astrocytic and GS function, respectively, abolished the effect of PILO on amygdaloid-kindling seizures. We conclude that one sub-threshold dose of a proconvulsant may enhance the progression of subsequent epilepsy and astrocytic GS may play a role in this phenomenon. Thus, a future therapy for epilepsy could be inhibition of astrocytes and/or GS.

  18. Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R: partial purification and properties of methionyl-transfer ribonucleic acid synthetase and methionyl-transfer ribonucleic acid formyltransferase.

    Science.gov (United States)

    Samuel, C E; Rabinowitz, J C

    1974-04-01

    The initiation of protein synthesis by Streptococcus faecalis R grown in folate-free culture occurs without N-formylation or N-acylation of methionyl-tRNA(f) (Met). Methionyl-tRNA synthetase and methionyl-tRNA formyltransferase were partially purified from S. faecalis grown under normal culture conditions in the presence of folate (plus-folate); the general properties of the enzymes were determined and compared with the properties of the enzymes purified from wild-type cells grown in the absence of folate (minus-folate). S. faecalis methionyl-tRNA synthetase displays optimal activity at pH values between 7.2 and 7.8, requires Mg(2+), and has an apparent molecular weight of 106,000, as determined by gel filtration, and 127,000, as determined by sucrose density gradient centrifugation. The K(m) values of plus-folate methionyl-tRNA synthetase for each of the three substrates in the aminoacylation reaction (l-methionine, adenosine triphosphate, and tRNA) are nearly identical to the respective substrate Michaelis constants of minus-folate methionyl-tRNA synthetase. Furthermore, both plus- and minus-folate S. faecalis methionyl-tRNA synthetases catalyze, at equal rates, the aminoacylation of tRNA(f) (Met) and tRNA(m) (Met) isolated from either plus-folate or minus-folate cells. S. faecalis methionyl-tRNA formyltransferase displays optimal activity at pH values near 7.0, is stimulated by Mg(2+), and has an apparent molecular weight of approximately 29,900 when estimated by sucrose density gradient centrifugation. The K(m) value of plus-folate formyltransferase for plus-folate Met-tRNA(f) (Met) does not differ significantly from that of minus-folate formyltransferase for minus-folate Met-tRNA(f) (Met). Both enzymes can utilize either 10-formyltetrahydrofolate or 10-formyltetrahydropteroyltriglutamate as the formyl donor; the Michaelis constant for the monoglutamyl pteroyl coenzyme is slightly less than that of the triglutamyl pteroyl coenzyme for both transformylases

  19. Leucine responsive regulatory protein is involved in methionine metabolism and polyamine homeostasis in acetic acid bacterium Komagataeibacter europaeus.

    Science.gov (United States)

    Ishii, Yuri; Akasaka, Naoki; Sakoda, Hisao; Hidese, Ryota; Fujiwara, Shinsuke

    2018-01-01

    The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 μM) and KGMA7203 (4.90±0.61 μM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 μM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Targeting CPS1 in the treatment of Carbamoyl phosphate synthetase 1 (CPS1) deficiency, a urea cycle disorder.

    Science.gov (United States)

    Diez-Fernandez, Carmen; Häberle, Johannes

    2017-04-01

    Carbamoyl phosphate synthetase 1 (CPS1) deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder (UCD), which can lead to life-threatening hyperammonemia. Unless promptly treated, it can result in encephalopathy, coma and death, or intellectual disability in surviving patients. Over recent decades, therapies for CPS1D have barely improved leaving the management of these patients largely unchanged. Additionally, in many cases, current management (protein-restriction and supplementation with citrulline and/or arginine and ammonia scavengers) is insufficient for achieving metabolic stability, highlighting the importance of developing alternative therapeutic approaches. Areas covered: After describing UCDs and CPS1D, we give an overview of the structure- function of CPS1. We then describe current management and potential novel treatments including N-carbamoyl-L-glutamate (NCG), pharmacological chaperones, and gene therapy to treat hyperammonemia. Expert opinion: Probably, the first novel CPS1D therapies to reach the clinics will be the already commercial substance NCG, which is the standard treatment for N-acetylglutamate synthase deficiency and has been proven to rescue specific CPS1D mutations. Pharmacological chaperones and gene therapy are under development too, but these two technologies still have key challenges to be overcome. In addition, current experimental therapies will hopefully add further treatment options.

  1. Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

    Science.gov (United States)

    Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

    2016-08-02

    Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

  2. Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily.

    Science.gov (United States)

    Reuter, K; Mofid, M R; Marahiel, M A; Ficner, R

    1999-12-01

    The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

  3. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    International Nuclear Information System (INIS)

    Bandaralage, Sahan P.S.; Farnaghi, Soheil; Dulhunty, Joel M.; Kothari, Alka

    2016-01-01

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  4. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    Energy Technology Data Exchange (ETDEWEB)

    Bandaralage, Sahan P.S. [Gold Coast Hospital and Health Service, Southport, Queensland (Australia); Griffith University, School of Medicine, Southport, Queensland (Australia); Farnaghi, Soheil [Caboolture Hospital, Caboolture, Queensland (Australia); Dulhunty, Joel M.; Kothari, Alka [Redcliffe Hospital, Redcliffe, Queensland (Australia); The University of Queensland, School of Medicine, Herston, Queensland (Australia)

    2016-03-15

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  5. Seryl-tRNA Synthetases in Translation and Beyond

    Directory of Open Access Journals (Sweden)

    Marko Močibob

    2016-06-01

    Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

  6. Allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase in Clostridium: another conserved generic characteristic.

    Science.gov (United States)

    Jensen, R A; Twarog, R

    1972-09-01

    Enzymological studies were done to characterize the allosteric control of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase in three species of Clostridium. Allosteric control was identified as feedback inhibition by phenylalanine and was qualitatively similar for the DAHP synthetases of C. butyricum, C. acetobutylicum, and C. tetanomorphum. Quantitative differences in the enzymology and kinetics of allosteric control distinguished C. tetanomorphum from C. butyricum and C. acetobutylicum. Crude extracts contained apparent proteolytic activity which could be fractionated from DAHP synthetase. The proteolytic activity was more labile than DAHP synthetase in extracts and was progressively inactivated by serial freeze-thaw treatments. Protease activity was at least partially inhibited by phenylmethylsulfonyl-fluoride. The method of comparative allostery of DAHP synthetase distinguishes the genera Bacillus and Clostridium, each having a strongly conserved pattern of regulation for DAHP synthetase. The data reinforce previous conclusions that allosteric control patterns governing the activity of DAHP synthetase are stable, reliable generic characteristics.

  7. Relative bioavailability of manganese in a manganese-methionine complex for broiler chicks.

    Science.gov (United States)

    Henry, P R; Ammerman, C B; Miles, R D

    1989-01-01

    The relative bioavailability of Mn from reagent grade Mn monoxide and feed grade Mn-methionine was compared with that from reagent grade Mn sulfate using 288 one-day-old male Cobb chicks. The basal corn-soybean meal diet (93 ppm Mn dry matter basis) was supplemented with 0, 700, 1,400, and 2,100 ppm Mn as Mn sulfate monohydrate, Mn oxide, or Mn-methionine. Additional diets contained 700, 1,400, and 2,100 ppm Mn as sulfate or oxide in combination with .16, .32, or .48% added DL-methionine, respectively, to equalize methionine concentrations in Mn-methionine-containing diets. Diets were fed ad libitum for 3 wk. Tibia and kidney Mn concentrations increased linearly (P less than .001) as dietary Mn increased. Addition of methionine to diets containing sulfate and oxide did not influence (P greater than .10) tissue Mn concentrations. Based on slope ratios from multiple linear regression of bone and kidney Mn concentrations on added dietary Mn from various sources, the respective relative bioavailability values were 96 and 86% from Mn oxide and 108 and 132% from Mn-methionine compared with 100% from Mn sulfate. Except for the first, all values were significantly different from 100%. Thus, Mn from Mn oxide is significantly less available and Mn from Mn-methionine is significantly more available than that from Mn sulfate monohydrate.

  8. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    Energy Technology Data Exchange (ETDEWEB)

    Berges, J [Universite Pierre et Marie Curie, UMR 7616, Laboratoire de Chimie Theorique, 75005 Paris (France); Trouillas, P [EA 4021 Faculte de Pharmacie, 2 Rue du Dr. Marcland, 87025 Limoges Cedex (France); Houee-Levin, C, E-mail: jb@lct.jussieu.fr, E-mail: patrick.trouillas@unilim.fr, E-mail: chantal.houee@u-psud.fr [Universite Paris Sud, UMR 8000, Laboratoire de Chimie Physique, 91405 Orsay (France) (France)

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH{sup -} elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S:X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  9. Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase.

    Science.gov (United States)

    Jin, Li-Qun; Li, Zong-Tong; Liu, Zhi-Qiang; Zheng, Yu-Guo; Shen, Yin-Chu

    2014-10-01

    Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.

  10. Influence of dietary methionine on the metabolism of selenomethionine in rats

    International Nuclear Information System (INIS)

    Butler, J.A.; Beilstein, M.A.; Whanger, P.D.

    1989-01-01

    To determine the influence of methionine on selenomethionine (SeMet) metabolism, weanling male rats were fed for 8 wk a basal diet marginally deficient in sulfur amino acids, containing 2.0 micrograms selenium (Se)/g as DL-SeMet and supplemented with 0, 0.3, 0.6 or 1.2% DL-methionine. Increased dietary methionine caused decreased selenium deposition in all tissues examined but increased glutathione peroxidase activity in testes, liver and lungs. A positive correlation was found between dietary methionine and the calculated percentage of selenium associated with GSHPx. In a second experiment, 75 SeMet was injected into weanling male rats which had been fed the basal diet containing 2.0 micrograms selenium as DL-SeMet with or without the addition of 1.0% methionine. The selenoamino acid content of tissues and the distribution of 75 Se in erythrocyte proteins were determined. In comparison to the rats fed the basal diet without added methionine, significantly more 75 Se-selenocysteine was found in liver and muscle, more 75 Se was found in erythrocyte GSHPx and less 75 Se was found in erythrocyte hemoglobin of rats fed 1.0% methionine. These data suggest that methionine diverts SeMet from incorporation into general proteins and enhances its conversion to selenocysteine for specific selenium-requiring proteins, such as GSHPx

  11. Comparison of the sustainability metrics of the petrochemical and biomass-based routes to methionine

    NARCIS (Netherlands)

    Sanders, J.P.M.; Sheldon, R.A.

    2015-01-01

    Sustainability metrics, based on material efficiency, energy input, land use and costs, of three processesfor the manufacture of methionine are compared. The petrochemical process affords dl-methionine whilethe two biomass-based routes afford the l-enantiomer. From the point of view of the major

  12. Genetic and biochemical differences in populations bred for extremes in maize grain methionine concentration.

    Science.gov (United States)

    Newell, Mark A; Vogel, Karla E; Adams, Marie; Aydin, Nevzat; Bodnar, Anastasia L; Ali, Muhammad; Lauter, Adrienne N Moran; Scott, M Paul

    2014-02-19

    Methionine is an important nutrient in animal feed and several approaches have been developed to increase methionine concentration in maize (Zea mays L.) grain. One approach is through traditional breeding using recurrent selection. Using divergent selection, genetically related populations with extreme differences in grain methionine content were produced. In order to better understand the molecular mechanisms controlling grain methionine content, we examined seed proteins, transcript levels of candidate genes, and genotypes of these populations. Two populations were selected for high or low methionine concentration for eight generations and 40 and 56% differences between the high and low populations in grain methionine concentration were observed. Mean values between the high and low methionine populations differed by greater than 1.5 standard deviations in some cycles of selection. Other amino acids and total protein concentration exhibited much smaller changes. In an effort to understand the molecular mechanisms that contribute to these differences, we compared transcript levels of candidate genes encoding high methionine seed storage proteins involved in sulfur assimilation or methionine biosynthesis. In combination, we also explored the genetic mechanisms at the SNP level through implementation of an association analysis. Significant differences in methionine-rich seed storage protein genes were observed in comparisons of high and low methionine populations, while transcripts of seed storage proteins lacking high levels of methionine were unchanged. Seed storage protein levels were consistent with transcript levels. Two genes involved in sulfur assimilation, Cys2 and CgS1 showed substantial differences in allele frequencies when two selected populations were compared to the starting populations. Major genes identified across cycles of selection by a high-stringency association analysis included dzs18, wx, dzs10, and zp27. We hypothesize that transcriptional

  13. Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.

    Science.gov (United States)

    Borrego, Stacey L; Fahrmann, Johannes; Datta, Rupsa; Stringari, Chiara; Grapov, Dmitry; Zeller, Michael; Chen, Yumay; Wang, Ping; Baldi, Pierre; Gratton, Enrico; Fiehn, Oliver; Kaiser, Peter

    2016-01-01

    The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as "methionine dependence" or "methionine stress sensitivity" in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8. Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential. This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive

  14. Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

    Directory of Open Access Journals (Sweden)

    Adam C. Mirando

    2014-12-01

    Full Text Available In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.

  15. Prediction of the Hydrogen Peroxide-Induced Methionine Oxidation Propensity in Monoclonal Antibodies.

    Science.gov (United States)

    Agrawal, Neeraj J; Dykstra, Andrew; Yang, Jane; Yue, Hai; Nguyen, Xichdao; Kolvenbach, Carl; Angell, Nicolas

    2018-01-08

    Methionine oxidation in therapeutic antibodies can impact the product's stability, clinical efficacy, and safety and hence it is desirable to address the methionine oxidation liability during antibody discovery and development phase. Although the current experimental approaches can identify the oxidation-labile methionine residues, their application is limited mostly to the development phase. We demonstrate an in silico method that can be used to predict oxidation-labile residues based solely on the antibody sequence and structure information. Since antibody sequence information is available in the discovery phase, the in silico method can be applied very early on to identify the oxidation-labile methionine residues and subsequently address the oxidation liability. We believe that the in silico method for methionine oxidation liability assessment can aid in antibody discovery and development phase to address the liability in a more rational way. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  16. Intestinal digestive enzyme activity under the influence of different dietary supplements methionine and lysine in the diet of Sparidentex hasta

    OpenAIRE

    Movahedian, R.; Zakeri, M.; Kochanian, P.; Mousavi, S.M.; Taghavi Moghadam, A.

    2016-01-01

    This study was conducted to determine the effects of dietary methionine and lysine supplementation on digestive enzymes activity in juvenile Sobaity, Sparidentex hasta. For this purpose, 180 juvenile fish with an initial average weight of 31.38 ± 1.4 g were distributed randomly in eighteen (300 L) polyethylene tanks. 6 experimental diets were prepared with different levels of methionine and lysine including control diet (without dietary methionine and lysine), Diet 1: 100% methionine; Diet 2:...

  17. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Edward M. [Univ. of Rochester, NY (United States). Dept. of Radiation Biology and Biophysics

    1980-01-01

    Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy+ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy+ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy+ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)

  18. Common peptides study of aminoacyl-tRNA synthetases.

    Directory of Open Access Journals (Sweden)

    Assaf Gottlieb

    Full Text Available BACKGROUND: Aminoacyl tRNA synthetases (aaRSs constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains. RESULTS: We utilized the Common Peptides (CPs framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS-class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA enzyme overlapping binding sites in both families. CONCLUSIONS: The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families.

  19. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  20. diet with l-asd d,l-methionine on the growth of yiudfish clar/as ...

    African Journals Online (AJOL)

    BSN

    Almough the meal contains all me essential amino acids in its protein, me amoW1t of memionine present ... more amino acids is to supplement the protein with appropriate amounts of the amino acid in question. ;.,:aneko (1969) ..... sulfur compounds by fingerlings channel catfish L-methionine, D-L-, methionine, methionine ...

  1. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte–neuron co-cultures

    Science.gov (United States)

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-01-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of 15NH4+ in alanine during acute hyperammonemia. We observed a fourfold increased amount of 15NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to 15NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of 15NH4 into alanine together with increased 15N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy. PMID:23673435

  2. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte-neuron co-cultures.

    Science.gov (United States)

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-08-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of (15)NH4(+) in alanine during acute hyperammonemia. We observed a fourfold increased amount of (15)NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to (15)NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of (15)NH4 into alanine together with increased (15)N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

  3. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase

    Science.gov (United States)

    Junker, Anne; Fischer, Juliane; Sichhart, Yvonne; Brandt, Wolfgang; Dräger, Birgit

    2013-01-01

    Putrescine N-methyltransferases (PMTs) are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-l-methionine (SAM) as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs), which are ubiquitous enzymes of polyamine metabolism. SPDSs use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in D. stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom. PMID:23908659

  4. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase.

    Directory of Open Access Journals (Sweden)

    Anne eJunker

    2013-07-01

    Full Text Available Putrescine N-methyltransferases (PMTs are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-L-methionine (SAM as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs, which are ubiquitous enzymes of polyamine metabolism. SPDS use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in Datura stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom.

  5. Polymorphism in folate- and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer.

    Science.gov (United States)

    Kang, Sokbom; Kim, Jae Weon; Kang, Gyeong Hoon; Park, Noh Hyun; Song, Yong Sang; Kang, Soon Beom; Lee, Hyo Pyo

    2005-01-01

    This study was conducted to explore the association between the CpG island hypermethylation of tumor-associated genes and the polymorphisms of methyl group metabolizing enzymes in uterine cervical cancer. We analyzed CpG island hypermethylation in 15 genes (APC, CDH1, COX2, DAPK, FHIT, GSTP1, HLTF1, hMLH1, MGMT, p14, p16, RASSF1A, RUNX3, THBS1, and TIMP3) and its association with the methylene-tetrahydrofolate reductase (MTHFR) C677T and A1298C and the methionine synthase (MS) A2756G polymorphisms in 82 Korean women with uterine cervical cancer. All uterine cervical cancer samples had at least one gene methylated. The average number of methylated genes was lower in patients with the heterozygous genotype of MTHFR and MS than in those with the common homozygous genotype, although this difference was not significant. The MTHFR 677 CT genotype was significantly associated with the decreased promoter hypermethylation of O(6)-methylguanine DNA methyltransferase (MGMT) (OR = 0.22, 95% confidence interval (CI) 0.07-0.70, P = 0.011). However, the MTHFR C677T and A1298C and the MS A2756G polymorphisms were not associated with an increased risk of uterine cervical cancer. These findings suggest that there is a possible interaction between epigenetic and genetic factors in uterine cervical cancer.

  6. Effects of Dietary Methionine Levels on Choline Requirements of Starter White Pekin Ducks

    Directory of Open Access Journals (Sweden)

    Z. G. Wen

    2016-12-01

    Full Text Available A 2×5 factorial experiment, using 2 dietary methionine levels (0.28% and 0.48% and 5 dietary choline levels (0, 394, 823, 1,239, and 1,743 mg/kg, was conducted to study the effects of dietary methionine status on choline requirements of starter white Pekin ducks from 7 to 28 days of age. Four hundred eighty 7-d-old male White Pekin ducks were randomly allotted to ten dietary treatments, each containing 6 replicate pens with 8 birds per pen. At 28 d of age, weight gain, feed intake, and feed/gain were measured and the legs of all ducks from each pen were examined for incidence of perosis. Perosis and growth depression were observed in choline-deficient ducks and supplementation of choline reduced perosis and significantly increased weight gain and feed intake regardless of dietary methionine levels (p<0.05. In addition, significant positive effects of dietary methionine supplementation on weight gain, feed intake, and feed/gain were observed at any choline level (p<0.05. Supplementation of 1,743 mg/kg choline in diets alleviated the depression of weight gain and feed intake caused by methionine deficiency at 0.28% methionine level. The interaction between choline and methionine influenced weight gain and feed intake of ducks (p<0.05. At 0.28% methionine level, 1,743 mg/kg choline group caused 4.92% and 3.23% amount of improvement in weight gain and feed intake compared with 1,239 mg/kg choline group, respectively. According to the broken-line regression, the choline requirements of starter Pekin ducks for weight gain and feed intake were 1,472 and 1,424 mg/kg at 0.28% methionine level and 946 and 907 mg/kg at 0.48% methionine level, respectively. It suggested the choline recommendations of starter Pekin ducks on a semi-purified diet were 1448 mg/kg at 0.28% methionine level and 927 mg/kg at 0.48% methionine level, respectively. Compared with the adequate methionine level, menthionine deficiency markedly increased the choline requirements of

  7. Studies towards the synthesis of ATP analogs as potential glutamine synthetase inhibitors

    CSIR Research Space (South Africa)

    Salisu, S

    2011-05-01

    Full Text Available In research directed at the development of adenine triphosphate (ATP) analogs as potential glutamine synthetase (GS) inhibitors, adenine and allopurinol derivatives have been synthesized either as novel ATP analogs or as scaffolds...

  8. Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes

    NARCIS (Netherlands)

    Lamers, W. H.; van Roon, M.; Mooren, P. G.; de Graaf, A.; Charles, R.

    1985-01-01

    A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic

  9. Аldosterone synthetase gene (CYP11B2 polymorphism and structural parameters of the left ventricle in patients with coronary heart disease, postinfarction cardiosclerosis

    Directory of Open Access Journals (Sweden)

    M. N. Dolzhenko

    2017-12-01

    Full Text Available Purpose of the work – to investigate the possible contribution of aldosterone synthetase gene (CYP11B2 polymorphism to the disease course and structural parameters of LV in patients with coronary heart disease, postinfarction cardiosclerosis. Materials and мethods. General clinical examination of 100 patients with postinfarction cardiosclerosis was done at the Cardiology Department of P. L Shupyk NMAPE. Genetic testing was performed by polymerase chain reaction in real time at the Bogomolets Institute of Physiology,Kyiv,Ukraine. Exclusion criteria were hemodynamically significant valvular heart diseases, chronic obstructive pulmonary diseases, permanent or temporary heart pacing, acute heart failure and implanted cardioverter-defibrillator, permanent atrial fibrillation. Statistical analysis of the results was performed using Microsoft Excel, the statistical program SPSS (version 20, US. The results obtained are presented as M ± σ. Results. The stenosis of the left main coronary artery was observed in 25.9 % of cases in the subgroup of the TT variant. It should be noted that in the TC subgroup of aldosterone synthase gene variant polymorphism the incidence of the left main coronary artery lesion was 13.9 %. There has been no single case of left main coronary artery lesion in the SS subgroup with little statistical significance in comparison with the subgroup of TT variant of the polymorphism (P = 0.048. In the analysis of clinical data the most marked manifestations of angina pectoris were in subgroups of TT and TC – 73.3 % and 72.7 %, respectively, compared with CC subgroup – 40 %, reliable for both subgroups (P1.2 = 0.95, P1.3 = 0.039, P2.3 = 0.029. In the analysis of LV morphological characteristics the smallest indices of the LV mass have been revealed in the CC subgroup of the polymorphism variant (190.5 ± 52.1 g, compared with the LV mass values in the TT subgroup (231.00 ± 55.21 g, P = 0.03 and TC (197.421 ± 63.15, P > 0.05. A

  10. Producing biofuels using polyketide synthases

    Science.gov (United States)

    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.

  11. Vibrational and thermal study of l-methionine nitrate polycrystals

    Energy Technology Data Exchange (ETDEWEB)

    Victor, F.M.S.; Ribeiro, L.H.L.; Facanha Filho, P.F.; Santos, C.A.S.; Soares, R.A.; Abreu, D.C.; Sousa, J.C.F.; Carvalho, J.O.; Santos, A.O. dos [Universidade Federal do Maranhao (UFMA), MA (Brazil)

    2016-07-01

    Full text: Intensified in studies of nonlinear optical materials has been observed over the past two decades for its wide application in telecommunications, optical modulation and optical signal processing. The goal of this work is the thermal and vibrational study of L-methionine nitrate polycrystalline. The polycrystals were obtained by the method of slow evaporation of solvent at ambient temperature of 25 ° C. The X-ray diffraction was performed to confirm the structure of the material, which has monoclinic structure (space group P21) with four molecules per unit cell structure. Refinement by Rietveld method has been optimized and good quality parameters Rwp = 7.97% , Rp = 5.74 and S = 1.92%. The thermal stability of the material was verified from Thermogravimetric analysis (TGA), Differential Thermal Analysis (DTA) and Differential Scanning Calorimetry (DSC). The measures showed a possible phase transition event at about 107°C before the melting point of the material, which took place at about 127°C. Thermogravimetric analysis showed two mass loss events of 61.5% and 30.4%. The vibrational modes of the L-methionine nitrate molecule were identified by Raman spectroscopy in the spectral range between 35cm-1 and 3500 cm-1, the scattering measurements were made from room temperature up to the melting temperature of the material (140 ° C ) in which the disappearance of bands was found in the region of normal modes at 130 ° C, thus demonstrating a irreversible structural phase transition, because the spectrum obtained after returning the sample to ambient temperature is typical of amorphous material. (author)

  12. Methionine restriction alters bone morphology and affects osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Amadou Ouattara

    2016-12-01

    Full Text Available Methionine restriction (MR extends the lifespan of a wide variety of species, including rodents, drosophila, nematodes, and yeasts. MR has also been demonstrated to affect the overall growth of mice and rats. The objective of this study was to evaluate the effect of MR on bone structure in young and aged male and female C57BL/6J mice. This study indicated that MR affected the growth rates of males and young females, but not aged females. MR reduced volumetric bone mass density (vBMD and bone mineral content (BMC, while bone microarchitecture parameters were decreased in males and young females, but not in aged females compared to control-fed (CF mice. However, when adjusted for bodyweight, the effect of MR in reducing vBMD, BMC and microarchitecture measurements was either attenuated or reversed suggesting that the smaller bones in MR mice is appropriate for its body size. In addition, CF and MR mice had similar intrinsic strength properties as measured by nanoindentation. Plasma biomarkers suggested that the low bone mass in MR mice could be due to increased collagen degradation, which may be influenced by leptin, IGF-1, adiponectin and FGF21 hormone levels. Mouse preosteoblast cell line cultured under low sulfur amino acid growth media attenuated gene expression levels of Col1al, Runx2, Bglap, Alpl and Spp1 suggesting delayed collagen formation and bone differentiation. Collectively, our studies revealed that MR altered bone morphology which could be mediated by delays in osteoblast differentiation. Keywords: Methionine restriction, Aged mice, Micro-computed tomography, Nanoindentation, MC3T3-E1 subclone 4

  13. REPRESSION BY ADENINE OF THE FORMYLTETRAHYDROFOLATE SYNTHETASE IN AN ANTIFOLIC-RESISTANT MUTANT OF STREPTOCOCCUS FAECALIS.

    Science.gov (United States)

    ALBRECHT, A M; HUTCHISON, D J

    1964-04-01

    Albrecht, Alberta M. (Sloan-Kettering Institute for Cancer Research, New York, N.Y.), and Dorris J. Hutchison. Repression by adenine of the formyltetrahydrofolate synthetase in an antifolic-resistant mutant of Streptococcus faecalis. J. Bacteriol. 87:792-798. 1964.-In an amethopterin-resistant mutant of Streptococcus faecalis ATCC 8043 under cultivation conditions requiring purine synthesis de novo, both the dihydrofolate reductase and the formyltetrahydrofolate synthetase were formed as constant fractions of the total protein synthesized during the exponential phase of growth. When excess adenine was added to the medium, the rate of formation of the synthetase was markedly decreased, i.e., repressed. Under these latter conditions, the synthesis of the reductase proceeded at a rate equal to that observed in the absence of adenine. The repressibility of the synthetase by adenine was demonstrated also by the decrease in rate of synthetase formation upon the addition of adenine to a culture actively synthesizing this enzyme. Guanine and hypoxanthine, like adenine, also repressed the synthetase; exogenous xanthine was less effective. Neither of the pyrimidines, thymine and uracil, at approximately 1 mug/ml, interfered with synthesis of the two enzymes.

  14. Gain-Of-Function Mutational Activation of Human TRNA Synthetase Procytokine

    Energy Technology Data Exchange (ETDEWEB)

    Yang, X.L.; Kapoor, M.; Otero, F.J.; Slike, B.M.; Tsuruta, H.; Frausto, R.; Bates, A.; Ewalt, K.L.; Cheresh, D.A.; Schimmel, P.; /Scripps Res. Inst. /SLAC, SSRL

    2009-04-30

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

  15. Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation

    Science.gov (United States)

    Aledo, Juan C.; Cantón, Francisco R.; Veredas, Francisco J.

    2015-01-01

    Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants. PMID:26597773

  16. Methionine-oxidized horse heart cytochrome c. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage.

    Science.gov (United States)

    Myer, Y P; Kumar, S

    1989-02-01

    The ascorbate reduction of the CT-cytochromes--two chemically generated forms of horse heart cytochrome c, FIII and FII, with both methionines, 80 and 65, as methionine sulfoxides, no iron-sulfur linkage, and potentiometric and physiological oxidoreduction properties distinct from those of the native protein and one another (J. Pande et al., 1987)--has been investigated using a stopped-flow technique. The reaction was monitored at 550 nm, and studies were conducted in 10 mM phosphate + 0.17 M NaCl buffer, pH 7.4. Both CT-cytochromes are reduced by triphasic profiles, a faster and an intermediate ascorbate-dependent reaction and a slow, ascorbate-independent process. Both CT-cytochromes contain three molecular forms in slow equilibrium, two reducing directly by reaction with ascorbate and a third through conversion to one of the reducible forms. Like the reaction of the native protein, the ascorbate dependence of both the rapid and the intermediate process is nonlinear, approaching saturation values at high concentrations. The ascorbate profiles of the pseudo-first-order reduction constants are typical of the model for the reduction reaction of the unmodified protein, binding followed by a first-order reduction reaction (Myer et al., 1980; Myer and Kumar, 1984), but with distinct kinetic parameters, the first-order reduction constants and the protein-ascorbate stability constants. It has been concluded that the functional-conformational differences between the two CT-cytochromes are not operational to any significant extent in the reduction reaction with ascorbate. The methionine-80-sulfur-iron linkage of the protein is not a crucial requirement for the ascorbate reduction of the protein. The mechanism of the reaction in the main is also insensitive to the replacement of Met-80-S from heme coordination and/or the associated conformational-oxidoreduction properties of the protein. Of the two aspects of the reaction, the efficiency of the electron-transfer reaction

  17. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

    Science.gov (United States)

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2017-06-01

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

  18. Dietary Methionine Requirement for Growth of Juvenile Humpback Grouper (Cromileptes altivelis

    Directory of Open Access Journals (Sweden)

    Nyoman Adiasmara Giri

    2006-12-01

    Full Text Available An experiment to find out amino acid methionine requirement for growth of juvenile humpback grouper has been conducted in 18 polycarbonate 100 L tanks. Each tank was equipped with flow-through water system. Twelve juveniles of humpback grouper (5.6 ± 0.7 g each were randomly selected and stocked in each tank. Juveniles humpback grouper for the experiment were purchased from back yard hatchery in Gondol. Fish fed test diets twice everyday at satiation level for 49 days. Test diets were prepared as dry pellet with casein and fish meal as the intact protein sources, supplemented with the mixture of crystalline L-amino acids to correspond to the amino acid pattern found in the whole body protein of the juvenile humpback grouper, except methionine. Basal diet (diet-1 containing 0.86% methionine was supplied from casein and fish meal. Graded level (0.3% of L-methionine was added to the basal diet to get the final methionine level in each test diet of 0.86%, 1.16%, 1.46%, 1.76%, 2.06%, and 2.36 %. The experiment was designed according to completely random design (CRD with 6 treatments (methionine levels and three replicates for each treatment. Result of the experiment showed that dietary methionine content influenced final weight, weight gain, specific growth rate, feed efficiency, protein retention, and body protein content of juvenile humpback grouper. Optimum dietary methionine for juvenile humpback grouper was calculated using broken line regression analysis.  Optimum dietary methionine requirement for growth of juvenile humpback grouper was 1.18% (2.41% of dietary protein and 1.16% (2.37% of dietary protein based on weight gain data and feed efficiency, respectively.

  19. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  20. Characterization of FdmV as an Amide Synthetase for Fredericamycin A Biosynthesis in Streptomyces griseus ATCC 43944*

    OpenAIRE

    Chen, Yihua; Wendt-Pienkowski, Evelyn; Ju, Jianhua; Lin, Shuangjun; Rajski, Scott R.; Shen, Ben

    2010-01-01

    Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the...

  1. Nitric oxide synthetase and Helicobacter pylori in patients undergoing appendicectomy.

    LENUS (Irish Health Repository)

    Kell, M R

    2012-02-03

    BACKGROUND: This study was designed to determine whether Helicobacter pylori forms part of the normal microenvironment of the appendix, whether it plays a role in the pathogenesis of acute appendicitis, and whether it is associated with increased expression of inducible nitric oxide synthetase (iNOS) in appendicular macrophages. METHODS: Serology for H. pylori was performed on 51 consecutive patients undergoing emergency appendicectomy. Appendix samples were tested for urease activity, cultured and stained for H. pylori, graded according to the degree of inflammatory infiltrate, and probed immunohistochemically for iNOS expression. RESULTS: The mean age of the patients was 21 (range 7-51) years. Seventeen patients (33 per cent) were seropositive for H. pylori but no evidence of H. pylori was found in any appendix specimen. However, an enhanced inflammatory cell infiltration was observed in seropositive patients (P < 0.04) and the expression of macrophage iNOS in the mucosa of normal and inflamed appendix specimens was increased (P < 0.01). CONCLUSION: H. pylori does not colonize the appendix and is unlikely to be a pathogenic stimulus for appendicitis. Priming effects on mucosal immunology downstream from the foregut may occur after infection with H. pylori.

  2. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    International Nuclear Information System (INIS)

    Das, S.; Gillin, F.D.

    1987-01-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of 3 H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei

  3. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Energy Technology Data Exchange (ETDEWEB)

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  4. Essential nontranslational functions of tRNA synthetases.

    Science.gov (United States)

    Guo, Min; Schimmel, Paul

    2013-03-01

    Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. Although these new functions were thought to be 'moonlighting activities', many are as critical for cellular homeostasis as their activity in translation. New roles have been associated with their cytoplasmic forms as well as with nuclear and secreted extracellular forms that affect pathways for cardiovascular development and the immune response and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. New architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. Although a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid-binding site for another purpose.

  5. Essential Non-Translational Functions of tRNA Synthetases

    Science.gov (United States)

    Guo, Min; Schimmel, Paul

    2013-01-01

    Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. While these new functions were thought to be ‘moonlighting activities’, many are as critical for cellular homeostasis as the activity in translation. New roles have been associated with cytoplasmic forms as well as with nuclear and secreted extracellular forms that impact pathways for cardiovascular development, the immune response, and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. Novel architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. While a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid binding site for another purpose. PMID:23416400

  6. The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2

    Energy Technology Data Exchange (ETDEWEB)

    Webb, G.C.; Vaska, V.L.; Ford, J.H. [Queen Elizabeth Hospital, Woodville (Australia)] [and others

    1995-12-10

    Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxoprolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization. 16 refs., 2 figs.

  7. The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

    Directory of Open Access Journals (Sweden)

    Ariel Alperstein

    Full Text Available The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

  8. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    Directory of Open Access Journals (Sweden)

    A Theron

    Full Text Available Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  9. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    Science.gov (United States)

    Theron, A; Roth, R L; Hoppe, H; Parkinson, C; van der Westhuyzen, C W; Stoychev, S; Wiid, I; Pietersen, R D; Baker, B; Kenyon, C P

    2017-01-01

    Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  10. Valyl-tRNA synthetase gene of Escherichia coli K12: Molecular genetic characterization and homology within a family of aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Heck, J.D. III.

    1988-01-01

    This work reports the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene encoding valyl-tRNA synthetase. The valS gene was subcloned from plasmid pLC26-22 by genetic complementation of a valS ts strain. The DNA region encoding the valS structural gene was determined by in vitro coupled transcription-translation assays. Cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase specific activity twelve-fold. DNA sequences flanking the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both [α- 32 P]labeled and [γ- 32 P]end-labeled in vitro transcription assays. The DNA sequence of the valS gene of Escherichia coli has been determined. Significant similarity at the primary sequence level was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. An extended open reading frame (ORF) encoded on the DNA strand opposite the valS structural gene is described

  11. Differential expression of argininosuccinate synthetase in serous and non‐serous ovarian carcinomas

    Science.gov (United States)

    Cheon, Dong‐Joo; Walts, Ann E; Beach, Jessica A; Lester, Jenny; Bomalaski, John S; Walsh, Christine S; Ruprecht Wiedemeyer, W; Karlan, Beth Y

    2014-01-01

    Abstract The current standard of care for epithelial ovarian cancer does not discriminate between different histologic subtypes (serous, clear cell, endometrioid and mucinous) despite the knowledge that ovarian carcinoma subtypes do not respond uniformly to conventional platinum/taxane‐based chemotherapy. Exploiting addictions and vulnerabilities in cancers with distinguishable molecular features presents an opportunity to develop individualized therapies that may be more effective than the current ‘one size fits all' approach. One such opportunity is arginine depletion therapy with pegylated arginine deiminase, which has shown promise in several cancer types that exhibit low levels of argininosuccinate synthetase including hepatocellular and prostate carcinoma and melanoma. Based on the high levels of argininosuccinate synthetase previously observed in ovarian cancers, these tumours have been considered unlikely candidates for arginine depletion therapy. However, argininosuccinate synthetase levels have not been evaluated in the individual histologic subtypes of ovarian carcinoma. The current study is the first to examine the expression of argininosuccinate synthetase at the mRNA and protein levels in large cohorts of primary and recurrent ovarian carcinomas and ovarian cancer cell lines. We show that the normal fallopian tube fimbria and the majority of primary high‐grade and low‐grade serous ovarian carcinomas express high levels of argininosuccinate synthetase, which tend to further increase in recurrent tumours. In contrast to the serous subtype, non‐serous ovarian carcinoma subtypes (clear cell, endometrioid and mucinous) frequently lack detectable argininosuccinate synthetase expression. The in vitro sensitivity of ovarian cancer cell lines to arginine depletion with pegylated arginine deiminase was inversely correlated with argininosuccinate synthetase expression. Our data suggest that the majority of serous ovarian carcinomas are not susceptible

  12. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  13. Ferulic acid depletion by cultured soybean seedlings under action of glucose and methionine

    Directory of Open Access Journals (Sweden)

    Herrig Vanessa

    2000-01-01

    Full Text Available Cultured soybean seedlings were used to investigate how glucose or methionine influenced depletion of ferulic acid. Three-day-old seedlings were grown in hydroponic solution containing ferulic acid plus glucose or methionine, and the level of the phenolic acid were monitored in the nutrient culture. The results showed that ferulic acid depletion was more rapid in the presence of those compounds. After 6 h, the increase caused by glucose (0.01 and 0.05 mM was more pronounced than methionine in the same concentrations. On the other hand, methionine (0.1 and 0.2 mM increased depletion more significantly than glucose. Results suggested that both compounds might to increase the allelopathic effects of ferulic acid in the seedlings.

  14. Simultaneous determination of plasma total homocysteine and methionine by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Jiang, Yi; Mistretta, Brandon; Elsea, Sarah; Sun, Qin

    2017-01-01

    The sulfur-containing amino acid homocysteine is a cardiac risk factor and a biomarker for several inborn errors of metabolism in methionine synthesis. A simple LC-MS/MS method was developed and validated for determination of homocysteine and methionine in human plasma. Rapid separation was achieved using a reverse phase liquid chromatography. Mass spectrometry identification was performed in positive electrospray ionization mode for homocysteine and methionine. Accuracy, precision, linearity, recovery and sample stability were evaluated in the method validation. The test is applied in diagnosis of homocystinuria and monitoring total homocysteine levels. Moreover, simultaneous measurement of methionine helps in the differentiation of homocystinuria and some cobalamin disorders (such as cblC and cblD defects) without additional amino acid testing. Lastly, this assay is sensitive to detect reduced total homocysteine levels that are possibly seen in sulfocysteinuria and molybdenum cofactor deficiencies. Copyright © 2016. Published by Elsevier B.V.

  15. miR-210 expression is associated with methionine-induced differentiation of trout satellite cells.

    Science.gov (United States)

    Latimer, Mary; Sabin, Nathalie; Le Cam, Aurélie; Seiliez, Iban; Biga, Peggy; Gabillard, Jean-Charles

    2017-08-15

    In fish, data on microRNAs (miRNAs) involved in myogenesis are scarce. In order to identify miRNAs involved in satellite cell differentiation, we used a methionine depletion/replenishment protocol to synchronize myogenic cell differentiation. Our results validated that methionine removal (72 h) from the medium strongly decreased myoD1 and myogenin expression, indicating differentiation arrest. In contrast, methionine replenishment rescued expression of myoD1 and myogenin , showing a resumption of differentiation. We performed a miRNA array analysis of myogenic cells under three conditions: presence of methionine for 72 h (control), absence of methionine for 72 h (Meth-) and absence of methionine for 48 h followed by 24 h of methionine replenishment (Meth-/+). A clustering analysis identified three clusters: cluster I corresponds to miRNA upregulated only in Meth-/+ conditions; cluster II corresponds to miRNA downregulated only in Meth-/+ conditions; cluster III corresponds to miRNAs with high expression in control, low expression in Meth- conditions and intermediate expression after methionine replenishment (Meth-/+). Cluster III was very interesting because it fitted with the data obtained for myoD1 and myogenin (supporting an involvement in differentiation) and contained seven miRNAs with muscle-related function (e.g. miR-133a) and one (miR-210) with unknown function. Based on our previously published miRNA repertoire ( Juanchich et al., 2016), we confirmed miR-133a was expressed only in white muscle and showed that miR-210 had strong expression in white muscle. We also showed that miR-210 expression was upregulated during differentiation of satellite cells, suggesting that miR-210 was potentially involved in the differentiation of satellite cells. © 2017. Published by The Company of Biologists Ltd.

  16. Safety of methionine, a novel biopesticide, to adult and larval honey bees (Apis mellifera L.).

    Science.gov (United States)

    Weeks, Emma N I; Schmehl, Daniel R; Baniszewski, Julie; Tomé, Hudson V V; Cuda, James P; Ellis, James D; Stevens, Bruce R

    2018-03-01

    Methionine is an essential/indispensible amino acid nutrient required by adult and larval honey bees (Apis mellifera L. [Hymenoptera: Apidae]). Bees are unable to rear broods on pollen deficient in methionine, and reportedly behaviorally avoid collecting pollen or nectar from florets deficient in methioinine. In contrast, it has been demonstrated that methionine is toxic to certain pest insects; thus it has been proposed as an effective biopesticide. As an ecofriendly integrated pest management agent, methionine boasts a novel mode of action differentiating it from conventional pesticides, while providing non-target safety. Pesticides that minimize collateral effects on bees are desirable, given the economic and ecological concerns about honey bee health. The aim of the present study was to assess the potential impact of the biopesticide methionine on non-target adult and larval honey bees. Acute contact adult toxicology bioassays, oral adult assessments and chronic larval toxicity assessments were performed as per U.S. Environmental Protection Agency (EPA) requirements. Our results demonstrated that methionine fits the U.S. EPA category of practically nontoxic (i.e. lethal dose to 50% mortality or LD 50 > 11µg/bee) to adult honey bees. The contact LD 50 was > 25µg/bee and the oral LD 50 was > 100µg/bee. Mortality was observed in larval bees that ingested DL-methionine (effective concentration to 50% mortality or EC 50 560µg/bee). Therefore, we conclude that methionine poses little threat to the health of the honey bee, due to unlikely exposure at concentrations shown to elicit toxic effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Utility of “1”1C -methionine PET/CT in neuro-oncology

    International Nuclear Information System (INIS)

    Casas Parera, I.; Igirio Gamero, J.L.; Báez, A.; Tafur Canabal, J.G.; Báez, M.; Kuchkaryan, V.; B lumenkrantz, Y.; Bruno, G.

    2013-01-01

    Positron emission tomography (PET) with “1”1C-methionine (“1”1C-methionine PET/CT) is a new technique used to evaluate primary central nervous system (CNS) tumors. We describe our experience regarding the first 4 patients with glial tumors and “1”1C-methionine PET/CT. This is a descriptive, observational and prospective study of 4 patients between 38-50 years of age, with different gliomas (WHO classification). MRI and “1”1C-methionine PET/CT were performed in all cases. Case 1, gliomatosis cerebri grade II post-radiotherapy. Case 2, oligodendroglioma grade II diagnosed and treated with radiotherapy in 1993. Case 3, glioblastoma grade IV post-radiotherapy + temozolomide. Case 4, anaplastic oligoastrocytoma grade III post-radiotherapy + temozolomide. The pattern of “1”1C-methionine uptake compared with MRI showed tumor progression in cases 1, 3 and 4, and in case 2 showed uptake although the final diagnosis was pseudoprogression. Unlike “1”8fluordeoxiglucose PET/TC, “1”1C-methionine uptake in normal brain tissue and pseudoprogression is low, and gliomas are displayed as metabolically active areas. The “1”1C-methionine PET/CT provided valuable information on the tumoral behavior and extension, although in one case presented did not differentiate tumor progression from pseudoprogression. “1”1C-methionine PET/CT could be a useful tool in the study and follow-up to patients with gliomas. (authors) [es

  18. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    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.

  19. Impact of methionine oxidation on calmodulin structural dynamics

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin [Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455 (United States); Moen, Rebecca J. [Chemistry and Geology Department, Minnesota State University, Mankato, MN 56001 (United States); Olenek, Michael J. [Biology Department, University of Wisconsin, La Crosse, WI 54601 (United States); Klein, Jennifer C., E-mail: jklein@uwlax.edu [Biology Department, University of Wisconsin, La Crosse, WI 54601 (United States); Thomas, David D., E-mail: ddt@umn.edu [Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455 (United States)

    2015-01-09

    Highlights: • We measured the distance distribution between two spin labels on calmodulin by DEER. • Two structural states, open and closed, were resolved at both low and high Ca. • Ca shifted the equilibrium toward the open state by a factor of 13. • Methionine oxidation, simulated by glutamine substitution, decreased the Ca effect. • These results have important implications for aging in muscle and other tissues. - Abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron–electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4 nm (closed) and another at ∼6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each

  20. Induction of carbamoyl phosphate synthetase III and glutamine synthetase mRNA during confinement stress in gulf toadfish (Opsanus beta).

    Science.gov (United States)

    Kong, H; Kahatapitiya, N; Kingsley, K; Salo, W L; Anderson, P M; Wang, Y S; Walsh, P J

    2000-01-01

    Gulf toadfish (Opsanus &bgr;) rapidly switch to excretion of urea as their main nitrogenous waste product under several laboratory conditions, including confinement to small volumes of water. Prior evidence suggested that the activities of two key enzymes of urea synthesis exhibited potentially different modes of upregulation during this switch, with carbamoyl phosphate synthethase III (CPSase III) activated allosterically by N-acetylglutamate, and glutamine synthetase (GSase) activated by increases in the concentration of protein. The present study was undertaken to examine additional aspects of the regulation of these enzymes. The sequence for O. beta CPSase III cDNA was obtained, and it was found to be similar to that of other piscine CPSases. The sequence also allowed us to develop riboprobes for CPSase III mRNA analysis using ribonuclease protection assays (RPAs). CPSase III mRNA was expressed in liver, muscle, kidney and intestine, in agreement with prior enzymatic measurements. Levels of CPSase III mRNA increased five- to tenfold (relative to beta-actin mRNA) in liver (but not muscle) following 48 h of confinement stress. Measured by western analysis using an antibody to chicken GSase, confined O. beta GSase protein concentrations increased eightfold over control levels, in agreement with prior and present measurements of increases in GSase activity. Furthermore, RPAs of GSase mRNA levels demonstrated an increase of fivefold during confinement.

  1. Methionine restriction activates the retrograde response and confers both stress tolerance and lifespan extension to yeast, mouse and human cells.

    Directory of Open Access Journals (Sweden)

    Jay E Johnson

    Full Text Available A methionine-restricted diet robustly improves healthspan in key model organisms. For example, methionine restriction reduces age-related pathologies and extends lifespan up to 45% in rodents. However, the mechanisms underlying these benefits remain largely unknown. We tested whether the yeast chronological aging assay could model the benefits of methionine restriction, and found that this intervention extends lifespan when enforced by either dietary or genetic approaches, and furthermore, that the observed lifespan extension is due primarily to reduced acid accumulation. In addition, methionine restriction-induced lifespan extension requires the activity of the retrograde response, which regulates nuclear gene expression in response to changes in mitochondrial function. Consistent with an involvement of stress-responsive retrograde signaling, we also found that methionine-restricted yeast are more stress tolerant than control cells. Prompted by these findings in yeast, we tested the effects of genetic methionine restriction on the stress tolerance and replicative lifespans of cultured mouse and human fibroblasts. We found that such methionine-restricted mammalian cells are resistant to numerous cytotoxic stresses, and are substantially longer-lived than control cells. In addition, similar to yeast, the extended lifespan of methionine-restricted mammalian cells is associated with NFκB-mediated retrograde signaling. Overall, our data suggest that improved stress tolerance and extension of replicative lifespan may contribute to the improved healthspan observed in methionine-restricted rodents, and also support the possibility that manipulation of the pathways engaged by methionine restriction may improve healthspan in humans.

  2. Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule.

    Science.gov (United States)

    Folzer, Emilien; Diepold, Katharina; Bomans, Katrin; Finkler, Christof; Schmidt, Roland; Bulau, Patrick; Huwyler, Jörg; Mahler, Hanns-Christian; Koulov, Atanas V

    2015-09-01

    Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  3. Influence of dietary protein and excess methionine on choline needs for young bobwhite quail

    Science.gov (United States)

    Serafin, J.A.

    1982-01-01

    Experiments were conducted with young Bobwhite quail (Colinus virginianus) to investigate the effect of differing dietary protein levels and nondetrimental amounts of excess methionine on choline needs. Growth and feed consumption of quail fed an adequate (27.3%) protein purified diet supplemented with 2000 mg/kg of choline were unaffected by increasing the level of excess methionine to 1.75%; however, greater amounts (2.0%, 2.25%) of excess methionine depressed growth (P less than .01), reduced feed consumption (P less than .01), and decreased feed utilization (P less than .05). Quail fed a purified diet containing 13.85% protein and 515 mg/kg of choline grew poorly. Growth was unaffected by additional choline in this diet. Growth was suboptimal among quail fed purified diets containing adequate or high (41.55%) levels of protein in which choline was limiting; however, a high level of protein did not in itself affect performance. Growth was improved by supplemental choline in these diets. Growth of quail fed purified diets with up to 1.35% excess methionine which were limiting (531 mg/kg) in choline was less than that of groups fed 2000 mg/kg of added dietary choline (P less than .01); however, excess methionine did not significantly influence growth of quail fed choline-deficient diets. These experiments indicate that neither high dietary protein nor excess methionine, fed at non-growth-depressing levels, increases dietary choline needs for young Bobwhite quail.

  4. Methionine oxidation activates a transcription factor in response to oxidative stress.

    Science.gov (United States)

    Drazic, Adrian; Miura, Haruko; Peschek, Jirka; Le, Yan; Bach, Nina C; Kriehuber, Thomas; Winter, Jeannette

    2013-06-04

    Oxidant-mediated antibacterial response systems are broadly used to control bacterial proliferation. Hypochlorite (HOCl) is an important component of the innate immune system produced in neutrophils and specific epithelia. Its antimicrobial activity is due to damaging cellular macromolecules. Little is known about how bacteria escape HOCl-inflicted damage. Recently, the transcription factor YjiE was identified that specifically protects Escherichia coli from HOCl killing. According to its function, YjiE is now renamed HypT (hypochlorite-responsive transcription factor). Here we unravel that HypT is activated by methionine oxidation to methionine sulfoxide. Interestingly, so far only inactivation of cellular proteins by methionine oxidation has been reported. Mutational analysis revealed three methionines that are essential to confer HOCl resistance. Their simultaneous substitution by glutamine, mimicking the methionine sulfoxide state, increased the viability of E. coli cells upon HOCl stress. Triple glutamine substitution generates a constitutively active HypT that regulates target genes independently of HOCl stress and permanently down-regulates intracellular iron levels. Inactivation of HypT depends on the methionine sulfoxide reductases A/B. Thus, microbial protection mechanisms have evolved along the evolution of antimicrobial control systems, allowing bacteria to survive within the host environment.

  5. When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

    Science.gov (United States)

    Roy, Hervé; Becker, Hubert Dominique; Reinbolt, Joseph; Kern, Daniel

    2003-01-01

    Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism. PMID:12874385

  6. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  7. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    International Nuclear Information System (INIS)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A.

    2012-01-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine

  8. Methionine Uptake and Required Radiation Dose to Control Glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Iuchi, Toshihiko, E-mail: tiuchi@chiba-cc.jp [Division of Neurological Surgery, Chiba Cancer Center, Chiba (Japan); Hatano, Kazuo [Division of Radiation Oncology, Tokyo Bay Advanced Imaging and Radiation Oncology Clinic, Makuhari, Chiba (Japan); Uchino, Yoshio [Division of Nuclear Medicine, Chiba Ryogo Center, Chiba (Japan); Itami, Makiko [Division of Surgical Pathology, Chiba Cancer Center, Chiba (Japan); Hasegawa, Yuzo; Kawasaki, Koichiro; Sakaida, Tsukasa [Division of Neurological Surgery, Chiba Cancer Center, Chiba (Japan); Hara, Ryusuke [Division of Radiation Oncology, Chiba Cancer Center, Chiba (Japan)

    2015-09-01

    Purpose: The purpose of this study was to retrospectively assess the feasibility of radiation therapy planning for glioblastoma multiforme (GBM) based on the use of methionine (MET) positron emission tomography (PET), and the correlation among MET uptake, radiation dose, and tumor control. Methods and Materials: Twenty-two patients with GBM who underwent MET-PET prior to radiation therapy were enrolled. MET uptake in 30 regions of interest (ROIs) from 22 GBMs, biologically effective doses (BEDs) for the ROIs and their ratios (MET uptake:BED) were compared in terms of whether the ROIs were controlled for >12 months. Results: MET uptake was significantly correlated with tumor control (odds ratio [OR], 10.0; P=.005); however, there was a higher level of correlation between MET uptake:BED ratio and tumor control (OR, 40.0; P<.0001). These data indicated that the required BEDs for controlling the ROIs could be predicted in terms of MET uptake; BED could be calculated as [34.0 × MET uptake] Gy from the optimal threshold of the MET uptake:BED ratio for tumor control. Conclusions: Target delineation based on MET-PET was demonstrated to be feasible for radiation therapy treatment planning. MET-PET could not only provide precise visualization of infiltrating tumor cells but also predict the required radiation doses to control target regions.

  9. Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

    Science.gov (United States)

    Kassmann, Mario; Hansel, Alfred; Leipold, Enrico; Birkenbeil, Jan; Lu, Song-Qing; Hoshi, Toshinori; Heinemann, Stefan H.

    2010-01-01

    Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion. PMID:18369661

  10. Expression of Four Methionine Sulfoxide Reductases in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Kuldeep Singh

    2012-01-01

    Full Text Available Staphylococcus aureus possesses three MsrA enzymes (MsrA1, MsrA2, MsrA3 that reduce the S-epimer of methionine sulfoxide (MetO and an MsrB enzyme that reduces R-MetO. The four msr genes are expressed from three different promoters. The msrA1/msrB genes are coexpressed. To determine the expression pattern of msr genes, three independent reporter strains were constructed where msr promoter was cloned in front of a promoterless lacZ and the resulting construct was integrated in the chromosome. Using these strains, it was determined that the msrA1/B expression is significantly higher in S. aureus compared to msrA2 or msrA3. Expression of msrA1/B was highest during stationary phase growth, but the expression of msrA2 and msrA3 was highest during the early to midexponential growth phase. Expression of msrA1/B was induced by oxacillin and the expression of msrA3 was upregulated by salt. Expression of msrA2 remained unchanged under all tested conditions.

  11. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    Science.gov (United States)

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids. PMID:28966840

  12. Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

    Science.gov (United States)

    Mathur, M; Satpathy, M; Sachar, R C

    1992-11-17

    Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments

  13. Identification of autoantibodies to tyrosil-tRNA synthetase in heart disfunctions

    Directory of Open Access Journals (Sweden)

    Ryabenko D. V.

    2010-09-01

    Full Text Available Aim. To investigate the levels of specific autoantibodies against tyrosyl-tRNA synthetase and its individual modules in the blood serum of people with heart failure caused by dilated cardiomyopathy, myocarditis and ischemic heart disease compared with healthy donors. Methods. Recombinant proteins were obtained using bacterial strains transformed with appropriate plasmid vectors and were purified by chromatography on Ni-NTA-agarose. The levels of specific autoantibodies were investigated by ELISA. Results. The increased levels of autoantibodies specific to tyrosyl-tRNA synthetase, its N-terminal catalytic module and non-catalytic C-module, were found in the blood serum of patients, compared with healthy donors. Conclusions. The results obtained demonstrate the possible role of tyrosyl-tRNA synthetase in adaptive changes of the myocardium in response to stress factors.

  14. Inhibition of Dihydropteroate Synthetase from Escherichia coli by Sulfones and Sulfonamides

    Science.gov (United States)

    McCullough, Jerry L.; Maren, Thomas H.

    1973-01-01

    The inhibitory action of various diphenylsulfones and sulfonamides on dihydropteroate synthetase partially purified from Escherichia coli was examined. 4,4′-Diaminodiphenylsulfone (DDS; I50 = 2 × 10−5 M) and the monosubstituted derivatives 4-amino-4′-formamidodiphenylsulfone (I50 = 5.8 × 10−5 M) and 4-amino-4′-acetamidodiphenylsulfone (I50 = 5.2 × 10−5 M) were effective inhibitors of dihydropteroate synthetase activity. Disubstitution of the arylamine groups of DDS (4,4′-diformamidodiphenylsulfone and 4,4′-diacetamidodiphenylsulfone) resulted in complete loss of inhibitory activity. Both DDS (KI = 5.9 × 10−6 M) and sulfadiazine (KI = 2.5 × 10−6 M) were found to be competitive inhibitors of dihydropteroate synthetase. These findings are discussed in regard to the Bell and Roblin theory of structure-activity relationships for p-aminobenzoic acid antagonists. PMID:4597736

  15. 1-13C; methyl-2H3 methionine kinetics in humans: Methionine conservation and cystine sparing

    International Nuclear Information System (INIS)

    Storch, K.J.; Wagner, D.A.; Burke, J.F.; Young, V.R.

    1990-01-01

    Methionine (Met) conservation in healthy young adult men (4/diet group) was explored by supplying one of the following three L-amino acid based diets: (1) adequate Met but no cystine; (2) neither Met nor cystine; or (3) no Met but cystine supplementation. After 5 days, subjects received a continuous intravenous infusion of L-[1-13C; methyl-2H3]Met for 5 h while the diet was given as small isocaloric isonitrogenous meals. Estimates were made of rates of Met incorporation into protein synthesis (S) and release from body proteins (B), transmethylation (TM), remethylation of homocysteine (RM), and transsulfuration (TS). For the adequate Met diet, the rates were S = 24 +/- 2, B = 18 +/- 1, TM = 12.4 +/- 1.7, RM = 4.7 +/- 1.1, and TS = 7.6 +/- 0.6 (SE) mumol.kg-1.h-1. The sulfur amino acid-devoid diet significantly (P less than 0.05) reduced S, TM, RM, and TS. Supplementation of this diet with cystine reduced Met oxidation (P = 0.05). Therefore, two loci are quantitatively important regulatory points in Met conservation in vivo: (1) the distribution of Met between the pathways of protein anabolism and TM (Met locus) and (2) the distribution of homocysteine between RM and TS (homocysteine locus)

  16. Diet- and hormone-induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus

    NARCIS (Netherlands)

    Moorman, A. F.; de Boer, P. A.; Charles, R.; Lamers, W. H.

    1990-01-01

    A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary-treated rats was carried out, using radioactively-labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line

  17. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  18. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end produc...

  19. Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma.

    Science.gov (United States)

    Kokkinakis, Demetrius M; Liu, Xiaoyan; Neuner, Russell D

    2005-09-01

    The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

  20. Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

    KAUST Repository

    Marondedze, Claudius

    2013-01-05

    Background: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. Findings. Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. Conclusions: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. 2013 Marondedze et al.; licensee BioMed Central Ltd.

  1. Fungal type III polyketide synthases.

    Science.gov (United States)

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

  2. Terpene synthases from Cannabis sativa.

    Directory of Open Access Journals (Sweden)

    Judith K Booth

    Full Text Available Cannabis (Cannabis sativa plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E-β-ocimene, (--limonene, (+-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  3. The influence of prenatal X-irradiation on the activity of SRNA-aminoacyl synthetases in the developing rabbit brain

    International Nuclear Information System (INIS)

    Wender, M.; Zgorzalewicz, B.

    1976-01-01

    The activities of sRNA-aminoacyl synthetases were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single x-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. The results indicate clearly that prenatal exposure to ionizing radiation also affects the steps of protein biosynthesis which depend on the activity of sRNA-aminoacyl synthetases. (author)

  4. Plasma methionine depletion and pharmacokinetic properties in mice of methionine γ-lyase from Citrobacter freundii, Clostridium tetani and Clostridium sporogenes.

    Science.gov (United States)

    Morozova, E A; Anufrieva, N V; Davydov, D Zh; Komarova, M V; Dyakov, I N; Rodionov, A N; Demidkina, T V; Pokrovsky, V S

    2017-04-01

    PK studies were carried out after a single i.v. administration of 500 and 1000 U/kg by measuring of MGL activity in plasma samples. L-methionine concentration was measured by mass spectrometry. After single i.v. injection of 500U/kg the circulating T 1/2 of enzymes in mice varies from 73 to 123min. The AUC 0-tinf values determined for MGL 500U/kg from C. freundii, C. tetani and C. sporogenes are 8.21±0.28, 9.04±0.33 and 13.88±0.39U/(ml×h), respectively. Comparison of PK parameters of three MGL sources in the dose of 500U/kg indicated the MGL C. sporogenes to have better PK parameters: clearance 0.83(95%CI: 0.779-0.871) - was lower than C. tetanii 1.27(95%CI: 1.18-1.36) and C. freundii 1.39(95%CI: 1.30-1.49). Mice plasma methionine decreased to undetectable level 10min after MGL 1000 U/kg injection. After MGL C. sporogenes 500U/kg injection plasma methionine level completely omitted after 10min till 6h, assuming the sustainability of negligible levels of methionine (tetani. There are no significant differences between methionine cleavage after MGL C. tetani and MGL C. sporogenes i.v. injection at all doses. MGL from C. sporogenes may be considered as promising enzyme for further investigation as potential anticancer agent. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    -utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5......-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate...

  6. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translatio...... of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions....

  7. The relative contribution of genes operating in the S-methylmethionine cycle to methionine metabolism in Arabidopsis seeds.

    Science.gov (United States)

    Cohen, Hagai; Salmon, Asaf; Tietel, Zipora; Hacham, Yael; Amir, Rachel

    2017-05-01

    Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine synthesis in seeds. In addition, mutual effects exist between the S -methylmethionine cycle and the aspartate family pathway in seeds. Methionine, a sulfur-containing amino acid, is a key metabolite in plant cells. The previous lines of evidence proposed that the S-methylmethionine (SMM) cycle contributes to methionine synthesis in seeds where methionine that is produced in non-seed tissues is converted to SMM and then transported via the phloem into the seeds. However, the relative regulatory roles of the S-methyltransferases operating within this cycle in seeds are yet to be fully understood. In the current study, we generated transgenic Arabidopsis seeds with altered expression of three HOMOCYSTEINE S-METHYLTRANSFERASEs (HMTs) and METHIONINE S-METHYLTRANSFERASE (MMT), and profiled them for transcript and metabolic changes. The results revealed that AtHMT1 and AtHMT3, but not AtHMT2 and AtMMT, are the predominant enzymes operating in seeds as altered expression of these two genes affected the levels of methionine and SMM in transgenic seeds. Their manipulations resulted in adapted expression level of genes participating in methionine synthesis through the SMM and aspartate family pathways. Taken together, our findings provide new insights into the regulatory roles of the SMM cycle and the mutual effects existing between the two methionine biosynthesis pathways, highlighting the complexity of the metabolism of methionine and SMM in seeds.

  8. Involvement of BcStr2 in methionine biosynthesis, vegetative differentiation, multiple stress tolerance and virulence in Botrytis cinerea.

    Science.gov (United States)

    Shao, Wenyong; Yang, Yalan; Zhang, Yu; Lv, Chiyuan; Ren, Weichao; Chen, Changjun

    2016-04-01

    The Str2 gene encodes a cystathionine γ-synthase that is a key enzyme in methionine (Met) biosynthesis in Saccharomyces cerevisiae. Met plays a critical role in protein synthesis and diverse cellular processes in both eukaryotes and prokaryotes. In this study, we characterized the Str2 orthologue gene BcStr2 in Botrytis cinerea. The BcStr2 mutant was unable to grow on minimal medium (MM). In addition, conidia of the mutant were unable to germinate in water-agar medium within 15 h of incubation. Supplementation with 1 mm Met or 0.5 mg/mL homocysteine, but not 1 mm cysteine or 0.5 mg/mL glutathione, rescued the defect in mycelial growth of the BcStr2 deletion mutant. These results indicate that the enzyme encoded by BcStr2 is involved in the conversion of cysteine into homocysteine. The mutant exhibited decreased conidiation and impaired sclerotium development. In addition, the BcStr2 mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall-damaging agents and thermal stress. The mutant demonstrated dramatically decreased virulence on host plant tissues. All of the defects were restored by genetic complementation of the mutant with wild-type BcStr2. Taken together, the results of this study indicate that BcStr2 plays a critical role in the regulation of various cellular processes in B. cinerea. © 2015 BSPP and John Wiley & Sons Ltd.

  9. Egg quality of quails fed low methionine diet supplemented with betaine

    Science.gov (United States)

    Ratriyanto, A.; Indreswari, R.; Dewanti, R.; Wahyuningsih, S.

    2018-03-01

    This experiment investigated the effect of betaine supplementation to low methionine diet on egg quality of quails. A total of 340 laying quails (Coturnix coturnix japonica) was divided into 4 dietary treatments with 5 replicates of 17 quails each. The experiment was assigned in a completely randomized design. The four dietary treatments were the low methionine diet (0.3% methionine) without betaine supplementation and the low methionine diet supplemented with 0.07, 0.14, and 0.21% betaine. The experimental diets were applied for 8 weeks and the egg quality traits were measured at the age of 16 and 20 weeks. The data were subjected to analysis of variance, and when the treatment indicated significant effect, it was continued to orthogonal polynomial test to determine the optimum level of betaine. Increasing dietary levels of betaine increased the fat content of the egg with the linear regression of y = 11.0949 + 4.1914x (R2 = 0.18). However, supplementation of betaine did not affect protein content, yolk, albumen, and eggshell percentage. It can be concluded that betaine supplementation up to 0.21% to low methionine diet only had little effect in improving the quality traits of quail eggs.

  10. Nuclear magnetic resonance studies on bacterial dihydrofolate reductase containing (methyl-/sup 13/C)methionine

    Energy Technology Data Exchange (ETDEWEB)

    Blakley, R.L.; Cocco, L.; London, R.E.; Walker, T.E.; Matwiyoff, N.A.

    1978-06-13

    (methyl-/sup 13/C)Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the /sup 13/C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T/sub 1/ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CH/sub 2/-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH/sub 2/-S bond is highly restricted so that the methyl group oscillates through a relatively narrow angular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably due to conformational transitions in the enzyme. However, NADP/sup +/ produced several changes,including movement of one resonance downfield by at least 1.7 ppM.

  11. Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides.

    Science.gov (United States)

    Schauwecker, F; Pfennig, F; Grammel, N; Keller, U

    2000-04-01

    Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.

  12. A Transient Upregulation of Glutamine Synthetase in the Dentate Gyrus Is Involved in Epileptogenesis Induced by Amygdala Kindling in the Rat.

    Directory of Open Access Journals (Sweden)

    Hong-Liu Sun

    Full Text Available Reduction of glutamine synthetase (GS function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl, a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated. It was found that low doses of MSO (5 or 10 µg significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×10(6 TU/2 µl of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.

  13. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  14. Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

    DEFF Research Database (Denmark)

    Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J

    1989-01-01

    Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine...

  15. Regulation of Amidase Formation in Mutants from Pseudomonas aeruginosa PAO Lacking Glutamine Synthetase Activity

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1982-01-01

    The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation

  16. Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

    DEFF Research Database (Denmark)

    Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus

    2016-01-01

    Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There ...

  17. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid se...

  18. Regulation of the spatiotemporal pattern of expression of the glutamine synthetase gene

    NARCIS (Netherlands)

    Lie-Venema, H.; Hakvoort, T. B.; van Hemert, F. J.; Moorman, A. F.; Lamers, W. H.

    1998-01-01

    Glutamine synthetase, the enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine, is expressed in a tissue-specific and developmentally controlled manner. The first part of this review focuses on its spatiotemporal pattern of expression, the factors that regulate

  19. Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: expression of glutamine synthetase and acetylornithine transaminase.

    NARCIS (Netherlands)

    Guan, C.; Ribeiro, A.; Akkermans, A.D.L.; Jing, Y.; Kammen, van A.; Bisseling, T.; Pawlowski, K.

    1996-01-01

    Two nodule cDNA clones representing genes involved in Alnus glutinosa nitrogen metabolism were analysed. ag11 encoded glutamine synthetase (GS), the enzyme responsible for ammonium assimilation, while ag118 encoded acetylornithine transaminase (AOTA), an enzyme involved in the biosynthesis of

  20. Primer Dependent and Independent Forms of Soluble Starch Synthetase from Developing Barley Endosperms

    DEFF Research Database (Denmark)

    Kreis, M.

    1980-01-01

    The activity of soluble starch synthetase (ADP-glucose: agr-1,4-glucan agr-4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase was purif......The activity of soluble starch synthetase (ADP-glucose: agr-1,4-glucan agr-4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase...... was purified by ammonium sulfate precipitation and DEAE-cellulose chromatography and separated into four fractions. In the absence of an added carbohydrate primer two of the four fractions catalized the synthesis of a methanol-precipitable agr-glucan when high concentrations of sodium citrate and bovine serum...... albumim were added. The rate of agr-glucan synthesis by the unprimed reaction was higher than for the primed reaction. The four enzyme fractions were active with ADP-Glc, but not with UDP-Glc, both in the primed and in the unprimed reaction....

  1. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  2. Synthesis, accumulation and turnover of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of embryonic rat hepatocytes

    NARCIS (Netherlands)

    van Roon, M. A.; Charles, R.; Lamers, W. H.

    1987-01-01

    Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such

  3. Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum

    NARCIS (Netherlands)

    Di Lorenzo, M.; Stork, M.; Naka, H.; Tolmasky, M.E.; Crosa, J.H.

    2008-01-01

    Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa

  4. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

    NARCIS (Netherlands)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.; Weiner, I. David

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of

  5. Glutamine Synthetase in Muscle Is Required for Glutamine Production during Fasting and Extrahepatic Ammonia Detoxification

    NARCIS (Netherlands)

    He, Youji; Hakvoort, Theodorus B. M.; Köhler, S. Eleonore; Vermeulen, Jacqueline L. M.; de Waart, D. Rudi; de Theije, Chiel; ten Have, Gabrie A. M.; van Eijk, Hans M. H.; Kunne, Cindy; Labruyere, Wilhelmina T.; Houten, Sander M.; Sokolovic, Milka; Ruijter, Jan M.; Deutz, Nicolaas E. P.; Lamers, Wouter H.

    2010-01-01

    The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with

  6. Expression pattern of glutamine synthetase marks transition from collecting into conducting hepatic veins

    NARCIS (Netherlands)

    Lamers, W. H.; Vermeulen, J. L.; Hakvoort, T. B.; Moorman, A. F.

    1999-01-01

    The expression of glutamine synthetase (GS) is confined to a rim of hepatocytes surrounding the efferent hepatic veins in all mammalian species investigated. In rat liver, a two- to three-cell thick layer of GS-positive (GS(+)) hepatocytes uniformly surrounds the two to four terminal branching

  7. Isolation and characterization of the rat glutamine synthetase-encoding gene

    NARCIS (Netherlands)

    van de Zande, L.; Labruyère, W. T.; Arnberg, A. C.; Wilson, R. H.; van den Bogaert, A. J.; Das, A. T.; van Oorschot, D. A.; Frijters, C.; Charles, R.; Moorman, A. F.

    1990-01-01

    From a rat genomic library in phage lambda Charon4A, a complete glutamine synthetase-encoding gene was isolated. The gene is 9.5-10 kb long, consists of seven exons, and codes for two mRNA species of 1375 nucleotides (nt) and 2787 nt, respectively. For both mRNAs, full-length cDNAs containing a

  8. Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

    Directory of Open Access Journals (Sweden)

    Zheng-xun JIN

    2007-09-01

    Full Text Available Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

  9. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  10. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne U.; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine...

  11. Concurrent overactivation of the cytosolic glutamine synthetase and the GABA shunt in the ABA-deficient sitiens mutant of tomato leads to resistance against Botrytis cinerea.

    Science.gov (United States)

    Seifi, Hamed Soren; Curvers, Katrien; De Vleesschauwer, David; Delaere, Ilse; Aziz, Aziz; Höfte, Monica

    2013-07-01

    Deficiency of abscisic acid (ABA) in the sitiens mutant of tomato (Solanum lycopersicum) culminates in increased resistance to Botrytis cinerea through a rapid epidermal hypersensitive response (HR) and associated phenylpropanoid pathway-derived cell wall fortifications. This study focused on understanding the role of primary carbon : nitrogen (C : N) metabolism in the resistance response of sitiens to B. cinerea. How alterations in C : N metabolism are linked with the HR-mediated epidermal arrest of the pathogen has been also investigated. Temporal alterations in the γ-aminobutyric acid (GABA) shunt, glutamine synthetase/glutamate synthase (GS/GOGAT) cycle and phenylpropanoid pathway were transcriptionally, enzymatically and metabolically monitored in both wild-type and sitiens plants. Virus-induced gene silencing, microscopic analyses and pharmacological assays were used to further confirm the data. Our results on the sitiens-B. cinerea interaction favor a model in which cell viability in the cells surrounding the invaded tissue is maintained by a constant replenishment of the tricarboxylic acid (TCA) cycle through overactivation of the GS/GOGAT cycle and the GABA shunt, resulting in resistance through both tightly controlling the defense-associated HR and slowing down the pathogen-induced senescence. Collectively, this study shows that maintaining cell viability via alterations in host C : N metabolism plays a vital role in the resistance response against necrotrophic pathogens. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  12. Fluoresence quenching of riboflavin in aqueous solution by methionin and cystein

    International Nuclear Information System (INIS)

    Droessler, P.; Holzer, W.; Penzkofer, A.; Hegemann, P.

    2003-01-01

    The fluorescence quantum distributions, fluorescence quantum yields, and fluorescence lifetimes of riboflavin in methanol, DMSO, water, and aqueous solutions of the sulphur atom containing amino acids methionin and cystein have been determined. In methanol, DMSO, and water (pH=4-8) only dynamic fluorescence reduction due to intersystem crossing and internal conversion is observed. In aqueous methionin solutions of pH=5.25-9 a pH independent static and dynamic fluorescence quenching occurs probably due to riboflavin anion-methionin cation pair formation. In aqueous cystein solutions (pH range from 4.15 to 9) the fluorescence quenching increases with rising pH due to cystein thiolate formation. The cystein thiol form present at low pH does not react with neutral riboflavin. Cystein thiolate present at high pH seems to react with neutral riboflavin causing riboflavin deprotonation (anion formation) by cystein thiolate reduction to the cystein thiol form

  13. Crystal structure of a new homochiral one-dimensional zincophosphate containing l-methionine

    Directory of Open Access Journals (Sweden)

    Nadjet Chouat

    2015-07-01

    Full Text Available catena-Poly[[(l-methionine-κOzinc]-μ3-(hydrogen phosphato-κ3O:O′:O′′], [Zn{PO3(OH}(C5H11NO2S]n, a new one-dimensional homochiral zincophosphate, was hydrothermally synthesized using l-methionine as a structure-directing agent. The compound consists of a network of ZnO4 and (HOPO3 tetrahedra that form ladder-like chains of edge-fused Zn2P2O4 rings propagating parallel to [100]. The chains are decorated on each side by zwitterionic l-methionine ligands, which interact with the inorganic framework via Zn—O coordination bonds. The structure displays interchain N—H...O and O—H...S hydrogen bonds.

  14. Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

    Science.gov (United States)

    Aboelnga, Mohamed M.

    Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre?transfer editing and post?transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyl-tRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates

  15. Effect of maternal methionine supplementation on the transcriptome of bovine preimplantation embryos.

    Directory of Open Access Journals (Sweden)

    Francisco Peñagaricano

    Full Text Available Maternal nutrition exclusively during the periconceptional period can induce remarkable effects on both oocyte maturation and early embryo development, which in turn can have lifelong consequences. The objective of this study was to evaluate the effect of maternal methionine supplementation on the transcriptome of bovine preimplantation embryos. Holstein cows were randomly assigned to one of two treatments differing in level of dietary methionine (1.89 Met vs. 2.43 Met % of metabolizable protein from calving until embryo flushing. High quality preimplantation embryos from individual cows were pooled and then analyzed by RNA sequencing. Remarkably, a subtle difference in methionine supplementation in maternal diet was sufficient to cause significant changes in the transcriptome of the embryos. A total of 276 genes out of 10,662 showed differential expression between treatments (FDR <0.10. Interestingly, several of the most significant genes are related to embryonic development (e.g., VIM, IFI6, BCL2A1, and TBX15 and immune response (e.g., NKG7, TYROBP, SLAMF7, LCP1, and BLA-DQB. Likewise, gene set enrichment analysis revealed that several Gene Ontology terms, InterPro entries, and KEGG pathways were enriched (FDR <0.05 with differentially expressed genes involved in embryo development and immune system. The expression of most genes was decreased by maternal methionine supplementation, consistent with reduced transcription of genes with increased methylation of specific genes by increased methionine. Overall, our findings provide evidence that supplementing methionine to dams prior to conception and during the preimplantation period can modulate gene expression in bovine blastocysts. The ramifications of the observed gene expression changes for subsequent development of the pregnancy and physiology of the offspring warrant further investigation in future studies.

  16. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara (SSRL); (Maryland)

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  17. Nutritional levels of digestible methionine + cystine to brown-egg laying hens from 50 to 66 weeks of age

    Directory of Open Access Journals (Sweden)

    Clauber Polese

    2012-07-01

    Full Text Available The objective of this study was to determine the requirement of digestible methionine + cystine of brown-eggs laying hens from 50 to 66 weeks age at the end of the first production cycle. The design was completely randomized, with 150 Brown Shaver hens, which were distributed in five treatments with six replications of five birds each. Birds received a basal diet with 2857 kcal/kg metabolizable energy and 15.97% crude protein, supplemented with 0.132; 0.174, 0.215, 0.256 and 0.298% DL-methionine (98%, in order to provide 0.572, 0.613, 0.653, 0.693 and 0.734% digestible methionine + cystine. The levels of digestible methionine + digestible cystine followed, respectively, the relations of 67, 72, 77, 81 and 86% with lysine fixed at 0.851%. Feed intake, methionine + cystine intake, feed conversion per dozen eggs, egg weigth and mass, percentage of egg components, internal egg quality and weight gain were evaluated. Methionine + cystine levels showed a quadratic effect on feed conversion per dozen eggs and egg weight, a linear effect on feed conversion per kilogram of eggs and percentage of albumen. There was also a positive linear effect on yolk percentage. The methionine + cystine requirement was estimated at 0.572%, corresponding to 682 mg of digestible methionine + cystine/bird/day.

  18. Methionine Induces LAT1 Expression in Dairy Cow Mammary Gland by Activating the mTORC1 Signaling Pathway.

    Science.gov (United States)

    Duan, Xiaoyu; Lin, Ye; Lv, He; Yang, Yang; Jiao, Hongtao; Hou, Xiaoming

    2017-12-01

    Methionine is the limiting amino acid for milk protein synthesis in dairy cows. The effect of methionine availability on milk protein synthesis is dependent on its active transport into cells through amino acid transporters. L-type amino acid transporter 1 (LAT1), which induces the transport of neutral amino acids, is highly expressed in lactating mammary gland. However, the effect of methionine on LAT1 expression and the mechanism governing this process in dairy cow mammary gland are poorly understood. In this study, we show that treatment of dairy cow mammary epithelial cells with increasing concentrations of methionine for 24 h resulted in increased expression of LAT1 and its associated protein 4F2 heavy chain (4F2hc). Maximal expression levels occurred after treatment with 0.6 mM methionine. Methionine treatment also increased cell viability and β-casein synthesis. Western blots showed that methionine induced LAT1 and 4F2hc expression by activating mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling by rapamycin or raptor siRNA prevented the upregulation of LAT1 and 4F2hc. These results indicate that methionine may activate the mTORC1 signaling pathway and further increase LAT1 and 4F2hc expression in dairy cow mammary gland, thus affecting milk protein synthesis.

  19. Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies.

    Science.gov (United States)

    Loew, Caroline; Knoblich, Constanze; Fichtl, Jürgen; Alt, Nadja; Diepold, Katharina; Bulau, Patrick; Goldbach, Pierre; Adler, Michael; Mahler, Hanns-Christian; Grauschopf, Ulla

    2012-11-01

    The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development. Copyright © 2012 Wiley Periodicals, Inc.

  20. N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment

    Directory of Open Access Journals (Sweden)

    Caldovic L

    2011-08-01

    Full Text Available Nicholas Ah Mew, Ljubica CaldovicCenter for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington DC, USAAbstract: The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1, requires an allosteric activator, N-acetylglutamate (NAG. The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS. An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.Keywords: urea cycle, urea cycle disorder, N-acetyl-L-glutamate, N-acetylglutamate synthase, hyperammonemia, N-carbamyl-L-glutamate

  1. Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase

    International Nuclear Information System (INIS)

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2012-01-01

    The bacterial selenocysteine synthase SelA from Aquifex aeolicus was crystallized and the diffraction resolution was improved by lysine-residue methylation, truncation of N-terminal region (ΔN), and Lys-to-Ala point mutations. Phases were determined by using a selenomethionine-substituted crystal of the ΔN mutant. Selenocysteine (Sec), the 21st amino acid, is synthesized on its specific tRNA (tRNA Sec ) via a multi-step process. In bacteria, tRNA Sec is ligated first with serine by seryl-tRNA synthetase, which is followed by Ser-to-Sec conversion by Sec synthase (SelA). To elucidate its structure and catalytic mechanism, Aquifex aeolicus SelA was crystallized. Although wild-type SelA crystals diffracted X-rays poorly (to up to 8 Å resolution), the resolution was improved by introducing a quadruple point mutation targeting the loop regions and by methylating the lysine residues, which yielded 3.9 Å resolution diffraction data from a full-length SelA crystal. Truncation of the N-terminal region (ΔN) also improved the resolution. A 3.3 Å resolution data set for phase determination was obtained from a crystal of selenomethionine-substituted Lys-methylated SelA-ΔN

  2. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  3. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.

    1986-01-01

    of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined......Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...

  4. Bioavailability of D-methionine relative to L-methionine for nursery pigs using the slope-ratio assay

    Directory of Open Access Journals (Sweden)

    Changsu Kong

    2016-09-01

    Full Text Available This experiment was conducted to determine the bioavailability of D-methionine (Met relative to L-Met for nursery pigs using the slope-ratio assay. A total of 50 crossbred barrows with an initial BW of 13.5 kg (SD = 1.0 were used in an N balance study. A Met-deficient basal diet (BD was formulated to contain an adequate amount of all amino acids (AA for 10–20 kg pigs except for Met. The two reference diets were prepared by supplementing the BD with 0.4 or 0.8 g L-Met/kg at the expense of corn starch, and an equivalent concentration of D-Met was added to the BD for the two test diets. The pigs were adapted to the experimental diets for 5 d and then total but separated collection of feces and urine was conducted for 4 d according to the marker-to-marker procedure. Nitrogen intakes were similar across the treatments. Fecal N output was not affected by Met supplementation regardless of source and consequently apparent N digestibility did not change. Conversely, there was a negative linear response (P < 0.01 to Met supplementation with both Met isomers in urinary N output, which resulted in increased retained N (g/4 d and N retention (% of intake. No quadratic response was observed in any of the N balance criteria. The estimated bioavailability of D-Met relative to L-Met from urinary N output (g/4 d and N retention (% of intake as dependent variables using supplemental Met intake (g/4 d as an independent variable were 87.6% and 89.6%, respectively; however, approximately 95% of the fiducial limits for the relative bioavailability estimates included 100%. In conclusion, with an absence of statistical significance, the present study indicated that the mean relative bioequivalence of D- to L-Met was 87.6% based on urinary N output or 89.6% based on N retention.

  5. Effect of the opioid methionine enkephalinamide on signal transduction in human T-lymphocytes

    DEFF Research Database (Denmark)

    Sørensen, A N; Claesson, Mogens Helweg

    1998-01-01

    T cell receptor (TCR/CD3) induced fluctuations in intracellular free ionizied calcium, [Ca2+]i, was analysed in the human T leukemia cell clone, Jurkat, cultured in the presence of the opioid methionine enkephalinamide (Met-Enk) in titrated concentrations (10[-7] to 10[-15] M) or saline (PBS...

  6. Mechanism of oxidation of L-methionine by iron(III)

    Indian Academy of Sciences (India)

    phenanthroline complex have been studied in perchloric acid medium. The reaction is first order each in iron(III) and methionine. Increase in [phenanthroline] increases the rate while increase in [HClO4] decreases it. While the reactive species of the ...

  7. Dietary intake of B vitamins and methionine and risk of lung cancer.

    Science.gov (United States)

    Bassett, J K; Hodge, A M; English, D R; Baglietto, L; Hopper, J L; Giles, G G; Severi, G

    2012-02-01

    B vitamins and related enzymes involved in one-carbon metabolism are necessary for DNA replication, DNA repair and regulation of gene expression. Disruption of one-carbon mechanism may affect cancer risk. We investigated prospectively the relationship between dietary intakes of methionine, B vitamins associated with one-carbon metabolism and risk of lung cancer. The Melbourne Collaborative Cohort Study recruited 41,514 men and women aged 40-69 years between 1990 and 1994. During follow-up of 14,595 men and 22,451 women for an average of 15 years, we ascertained 348 incident lung cancers. Dietary intake of B vitamins and methionine was estimated from a 121-item food frequency questionnaire. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression. In current smokers, dietary intake of riboflavin was inversely associated with lung cancer risk (HR=0.53; 95% CI: 0.29-0.94, fifth versus first quintile; P-linear trend=0.01). No associations were found for former or never smokers or for dietary intake of any of the other B vitamins or methionine. Overall, we found little evidence of an association between B vitamins or methionine and lung cancer risk. The weak inverse association between riboflavin and lung cancer risk in current smokers needs further investigation.

  8. The First International Mini-Symposium on Methionine Restriction and Lifespan

    Directory of Open Access Journals (Sweden)

    Gene eAbles

    2014-05-01

    Full Text Available It has been 20 years since the Orentreich Foundation for the Advancement of Science, under the leadership Dr. Norman Orentreich, first reported that low methionine (Met ingestion by rats extends lifespan [1]. Since then, several studies have replicated the effects of dietary methionine restriction (MR in delaying age-related diseases [2–5]. We report the abstracts from the First International Mini-Symposium on Methionine Restriction and Lifespan held in Tarrytown, NY last September 2013. The goals were 1 to gather researchers with an interest in methionine restriction and lifespan, 2 to exchange knowledge, 3 to generate ideas for future investigations, and 4 to strengthen relationships within this community. The presentations highlighted the importance of research on cysteine, growth hormone (GH, and ATF4 in the paradigm of aging. In addition, the effects of dietary restriction or MR in the kidneys, liver, bones and the adipose tissue were discussed. The symposium also emphasized the value of other species, e.g. the naked mole rat, Brandt’s bat and drosophila in aging research. Overall, the symposium consolidated scientists with similar research interests and provided opportunities to conduct future collaborative studies.

  9. Investigation of the metal binding site in methionine aminopeptidase by density functional theory

    DEFF Research Database (Denmark)

    Jørgensen, Anne Techau; Norrby, Per-Ola; Liljefors, Tommy

    2002-01-01

    All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions...

  10. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    Science.gov (United States)

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  11. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    Science.gov (United States)

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASEStephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

  12. Acute Administration of Methionine Affects Performance of Swiss Mice in Learning and Memory Paradigms.

    Science.gov (United States)

    Abi, I; Magaji, R A; Magaji, M G

    2015-12-20

    Methionine, an essential amino acid, plays an essential role in the central nervous system CNS development. It serves as a crucial intermediate in the methylation, trans-sulfuration and amino- phosphorylationpathways,necessary for the synthesis of nucleic acids, phospholipids, hormones, neurotransmitters, antioxidants, polyamines, catecholamines and other biogenic amines. The effect of methionine on learning and memory in mice was investigated using Morris water maze (MWM), Elevated plus maze(EPM) and Y maze (YM). Animals were administered with distilled water (control), methionine (1,700mg/kg); folate (3mg/kg) or methionine (1700mg/kg) plus folate (3mg/kg) for 14 days. Escape latency and time spent in target quadrants; transfer latency and percentage spontaneous alternations were measured in the MWM, EPM and YM respectively. The animals were anaesthetized with inhalational chloroform and their brains subsequently harvested, homogenized and assayed for acetylcholinesterase24 hours after the experiment.Folate significantly(pmemory enhancement. However, a short course folate supplementation impairslearning and working memory especially when combined with methioninewhich may be as a result of sudden overwhelming of the methylation cycle, leading to homocysteinemia which is pro-dementia.

  13. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    Directory of Open Access Journals (Sweden)

    Honek John F

    2005-10-01

    Full Text Available Abstract Background The alkaline protease from Pseudomonas aeruginosa (AprA is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM, into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.

  14. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    Science.gov (United States)

    Walasek, Paula; Honek, John F

    2005-01-01

    Background The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease. PMID:16221305

  15. Mechanism of oxidation of L-methionine by iron(III)-1,10 ...

    Indian Academy of Sciences (India)

    Unknown

    stand for 2 h at 50°C. Thereafter 10 cm3 of 0⋅4 mol dm–3 sodium bicarbonate was added and the solution stirred vigorously, followed by dropwise addition of benzoyl chloride solution until precipitation was complete. The precipitate obtained was identified as N- benzoyl methionine sulphoxide (m.p. 183°C), a derivative of ...

  16. Independent and additive effects of glutamic acid and methionine on yeast longevity.

    Science.gov (United States)

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

    2013-01-01

    It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan.

  17. Effect of herbal choline and rumen-protected methionine on lamb ...

    African Journals Online (AJOL)

    Elizabeth A Mendoza B MD

    2018-01-30

    Jan 30, 2018 ... Effect of herbal choline and rumen-protected methionine on lamb performance and ... successful, presumably because the diets were based on low-quality forage with inadequate lysine (Lys) and ... experiments with dairy cows showed that dietary supplementation with RPM and RPC alleviated negative ...

  18. Mechanism of oxidation of L-methionine by iron (III)-1, 10 ...

    Indian Academy of Sciences (India)

    phenanthroline complex have been studied in perchloric acid medium. The reaction is first order each in iron(III) and methionine. Increase in [phenanthroline] increases the rate while increase in [HClO4] decreases it. While the reactive species of the ...

  19. Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

    DEFF Research Database (Denmark)

    Hartmann, R; Norby, P L; Martensen, P M

    1998-01-01

    A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range....... Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro......-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel...

  20. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

    CSIR Research Space (South Africa)

    Theron, Anjo

    2017-10-01

    Full Text Available studies indicating an alternative mechanism via the cytochrome cytochrome bc1 complex impacting on the homeostasis of ATP synthesis [39]. The inhibition of glutamine synthetase may also impact the ATP homeostasis as the resultant accumulation of α...

  1. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...

  2. Methionine adenosyltransferases in cancers: Mechanisms of dysregulation and implications for therapy.

    Science.gov (United States)

    Maldonado, Lauren Y; Arsene, Diana; Mato, José M; Lu, Shelly C

    2018-01-01

    Methionine adenosyltransferase genes encode enzymes responsible for the biosynthesis of S-adenosylmethionine, the principal biological methyl donor and precursor of polyamines and glutathione. Mammalian cells express three genes - MAT1A, MAT2A, and MAT2B - with distinct expression and functions. MAT1A is mainly expressed in the liver and maintains the differentiated states of both hepatocytes and bile duct epithelial cells. Conversely, MAT2A and MAT2B are widely distributed in non-parenchymal cells of the liver and extrahepatic tissues. Increasing evidence suggests that methionine adenosyltransferases play significant roles in the development of cancers. Liver cancers, namely hepatocellular carcinoma and cholangiocarcinoma, involve dysregulation of all three methionine adenosyltransferase genes. MAT1A reduction is associated with increased oxidative stress, progenitor cell expansion, genomic instability, and other mechanisms implicated in tumorigenesis. MAT2A/MAT2B induction confers growth and survival advantage to cancerous cells, enhancing tumor migration. Highlighted examples from colon, gastric, breast, pancreas and prostate cancer studies further underscore methionine adenosyltransferase genes' role beyond the liver in cancer development. In this subset of extra-hepatic cancers, MAT2A and MAT2B are induced via different regulatory mechanisms. Understanding the role of methionine adenosyltransferase genes in tumorigenesis helps identify attributes of these genes that may serve as valuable targets for therapy. While S-adenosylmethionine, and its metabolite, methylthioadenosine, have been largely explored as therapeutic interventions, targets aimed at regulation of MAT gene expression and methionine adenosyltransferase protein-protein interactions are now surfacing as potential effective strategies for treatment and chemoprevention of cancers. Impact statement This review examines the role of methionine adenosyltransferases (MATs) in human cancer development

  3. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

    OpenAIRE

    Gonzalez Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J.; Faraldos, Juan A.; Allemann, Rudolf Konrad

    2014-01-01

    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

  4. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  5. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  6. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  7. Producing alpha-olefins using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.; Keasling, Jay D.

    2018-01-02

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

  8. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  9. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  10. One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar.

    Science.gov (United States)

    Irmisch, Sandra; Müller, Andrea T; Schmidt, Lydia; Günther, Jan; Gershenzon, Jonathan; Köllner, Tobias G

    2015-10-28

    Labdane-related diterpenoids form the largest group among the diterpenes. They fulfill important functions in primary metabolism as essential plant growth hormones and are known to function in secondary metabolism as, for example, phytoalexins. The biosynthesis of labdane-related diterpenes is mediated by the action of class II and class I diterpene synthases. Although terpene synthases have been well investigated in poplar, little is known about diterpene formation in this woody perennial plant species. The recently sequenced genome of Populus trichocarpa possesses two putative copalyl diphosphate synthase genes (CPS, class II) and two putative kaurene synthase genes (KS, class I), which most likely arose through a genome duplication and a recent tandem gene duplication, respectively. We showed that the CPS-like gene PtTPS17 encodes an ent-copalyl diphosphate synthase (ent-CPS), while the protein encoded by the putative CPS gene PtTPS18 showed no enzymatic activity. The putative kaurene synthases PtTPS19 and PtTPS20 both accepted ent-copalyl diphosphate (ent-CPP) as substrate. However, despite their high sequence similarity, they produced different diterpene products. While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane. Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20. A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles. Gene expression analysis revealed that the diterpene synthase genes characterized showed organ-specific expression with the highest abundance of PtTPS17 and PtTPS20 transcripts in poplar roots. The poplar diterpene synthases PtTPS17, PtTPS19, and PtTPS20 contribute to the production of ent-kaurene and 16

  11. Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

    Science.gov (United States)

    Anderson, J Christopher [San Francisco, CA; Wu, Ning [Brookline, MA; Santoro, Stephen [Cambridge, MA; Schultz, Peter G [La Jolla, CA

    2009-08-18

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

  12. Methionine- and choline-deficient diet induces hepatic changes characteristic of non-alcoholic steatohepatitis

    Directory of Open Access Journals (Sweden)

    Éder Marcolin

    2011-03-01

    Full Text Available CONTEXT: Non-alcoholic steatohepatitis is a disease with a high incidence, difficult diagnosis, and as yet no effective treatment. So, the use of experimental models for non-alcoholic steatohepatitis induction and the study of its routes of development have been studied. OBJECTIVES: This study was designed to develop an experimental model of non-alcoholic steatohepatitis based on a methionine- and choline-deficient diet that is manufactured in Brazil so as to evaluate the liver alterations resulting from the disorder. METHODS: Thirty male C57BL6 mice divided in two groups (n = 15 were used: the experimental group fed a methionine- and choline-deficient diet manufactured by Brazilian company PragSoluções®, and the control group fed a normal diet, for a period of 2 weeks. The animals were then killed by exsanguination to sample blood for systemic biochemical analyses, and subsequently submitted to laparotomy with total hepatectomy and preparation of the material for histological analysis. The statistical analysis was done using the Student's t-test for independent samples, with significance level of 5%. RESULTS: The mice that received the methionine- and choline-deficient diet showed weight loss and significant increase in hepatic damage enzymes, as well as decreased systemic levels of glycemia, triglycerides, total cholesterol, HDL and VLDL. The diagnosis of non-alcoholic steatohepatitis was performed in 100% of the mice that were fed the methionine- and choline-deficient diet. All non-alcoholic steatohepatitis animals showed some degree of macrovesicular steatosis, ballooning, and inflammatory process. None of the animals which were fed the control diet presented histological alterations. All non-alcoholic steatohepatitis animals showed significantly increased lipoperoxidation and antioxidant enzyme GSH activity. CONCLUSION: The low cost and easily accessible methionine- and choline-deficient diet explored in this study is highly effective in

  13. [11C] Methionine as PET radiopharmaceutical produced at CDTN/CNEN

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, Marina B.; Ferreira, Soraya Z.; Carvalho, Tiago F.; Silva, Juliana B. da, E-mail: mbs@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Unidade de Pesquisa e Producao de Radiofarmacos

    2013-07-01

    Carbon-11 ({sup 11}C) is an attractive radionuclide used in positron emission tomography (PET) since carbon is a ubiquitous element in biomolecules. Positron emitter-labeled amino acids are being widely used as indicators of tumor activity due to enhanced expression of amino acid transporter systems in cancer cells. L-[Methyl-({sup 11}C)] Methionine or [{sup 11}C]Methionine is being used in neuro-oncology and, unlike 2-[{sup 18}F]fluoro-2-deoxy-D-glucose ({sup 18}FDG), gives more contrast images and improves brain tumor diagnosis. The aim of this work was to develop the synthesis and quality control of [{sup 11}C]Methionine at the Radiopharmaceuticals Research and Production Facility (UPPR) of CDTN/CNEN. The synthesis of [{sup 11}C] Methionine was performed using two Sep-Pak tC18 plus cartridges one as solid support for the {sup 11}C-methylation of the precursor L-homocysteine thiolactone hydrochloride and another for purification. The pH, radionuclidic identity and purity, residual solvents, radiochemical and chemical purity of the final product were evaluated as described on the European Pharmacopoeia 7.0 monograph. Total synthesis time was 18 minutes, the radiochemical yield was approximately 15% (non-decay corrected) and radiochemical purity was greater than 95%. [{sup 11}C]Methionine was successfully synthesized at CDTN using the described procedures and complied with quality requirements. Due to the rapid growth of oncologic PET scans in last decade, {sup 11}C labelling holds great promises in the next few years with the application of other tracers beyond {sup 18}FDG. This pioneering work of UPPR/CDTN represents a response to the demands of a growing nuclear medicine in the country focused on achieving better diagnostic imaging. (author)

  14. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    International Nuclear Information System (INIS)

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-01-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 o C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  15. Antioxidants L-carnitine and D-methionine modulate neuronal activity through GABAergic inhibition.

    Science.gov (United States)

    Wu, Calvin; Gopal, Kamakshi V; Moore, Ernest J; Gross, Guenter W

    2014-07-01

    Antioxidants are well known for their neuroprotective properties against reactive oxygen species in cortical neurons and auditory cells. We recently identified L-carnitine and D-methionine to be among agents that provide such protection. Here, we investigated their neuronal modulatory actions. We used cultured neuronal networks grown on microelectrode arrays to assess the effects of L-carnitine and D-methionine on network function. Spike production and burst properties of neuronal networks were used as parameters to monitor pharmacological responses. L-Carnitine and D-methionine reduced spike activity with 100% efficacy with EC50 values of 0.22 (± 0.01) mM and 1.06 (± 0.05) mM, respectively. In the presence of 1.0-40 μM of the GABAA antagonist bicuculline, the sigmoidal concentration-response curves of both compounds exhibited stepwise shifts, without a change in efficacy. Under a maximal bicuculline concentration of 40 μM, the EC50 increased to 3.57 (± 0.26) mM for L-carnitine and to 10.52 (± 0.97) mM for D-methionine, more than a tenfold increase. The agonist-antagonist interactions with bicuculline were estimated by Lineweaver-Burk plot analyses to be competitive, corroborated by the computed dissociation constants of bicuculline. For both compounds, the effects on the network burst pattern, activity reversibility, and bicuculline antagonism resembled that elicited by the GABAA agonist muscimol. We showed that the antioxidants L-carnitine and D-methionine modulate cortical electrical spike activity primarily through GABAA receptor activation. Our findings suggest the involvement of GABAergic mechanisms that perhaps contribute to the protective actions of these compounds.

  16. Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

    Science.gov (United States)

    Mirando, Adam C; Fang, Pengfei; Williams, Tamara F; Baldor, Linda C; Howe, Alan K; Ebert, Alicia M; Wilkinson, Barrie; Lounsbury, Karen M; Guo, Min; Francklyn, Christopher S

    2015-08-14

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

  17. Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

    Science.gov (United States)

    Lee, Eun-Young; Lee, Hyun-Cheol; Kim, Hyun-Kwan; Jang, Song Yee; Park, Seong-Jun; Kim, Yong-Hoon; Kim, Jong Hwan; Hwang, Jungwon; Kim, Jae-Hoon; Kim, Tae-Hwan; Arif, Abul; Kim, Seon-Young; Choi, Young-Ki; Lee, Cheolju; Lee, Chul-Ho; Jung, Jae U; Fox, Paul L; Kim, Sunghoon; Lee, Jong-Soo; Kim, Myung Hee

    2016-01-01

    The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/−) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection. PMID:27595231

  18. Functional identification of APIP as human mtnB, a key enzyme in the methionine salvage pathway.

    Directory of Open Access Journals (Sweden)

    Camille Mary

    Full Text Available The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28, 5'-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23, 5'-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109, 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77, aci-reductone dioxygenase (mtnD, EC 1.13.11.54 and 4-methylthio-2-oxo-butanoate (MTOB transaminase (EC 2.6.1.-. The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.

  19. Functional identification of APIP as human mtnB, a key enzyme in the methionine salvage pathway.

    Science.gov (United States)

    Mary, Camille; Duek, Paula; Salleron, Lisa; Tienz, Petra; Bumann, Dirk; Bairoch, Amos; Lane, Lydie

    2012-01-01

    The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5'-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5'-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.

  20. Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

    Science.gov (United States)

    Krishnamoorthy, Ezhilarasi; Hassan, Sameer; Hanna, Luke Elizabeth; Padmalayam, Indira; Rajaram, Rama; Viswanathan, Vijay

    2017-05-07

    Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

    Science.gov (United States)

    Sharma, Arvind; Sharma, Amit

    2015-02-01

    The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

  2. Bio-efficacy comparison of herbal-methionine and DL-methionine based on performance and blood parameters of broiler chickens

    Directory of Open Access Journals (Sweden)

    Sheila Hadinia

    2014-06-01

    Full Text Available This study was conducted to compare the bio-efficacy of herbal methionine (H-Met relative to DL-methionine (DL-Met on 160 “Ross 308” broiler chickens. DL-Met and H-Met were added to the basal diet in eight experimental treatments with three and four concentrations respectively in starter, grower and finisher period. Blood parameters which were measured at 24 and 42 days of age consisted of: serum proteins (total protein, albumin and globulin, serum uric acid, serum fats (low density lipoprotein, high density lipoprotein, triglyceride and cholesterol and serum enzymes (alanine amino transaminase and aspartate amino transaminase. Completely randomized design, multi-exponential and multilinear regressions were used to determine bio-efficacy of H-Met in terms of performance and blood parameters of broilers. The results showed that supplemented methionine (Met sources had no significant effect on blood parameters at 24 day of age. At 42 day of age the amounts of globulin and serum high density lipoprotein (HDL increased with supplemented Met, (p < 0.05. Regression analysis revealed that H-Met was 55.00, 71.00, 78.00, 47.00, 58.00 and 73.00% as efficacious as DL-Met for body weight gain, feed intake, feed conversion ratio, albumin, globulin and high density lipoprotein criteria, respectively. The average of bio-efficacy of H-Met compared to DL-Met was 67.00% and 59.00% on average across performance criteria and blood criteria respectively and was 63.00% across these two criteria tested. The results of the present study indicated that H-Met can be administered as a new and a natural source of Met in poultry industry.

  3. Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

    Science.gov (United States)

    Gebril, Sayed; Seger, Mark; Villanueva, Fabiola Muro; Ortega, Jose Luis; Bagga, Suman; Sengupta-Gopalan, Champa

    2015-10-01

    Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

  4. Enzyme kinetic modelling and analytical solution of nonlinear rate equation in the transformation of D-methionine into L-methionine in batch reactor using the new homotopy perturbation method

    OpenAIRE

    Sivasamy, Pavithra; Ganapathy, Jansi Rani Palaniyandi; Thinakaran, Iswarya; Lakshmanan, Rajendran

    2016-01-01

    A mathematical model of biotransformation of D-methionine into L-methionine in the cascade of the enzymes such as, D-amino acid oxidase (D-AAO), L-phenylalanine dehydrogenase (L-PheDH) and formate dehydrogenase (FDH) is discussed. The model is based on a system of coupled nonlinear reaction equations under non steady-state conditions for biochemical reactions occurring in the batch reactor that describes the substrate and product concentration within the catalyst. Simple analytical expression...

  5. Engineering of plant type III polyketide synthases.

    Science.gov (United States)

    Wakimoto, Toshiyuki; Morita, Hiroyuki; Abe, Ikuro

    2012-01-01

    Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. The lack of effect of a pyridoxine deficiency on the utilization of the hydroxyl analogue of methionine by the chick.

    Science.gov (United States)

    Saroka, J M; Combs, G F

    1986-04-01

    The purpose of this study was to determine whether a vitamin B6 deficiency affects the utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB), an analogue of methionine. The basal diet used for this purpose was based on peas, peanut meal, and glucose monohydrate and was low in both methionine and pyridoxine. Supplemental pyridoxine hydrochloride and L-methionine or HMB resulted in significant increases in growth rate and efficiency of feed utilization; supplemental pyridoxine also produced significant increases in the activity of glutamic-oxaloacetic transaminase (PGOT, EC 2.6.1.1.) in plasma. There was no discernible effect of change in vitamin B6 status on the utilization of HMB as compared with L-methionine.

  7. The browning value changes and spectral analysis on the Maillard reaction product from glucose and methionine model system

    Science.gov (United States)

    Al-Baarri, A. N.; Legowo, A. M.; Widayat

    2018-01-01

    D-glucose has been understood to provide the various effect on the reactivity in Maillard reaction resulting in the changes in physical performance of food product. Therefore this research was done to analyse physical appearance of Maillard reaction product made of D-glucose and methionine as a model system. The changes in browning value and spectral analysis model system were determined. The glucose-methionine model system was produced through the heating treatment at 50°C and RH 70% for 24 hours. The data were collected for every three hour using spectrophotometer. As result, browning value was elevated with the increase of heating time and remarkably high if compare to the D-glucose only. Furthermore, the spectral analysis showed that methionine turned the pattern of peak appearance. As conclusion, methionine raised the browning value and changed the pattern of spectral analysis in Maillard reaction model system.

  8. Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.

    Science.gov (United States)

    Yoshikuni, Yasuo; Martin, Vincent J J; Ferrin, Thomas E; Keasling, Jay D

    2006-01-01

    The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

  9. Activities of methionine-γ-lyase in the acidophilic archaeon “Ferroplasma acidarmanus” strain fer1

    Directory of Open Access Journals (Sweden)

    Khan MA

    2013-04-01

    Full Text Available M A Khan,1 Madeline M López-Muñoz,2 Charles W Kaspar,3 Kai F Hung1 1Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA; 2Department of Biology, Universidad de Puerto Rico, Mayaguez, Puerto Rico; 3Bacteriology Department, University of Wisconsin, Madison, WI, USA Abstract: Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1's ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine-γ-lyase (EC 4.4.1.11, an enzyme known to carry out α, γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5'-phosphate (PLP binding domain and a partially conserved catalytic domain in both putative sequences. Detection of PLP-dependent and L-methionine-dependent production of α-keto compounds and thiol groups in fer1 confirmed the presence of methionine-γ-lyase activity. Further, fer1 lysate was capable of processing related substrates, including D-methionine, L-cysteine, L-cystathionine, and L/D-homocysteine. When the two putative fer1 methionine-γ-lyase gene-coded proteins were expressed in Escherichia coli cells, one sequence demonstrated an ability to carry out α, γ-elimination activity, while the other exhibited γ-replacement activity. These fer1 methionine-γ-lyases also exhibited optimum pH, substrate specificity, and catalytic preferences that are different from methionine-γ-lyases from other organisms. These differences are discussed in the context of molecular phylogeny constructed using a maximum

  10. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    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.

  11. Synergistic Effect of L-Methionine and KI on Copper Corrosion Inhibition in HNO3 (1M)

    OpenAIRE

    Amel SEDIK; Sihem ABDERRAHMANE; Said BOUKERCHE; Abdelaziz HIMOUR; Amel GHARBI

    2014-01-01

    L-Methionine (L-Met) efficiency as a non-toxic corrosion inhibitor for copper in 1M HNO3 has been studied by using electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization. Copper corrosion rate significant decrease was observed in the presence of L-Met at 10-4M. The Obtained Results from potentiodynamic polarization and impedance measurements are in good agreement. L-Methionine adsorption on copper surface follows Langmuir isotherm. L-Met free energy adsorption on copper...

  12. Local cerebral metabolic rate of 11C-L-Methionine in early stages of dementia, schizophrenia, Parkinson's disease

    International Nuclear Information System (INIS)

    Bustany, P.; Henry, J.F.; de Rotrou, J.; Signoret, J.L.; Ziegler, M.; Zarifian, E.; Soussaline, F.; Comar, D.

    1983-06-01

    A dynamic three-compartment model of methionine metabolism in brain was applied in human patients using 11 C-L-Methionine and positron emission tomography (P.E.T). Psychometric evaluations of demented patients were correlated with a significant diminution of protein synthesis in the frontal area. This diminution was lower in ebephrenic patients (-17%) but was consistent with the results obtained with 18 F glucose. No significant abnormality was detected in patients with Parkinson disease

  13. Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

    Science.gov (United States)

    Yermolaieva, Olena; Xu, Rong; Schinstock, Carrie; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2004-02-01

    Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of methionine in proteins involving the enzyme peptide methionine sulfoxide reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

  14. Evaluation of Herbal Methionine and Mangifera Indica Against Lead-induced Organ Toxicity in Broilers

    Science.gov (United States)

    Lakshmi, D. Udaya; Adilaxmamma, K.; Reddy, A. Gopala; Rao, V. Vykunta

    2011-01-01

    Lead toxicity was studied in male broiler chicks (Cobb strain) of a day-old age. The chicks were randomly divided into six groups consisting of 15 in each group. Group 1 was maintained as basal diet control and group 2 was kept on lead at 300 ppm in feed throughout 5 wk as toxic control without any treatment. Groups 3 and 4 were maintained on herbal methionine at 1.4 g/kg feed + Mangifera Indica at 0.1% in feed, respectively. Groups 5 and 6 were treated with lead + herbal methionine and lead + M. indica, respectively, for the 5 wk. The concentration of thiobarbituric acid reactive substances (TBARS) and protein carbonyls, and the activities of superoxide dismutase (SOD) and catalase in liver and kidney revealed a significant (Pindica may be attributed to their antioxidant, anti-stress and hepatoprotective principles. PMID:21430924

  15. Stabilization of methionine-rich protein in Saccharomyces cerevisiae: targeting of BZN protein into the peroxisome.

    Science.gov (United States)

    Nicaud, J M; Raynal, A; Beyou, A; Merkamm, M; Ito, H; Labat, N

    1994-01-01

    We have constructed a gene coding for the 12-kDa intermediate form of the 2s methionine-rich protein from Bertholletia excelsa seeds. This protein, expressed intracellularly in yeast, is characterised by a 20-min half-life. By adding 11 amino acids corresponding to the peroxisome-targeting sequence (PTSc) of luciferase, we have significantly increased its half-life. This stabilization allowed accumulation of the BZN protein into the peroxisome as judged by cell fractionation. Accumulation of the 12-kDa protein results in a significant increase of the total methionine content in yeast cells (30%) indicating that such a microorganism could represent a practicable protected shuttle for an animal-feed additive.

  16. Structural Transitions Induced by a Recombinant Methionine-Trigger in Silk Spidroin

    Science.gov (United States)

    Wilson, Donna; Winkler, Stefan; Valluzzi, Regina; Kaplan, David

    2000-03-01

    Control of beta sheet formation is an important factor in the understanding and prediction of structural transitions and protein folding. In genetically engineered silk proteins this control has been achieved using oxidative triggers. A genetically engineered variant of a spider silk protein, and a peptide analog, based on the consensus sequence of Nephila clavipes dragline silk, were modified to include methionines flanking the beta sheet forming polyalanine regions. These methionines could be selectively reduced and oxidized, altering the bulkiness and charge of the sulfhydryl group to control beta sheet formation by steric hindrance. Biophysical characterization and monitoring of structural transitions and intermediates were accomplished through attenuated total reflectance infrared spectroscopy (ATR-IR) for solution state structures in both oxidized and reduced forms. For solid state structural characterization, IR microscopy and reflectance IR experiments were performed. Electron diffraction data as well as circular dichroism studies provide structural corroboration for all experiments in which reproducible sample preparation was achieved.

  17. Enzymatic activity of methionine adenosyltransferase variants identified in patients with persistent hypermethioninemia

    OpenAIRE

    Fernandez-Irigoyen, J. (Javier); Santamaria, E. (Enrique); Chien, Y-H (Yin-Hsiu); Hwu, W.L. (Wuh-Liang); Korman, S.H. (Stanley H.); Faghfoury, H. (Hanna); Schulze, A. (Andreas); Hoganson, G.E. (George E.); Stabler, S.P. (Sally P.); Allen, R.H. (Robert H.); Wagner, C. (Conrad); Mudd, S.H. (S. Harvey); Corrales, F.J. (Fernando José)

    2010-01-01

    Methionine adenosyltransferases (MAT's) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte prolife...

  18. Unusual activity pattern of leucine aminopeptidase inhibitors based on phosphorus containing derivatives of methionine and norleucine

    Czech Academy of Sciences Publication Activity Database

    Pícha, Jan; Liboska, Radek; Buděšínský, Miloš; Jiráček, Jiří; Pawelczak, M.; Mucha, A.

    2011-01-01

    Roč. 26, č. 2 (2011), s. 155-161 ISSN 1475-6366 R&D Projects: GA ČR GA203/06/1405; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : aminophosphonates * aminophospinates * methionine * norleucine * phosphorus containing dipeptides * cytosolic leucine aminopeptidase * inhibitors Subject RIV: CC - Organic Chemistry Impact factor: 1.617, year: 2011

  19. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    Energy Technology Data Exchange (ETDEWEB)

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W. (Cornell); (Iowa State)

    2010-09-23

    Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

  20. Mechanism of oxidation of L-methionine by iron(III)-1,10 ...

    Indian Academy of Sciences (India)

    Unknown

    phenanthroline and iron(III) 2,2′-bipyridyl oxidize methionine to sulphoxide, oxidation with the former oxidant is much faster compared to the latter. This is because of the higher oxidation potential of the iron(III)–phenanthroline complex compared to the iron(III)–2,2′-bipyridyl. Hence the intimate mechanism of oxidation of ...

  1. The two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Wang YiPing

    2005-10-01

    Full Text Available Abstract Background Two putative methionine aminopeptidase genes, map (essential and yflG (non-essential, were identified in the genome sequence of Bacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in B. subtilis. Results In silico analysis of MAP evolution uncovered a coordinated pattern of MAP and deformylase that did not correlate with the pattern of 16S RNA evolution. Biochemical assays showed that both MAP (MAP_Bs and YflG (YflG_Bs from B. subtilis overproduced in Escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. Compared with MAP_Bs, YflG_Bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. Both map and yflG genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both E. coli and B. subtilis. In contrast, lacZ gene transcriptional fusions showed that the promoter activity of map was 50 to 100-fold higher than that of yflG. Primer extension analysis detected the transcription start site of the yflG promoter. Further work identified that YvoA acted as a possible weak repressor of yflG expression in B. subtilis in vivo. Conclusion Both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro and in vivo. The high expression level of map and low expression level of yflG may account for their essentiality and dispensality in B. subtilis, respectively, when cells are grown under laboratory conditions. Their difference in activity on synthetic substrates suggests that they have different protein targets in vivo.

  2. Rapid Method for Quantifying the Extent of Methionine Oxidation in Intact Calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Galeva, Nadezhda A.; Esch, S Wynn; Williams, Todd D.; Markillie, Lye MENG.; Squier, Thomas C.

    2005-09-01

    We have developed a method for rapidly quantifying the extent to which the functionally important Met144 and Met145 residues near the C-terminus of calmodulin (CaM) are converted to the corresponding sulfoxides, Met(O). The method utilizes a whole protein collision induced dissociation (CID) approach on an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer. Using standards of CaM oxidized by hydrogen peroxide (H2O2) or peroxynitrite (ONOO-), we demonstrated that CID fragmentation of the protein ions resulted in a series of C-terminal singly charged y1?y15 ions. Fragments larger than y4 exhibited mass shifts of +16 or +32 Da, corresponding to oxidation of one or two methionines, respectively. To assess the extent of oxidative modification for Met144 and Met145 to Met(O), we averaged the ratio of intensities for yn, yn +16, and yn +32 ions, where n = 6?9. By alternating MS and CID scans at low and high collision energies, this technique allowed us to rapidly determine both the distribution of intact CaM oxiforms and the extent of oxidative modification in the C-terminal region of the protein in a single run. We have applied the method to studies of the repair of fully oxidized CaM by methionine sulfoxide reductases (MsrA and MsrB), which normally function in concert to reduce the S and R stereoisomers of methionine sulfoxide. We found that repair of Met(O)144 and Met(O)145 did not go to completion, but was more efficient than average Met repair. Absence of complete repair is consistent with previous studies showing that accumulation of methionine sulfoxide in CaM can occur during aging.

  3. Effect of methionine and lactic acid bacteria as aflatoxin binder on broiler performance

    Science.gov (United States)

    Istiqomah, Lusty; Damayanti, Ema; Julendra, Hardi; Suryani, Ade Erma; Sakti, Awistaros Angger; Anggraeni, Ayu Septi

    2017-06-01

    The use of aflatoxin binder product based amino acids, lacic acid bacteria, and natural product gived the opportunity to be an alternative biological decontamination of aflatoxins. A study was conducted to determine the efficacy of aflatoxin binder administration (amino acid methionine and lactic acid bacteria (Lactobacillus plantarum G7)) as feed additive on broiler performance. In this study, 75 Lohmann unsexed day old chicks were distributed randomly into 5 units of cages, each filled with 15 broilers. Five cages were assigned into 5 treatments groups and fed with feed contained aflatoxin. The treatments as follow: P1 (aflatoxin feed without aflatoxin binder), P3 (aflatoxin feed + 0.8% of methionine + 1% of LAB), P4 (aflatoxin feed + 1.2% of methionine + 1% of LAB), P5 (aflatoxin feed + 1% of LAB), and K0 (commercial feed). The measurement of aflatoxin content in feed was performed by Enzyme Linked Immunosorbent Assay method using AgraQuant® Total Aflatoxin Assay Romer Labs procedure. The experimental period was 35 days with feeding and drinking ad libitum. LAB was administered into drinking water, while methionine into feed. Vaccination program of Newcastle Disease (ND) was using active vaccine at 4 and 18 day old, while Infectious Bursal Disease (IBD) was given at 8 day old. Parameter of body weight was observed weekly, while feed consumption noted daily. The result showed that aflatoxin in feed for 35 days period did not significantly affect the body weight gain and feed conversion. The lowest percentage of organ damage at 21 day old was found in P5 treatment (55%), while at 35day old was found in P4 treatment (64%). It could be concluded that technological process of detoxifying aflatoxin could be applied in an attempt to reduce the effect on the toxicity of aflatoxin in poultry feed.

  4. Free methionine supplementation limits alcohol-induced liver damage in rats

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Bode, C.; Bode, J.C.

    1998-01-01

    .3 to 42.1 +/- 7.2 micromol/g of protein in the alcohol group). Hepatic adenosine triphosphate content increased significantly with higher methionine consumption (13.5 +/- 0.8 vs. 26.9 +/- 2.8 micromol/g of protein in the control group and 11.9 +/- 1.4 vs. 20.5 +/- 2.5 micromol/g of protein in the alcohol...

  5. Choline and methionine differentially alter methyl carbon metabolism in bovine neonatal hepatocytes.

    Science.gov (United States)

    Chandler, Tawny L; White, Heather M

    2017-01-01

    Intersections in hepatic methyl group metabolism pathways highlights potential competition or compensation of methyl donors. The objective of this experiment was to examine the expression of genes related to methyl group transfer and lipid metabolism in response to increasing concentrations of choline chloride (CC) and DL-methionine (DLM) in primary neonatal hepatocytes that were or were not exposed to fatty acids (FA). Primary hepatocytes isolated from 4 neonatal Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (61, 128, 2028, and 4528 μmol/L) and DLM (16, 30, 100, 300 μmol/L), with or without a 1 mmol/L FA cocktail in a factorial arrangement. After 24 h of treatment, media was collected for quantification of reactive oxygen species (ROS) and very low-density lipoprotein (VLDL), and cell lysates were collected for quantification of gene expression. No interactions were detected between CC, DLM, or FA. Both CC and DLM decreased the expression of methionine adenosyltransferase 1A (MAT1A). Increasing CC did not alter betaine-homocysteine S-methyltranferase (BHMT) but did increase 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylenetetrahydrofolate reductase (MTHFR) expression. Increasing DLM decreased expression of BHMT and MTR, but did not affect MTHFR. Expression of both phosphatidylethanolamine N-methyltransferase (PEMT) and microsomal triglyceride transfer protein (MTTP) were decreased by increasing CC and DLM, while carnitine palmitoyltransferase 1A (CPT1A) was unaffected by either. Treatment with FA decreased the expression of MAT1A, MTR, MTHFR and tended to decrease PEMT but did not affect BHMT and MTTP. Treatment with FA increased CPT1A expression. Increasing CC increased secretion of VLDL and decreased the accumulation of ROS in media. Within neonatal bovine hepatocytes, choline and methionine differentially regulate methyl carbon pathways and suggest that choline may play a critical role in

  6. Partitioning of One-Carbon Units in Folate and Methionine Metabolism Is Essential for Neural Tube Closure

    Directory of Open Access Journals (Sweden)

    Kit-Yi Leung

    2017-11-01

    Full Text Available Summary: Abnormal folate one-carbon metabolism (FOCM is implicated in neural tube defects (NTDs, severe malformations of the nervous system. MTHFR mediates unidirectional transfer of methyl groups from the folate cycle to the methionine cycle and, therefore, represents a key nexus in partitioning one-carbon units between FOCM functional outputs. Methionine cycle inhibitors prevent neural tube closure in mouse embryos. Similarly, the inability to use glycine as a one-carbon donor to the folate cycle causes NTDs in glycine decarboxylase (Gldc-deficient embryos. However, analysis of Mthfr-null mouse embryos shows that neither S-adenosylmethionine abundance nor neural tube closure depend on one-carbon units derived from embryonic or maternal folate cycles. Mthfr deletion or methionine treatment prevents NTDs in Gldc-null embryos by retention of one-carbon units within the folate cycle. Overall, neural tube closure depends on the activity of both the methionine and folate cycles, but transfer of one-carbon units between the cycles is not necessary. : Leung at al. find that embryonic neural tube closure depends both on the supply of one-carbon units to the folate cycle from glycine cleavage and on the methionine cycle. In contrast, transfer of one-carbon units from the folate cycle to the methionine cycle by MTHFR is dispensable. Keywords: one-carbon metabolism, folic acid, neural tube defects, spina bifida, glycine cleavage system, non-ketotic hyperglycinemia, eye, Mthfr, Gldc

  7. Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite.

    Science.gov (United States)

    Lee, Warren L; Gold, Benjamin; Darby, Crystal; Brot, Nathan; Jiang, Xiuju; de Carvalho, Luiz Pedro S; Wellner, Daniel; St John, Gregory; Jacobs, William R; Nathan, Carl

    2009-02-01

    Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.

  8. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

    Science.gov (United States)

    Pan, Hai; Chen, Kenneth; Chu, Liping; Kinderman, Francis; Apostol, Izydor; Huang, Gang

    2009-01-01

    Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements. PMID:19165723

  9. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

    DEFF Research Database (Denmark)

    Brinch-Pedersen, H.; Galili, G.; Sørensen, K.

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance......, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids...... as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine....

  10. Role of Ginkgo Biloba in Hyperhomocysteinemia Induced in Rats By L-Methionine and Gamma Irradiation

    International Nuclear Information System (INIS)

    Mansour, S.Z.

    2011-01-01

    The objective of this study is to evaluate the role of Ginkgo biloba in hyperhomocysteinemia and oxidative stress. Methionine was supplied orally to adult male albino rats with a dose of 1.7 g/kg/day during 4 weeks. Irradiation was applied to rats by whole body gamma irradiation with a dose of 2 Gy/week up to a total dose of 8 Gy. Ginkgo biloba (100 mg/kg/day) was supplemented orally to rats, daily, during the period of methionine administration and/or radiation exposure. Biochemical analysis in blood and brain tissues showed that methionine and/or gamma irradiation produced significant increases in homocysteine and acetylcholine esterase levels and significant decrease in nitric oxide (NO). Significant increase in malondialdehyde (MDA) with significant decreases in glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase levels were observed and alteration in plasma lipid profile was also recorded. Ginkgo biloba supplementation has significantly decreased homocysteine and acetylcholine esterase levels and increased NO while was associated with significant improvement of oxidative stress and lipid profile. It could be concluded that the protective effect of Gingko biloba against hyperhomocysteinemia and oxidative stress is attributed to its antioxidant and free radicals scavenging properties.

  11. Evidence for participation of the methionine sulfoxide reductase repair system in plant seed longevity.

    Science.gov (United States)

    Châtelain, Emilie; Satour, Pascale; Laugier, Edith; Ly Vu, Benoit; Payet, Nicole; Rey, Pascal; Montrichard, Françoise

    2013-02-26

    Seeds are in a natural oxidative context leading to protein oxidation. Although inevitable for proper progression from maturation to germination, protein oxidation at high levels is detrimental and associated with seed aging. Oxidation of methionine to methionine sulfoxide is a common form of damage observed during aging in all organisms. This damage is reversible through the action of methionine sulfoxide reductases (MSRs), which play key roles in lifespan control in yeast and animal cells. To investigate the relationship between MSR capacity and longevity in plant seeds, we first used two Medicago truncatula genotypes with contrasting seed quality. After characterizing the MSR family in this species, we analyzed gene expression and enzymatic activity in immature and mature seeds exhibiting distinct quality levels. We found a very strong correlation between the initial MSR capacities in different lots of mature seeds of the two genotypes and the time to a drop in viability to 50% after controlled deterioration. We then analyzed seed longevity in Arabidopsis thaliana lines, in which MSR gene expression has been genetically altered, and observed a positive correlation between MSR capacity and longevity in these seeds as well. Based on our data, we propose that the MSR repair system plays a decisive role in the establishment and preservation of longevity in plant seeds.

  12. Synthesis and study of catalytic application of l-methionine protected gold nanoparticles

    Science.gov (United States)

    Raza, Akif; Javed, Safdar; Qureshi, Muhammad Zahid; khan, Muhammad Usman; Khan, Muhammad Saleem

    2017-10-01

    Gold nanoparticle is growing class of nanotechnology due to large number of uses. We synthesized stable l-methionine protected gold nanoparticles (AuNps) by in situ reduction of HAuCl4 using sodium borohydrate as reducing and l-methionine as stabilizing agent in an aqueous medium. Different parameters (pH, capping agent, precursor salt, and heating time) were optimized to see the effect on the size of particles. Double beam spectrophotometer was used to carry out the spectroscopic studies. It was observed that pH and concentration of reducing salt are deciding factors in controlling the size and morphology of AuNps. Scanning electron microscopy (SEM) verified the formation of AuNPs as predicted by UV-Vis spectra. The interaction of AuNPs with l-methionine was confirmed by Fourier Transform Infrared (FTIR). The reduction of 4-nitrophenol acted as standard of reaction to check the response of AuNps catalyst. Complete reduction of 4-nitrophenol was accomplished by AuNps sol in just 60 s. Fastest reduction rate was observed with smaller spherical particles. This study concluded that size and shape of AuNps can be monitored by controlling the pH, concentration of capping and reducing agent. It also provides an economical solution to aquatic environment in terms of time saving and use of small volume of catalytic solution for reduction of several other toxic organic pollutants.

  13. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    Science.gov (United States)

    Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

    2016-01-01

    Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

  14. Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

    Directory of Open Access Journals (Sweden)

    Cabrera-Luque Juan

    2008-09-01

    Full Text Available Abstract Background The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG, an essential allosteric activator of carbamylphosphate synthetase I (CPSI in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS, which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS. Results Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine. Conclusion Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine

  15. Draft Genome Sequence of Saccharomonospora sp. Strain LRS4.154, a Moderately Halophilic Actinobacterium with the Biotechnologically Relevant Polyketide Synthase and Nonribosomal Peptide Synthetase Systems

    Science.gov (United States)

    Alonso-Carmona, Scarlett; Vera-Gargallo, Blanca; de la Haba, Rafael R.; Ventosa, Antonio; Sandoval-Trujillo, Horacio

    2017-01-01

    ABSTRACT The draft genome sequence of Saccharomonospora sp. strain LRS4.154, a moderately halophilic actinobacterium, has been determined. The genome has 4,860,108 bp, a G+C content of 71.0%, and 4,525 open reading frames (ORFs). The clusters of PKS and NRPS genes, responsible for the biosynthesis of a large number of biomolecules, were identified in the genome. PMID:28546487

  16. Lack of cytosolic glutamine synthetase1;2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings.

    Science.gov (United States)

    Ohashi, Miwa; Ishiyama, Keiki; Kusano, Miyako; Fukushima, Atsushi; Kojima, Soichi; Hanada, Atsushi; Kanno, Keiichi; Hayakawa, Toshihiko; Seto, Yoshiya; Kyozuka, Junko; Yamaguchi, Shinjiro; Yamaya, Tomoyuki

    2015-01-01

    The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the phloem companion cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  17. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture.

    Science.gov (United States)

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V; Carvalho, Helena G; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-04-01

    The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  18. Effect of Mini-Tyrosyl-tRNA Synthetase/Mini-Tryptophanyl-tRNA Synthetase on Angiogenesis in Rhesus Monkeys after Acute Myocardial Infarction.

    Science.gov (United States)

    Zeng, Rui; Wang, Mian; You, Gui-ying; Yue, Rong-zheng; Chen, Yu-cheng; Zeng, Zhi; Liu, Rui; Qiang, Ou; Zhang, Li

    2016-02-01

    The purpose of this study was to clarify the effect of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase (mini-TyrRS/mini-TrpRS) in ischemic angiogenesis in rhesus monkeys with acute myocardial infarction (AMI). A 27-gauge needle was incorporated percutaneously into the left ventricular myocardium of rhesus monkeys with AMI. All monkeys were randomized to receive adenoviral vector mini-TyrRS/mini-TrpRS, which was administered as five injections into the infarcted myocardium, or saline or ad-null (control groups). The injections were guided by EnSite NavX left ventricular electroanatomical mapping. Mini-TyrRS/mini-TrpRS proteins were detected by Western blot and immunoprecipitation analyses. Microvessel density (MVD) per section was measured using immunostaining with a CD34 monoclonal antibody. Proliferating cardiomyocytes were identified through histological and immunohistochemical analyses. Myocardial perfusion and cardiac function were estimated by G-SPECT. Infarction size was also measured. Western blot analyses showed that compared to the normal zone, the expression level of mini-TyrRS/mini-TrpRS was significantly different in the infarction zone. G-SPECT analysis indicated that the mini-TyrRS group had better cardiac function and myocardial perfusion after the injection of ad-mini-TyrRS than before, while mini-TrpRS injection had a totally opposite effect. After mini-TyrRS was administered, there was less of an infarction zone and more proliferating cardiomyocytes and capillaries in the mini-TyrRS group compared to both of the control groups, and the ad-mini-TrpRS group had a totally opposite effect. These results indicated that angiogenesis could be either stimulated by mini-TyrRS or inhibited by mini-TrpRS. © 2015 John Wiley & Sons Ltd.

  19. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...

  20. Functional plasticity of paralogous diterpene synthases involved in conifer defense

    OpenAIRE

    Keeling, Christopher I.; Weisshaar, Sabrina; Lin, Roy P. C.; Bohlmann, Jörg

    2008-01-01

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant–herbivore, plant–pollinator, and plant–pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin...

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

    Science.gov (United States)

    Croteau, Rodney Bruce [Pullman, WA; Burke, Charles Cullen [Moscow, ID

    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.

  2. Addition of methionine and low cultivation temperatures increase palmitoleic acid production by engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Yamaoka, Masakazu

    2015-01-01

    Palmitoleic acid (POA) has recently gained attention for its health benefits and as a potential resource for industrial feedstock. This study focused on the use of Saccharomyces cerevisiae, which has a high POA content but low lipid content, for POA production. We created an oleaginous S. cerevisiae as a dga1 mutant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np). This was performed to further increase POA content in the oleaginous S. cerevisiae through optimization of culture conditions and genetic modifications. We found that high concentrations of methionine (2.0 g/l) increased POA production in a concentration-dependent way, while other amino acids such as cysteine, glycine, and glutamine showed no effect. It was not clear if the effect of methionine was mediated through S-adenosylmethionine, mainly because its addition did not increase POA content as did the addition of methionine. We increased POA content up to 55% by incubation of the dga1 transformant in a medium containing 2 g/l methionine at lower than normal temperatures ranging from 20 to 25 °C. Cultivation at such temperatures increased dry cell weight, but did not affect the lipid content, thereby increasing total POA production. The effects of methionine and low temperatures (20-25 °C) on POA content were more apparent in the strains overexpressing Dga1∆Np than those harboring empty vectors, which was consistent with the observation that POA was enriched in triacylglycerol. Overexpression of Ole1p, the enzyme responsible for POA production, did not increase POA content of the dga1 mutant overexpressing Dga1∆Np, but increased that of the wild-type strain overexpressing Dga1∆Np. The results suggested that genomic Ole1p in the dga1 mutant was active enough to achieve the optimal POA production under these conditions. Finally, the POA production by the S. cerevisiae transformant was increased 2.5-fold, which demonstrates that oleaginous S. cerevisiae is a potential source

  3. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    International Nuclear Information System (INIS)

    Kaur, Jatinder; Sawhney, Meenakshi; DattaGupta, Siddartha; Shukla, Nootan K; Srivastava, Anurag; Ralhan, Ranju

    2010-01-01

    We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

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

    Science.gov (United States)

    Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

    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.

  5. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    Science.gov (United States)

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

  6. Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: A fluorine-19 NMR study

    International Nuclear Information System (INIS)

    Chu, Wenchy; Horowitz, J.

    1991-01-01

    Interactions of 5-fluorouracil-substituted Escherichia coli tRNA Val with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene. Apparent K M and V max values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA Val . Binding of VRS to (FUra)tRNA Val induces structural perturbations that are reflected in selective changes in the 19 F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNA Val along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNA Val , suggesting conformational changes in this part of the molecule. No 19 F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA Val that has been proposed as a common intermediate in the aminoacylation reaction

  7. ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds.

    Science.gov (United States)

    Gaufichon, Laure; Marmagne, Anne; Belcram, Katia; Yoneyama, Tadakatsu; Sakakibara, Yukiko; Hase, Toshiharu; Grandjean, Olivier; Clément, Gilles; Citerne, Sylvie; Boutet-Mercey, Stéphanie; Masclaux-Daubresse, Céline; Chardon, Fabien; Soulay, Fabienne; Xu, Xiaole; Trassaert, Marion; Shakiebaei, Maryam; Najihi, Amina; Suzuki, Akira

    2017-08-01

    Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter Ca MV 35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  8. Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatanus).

    Science.gov (United States)

    Dalessandro, G; Northcote, D H

    1981-01-01

    Particulate enzymic preparations obtained from homogenates of differentiated xylem cells isolated from sycamore trees, catalyzed the formation of a radioactive xylan in the presence of UDP-D-[U-(14)C]xylose as substrate. The synthesized xylan was not dialyzable through Visking cellophane tubing. Successive extraction with cold water, hot water and 5% NaOH dissolved respectively 15, 5 and 80% of the radioactive polymer. Complete acid hydrolysis of the water-insoluble polysaccharide synthesized from UDP-D-[U-(14)C]xylose released all the radioactivity as xylose. β-1,4-Xylodextrins, degree of polymerization 2, 3, 4, 5 and 6, were obtained by partial acid hydrolysis (fuming HCl or 0.1 M HCl) of radioactive xylan. The polymer was hydrolysed to xylose, xylobiose and xylotriose by Driselase which contains 1,4-β xylanase activities. Methylation and then hydrolysis of the xylan released two methylated sugars which were identified as di-O-methyl[(14)C]xylose and tri-O-methyl-[(14)C]xylose, suggesting a 1→4-linked polymer. The linkage was confirmed by periodate oxidation studies. The apparent Km value of the synthetase for UDP-D-xylose was 0.4 mM. Xylan synthetase activity was not potentiated in the presence of a detergent. The enzymic activity was stimulated by Mg(2+) and Mn(2+) ions, although EDTA in the range of concentrations between 0.01 and 1 mM did not affect the reaction rate. It appears that the xylan synthetase system associated with membranes obtained from differentiated xylem cells of sycamore trees may serve for catalyzing the in vivo synthesis of the xylan main chain during the biogenesis of the plant cell wall.

  9. Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater.

    Directory of Open Access Journals (Sweden)

    Caroline Hoff-Risseti

    Full Text Available The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX, while cylindrospermopsin (CYN, which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

  10. Pre-operative localisation of hyperfunctional parathyroid tissue with {sup 11}C-methionine PET

    Energy Technology Data Exchange (ETDEWEB)

    Otto, D.; Boerner, A.R.; Hofmann, M.; Brunkhorst, T.; Meyer, G.J.; Petrich, T.; Knapp, W.H. [Hannover University Medical School, Department of Nuclear Medicine, Hannover (Germany); Scheumann, G.F. [Hannover University Medical School, Department of Visceral and Transplant Surgery, Hannover (Germany)

    2004-10-01

    Previous studies have shown high sensitivity of positron emission tomography (PET) with {sup 11}C-methionine in the pre-operative localisation of parathyroid adenoma and hyperplasia. Nonetheless, in secondary and tertiary hyperparathyroidism (HPT) and in patients with recurrent disease, pre-operative localisation of adenomatous (PTA) or hyperplastic tissue is still a problem with all available methods. The aim of this study was to define the optimal imaging protocol and to compare the diagnostic value of {sup 11}C-methionine PET and {sup 99m}Tc-methoxyisobutylisonitrile (MIBI) single-photon emission computed tomography (SPECT): in particular, we wished to define the benefit of {sup 11}C-methionine in those patients with inconclusive or negative conventional imaging. Thirty highly pre-selected patients with HPT were enrolled. Sixteen patients had primary HPT, 12 patients had secondary HPT, and two patients had recurrences of parathyroid carcinomas. All patients had ultrasound of the neck, dual-phase scintigraphy with {sup 99m}Tc-MIBI and PET with {sup 11}C-methionine. SUV{sub parathyroid}/SUV{sub cervical} {sub soft} {sub tissue} (target-to-background) and SUV{sub parathyroid} {sub tissue}/SUV{sub thyroid} {sub tissue} (target-to-non-target) ratios were calculated. After surgery, histology of specimens was obtained in all patients but one. In 12 patients with secondary or tertiary HPT, 36 hyperplastic parathyroid glands were histologically verified. Twenty-five of 36 lesions (69%) were detected with {sup 11}C-methionine PET and 17 (47%) with {sup 99m}Tc-MIBI scintigraphy. PET studies were positive in 17/18 (94%) cases in which HPT was related to adenomas or carcinomas. {sup 99m}Tc-MIBI scintigraphy/SPECT yielded pathological lesions in 9/18 cases (50%). All eight atypical localisations of parathyroid glands were detected with PET but only six of the eight were detected with {sup 99m}Tc-MIBI scintigraphy/SPECT. In 10/11 patients with recurrent HPT and non

  11. Methionine-hydroxy analogue was found to be significantly less bioavailable compared to dl-methionine for protein deposition in growing pigs.

    Science.gov (United States)

    Shoveller, A K; Moehn, S; Rademacher, M; Htoo, J K; Ball, R O

    2010-01-01

    When methionine (Met) is limiting in swine diets, it is commonly supplemented by using anhydrous dl-methionine (DLM, 99% purity) or liquid dl-methionine-hydroxy analogue free acid (MHA-FA, 88% purity). The objective of this experiment was to test the null hypothesis that the bioavailability of DLM and MHA-FA were not different for growing pigs, using the indicator amino acid (AA) (phenylalanine, Phe) oxidation (IAAO) method in a slope-ratio assay. Six barrows (mean BW during study: 21.1 kg) received seven dietary treatments with all pigs receiving all diets in random order at an intake of 95 g/kg BW0.75. The basal diet (BD) contained analyzed content of 15.1% CP, 0.20% Met, 0.73% Phe and all other AA in excess of requirement. The BD was supplemented with three graded levels of DLM or MHA-FA on an equimolar basis. Dietary treatments only varied in Met content and included: (i) BD, (ii) BD + 0.034% DLM, (iii) BD + 0.054% DLM, (iv) BD + 0.086% DLM, (v) BD + 0.029% MHA-FA, (vi) BD + 0.078% MHA-FA and (vii) BD + 0.107% MHA-FA, as analyzed. Indicator AA oxidation was determined during 4 h studies, where pigs were fed half-hourly meals each equal to 1/32 of their daily feed allowance. Each meal was mixed with 258.7 kBq (s.e. 2.6) of l-[1-14C]Phe with a prime of 3.5 times the half-hourly dose added to the first meal. The slope of the decrease in IAAO calculated by linear regression analysis was greater (P = 0.012) for DLM supplementation (9.87 ± 1.450 per g, 1.488 ± 0.215% per mmol) than for MHA-FA (6.48 ± 0.89 per g, 1.107 ± 0.152% per mmol). The ratio of slopes indicated a bioavailability of MHA-FA on a product basis, relative to DLM, of 65.7%. Bioavailability on an equimolar Met basis, calculated from the ratio of the slopes was 74.4% for MHA-FA, relative to DLM. In conclusion, these results indicate that the metabolic bioavailability of MHA-FA for growing pigs is appreciably lower than that of DLM on both an equimolar and a product basis.

  12. Evolution of the 2'-5'-Oligoadenylate Synthetase family in eukaryotes and bacteria

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Poulsen, Jesper Buchhave; Reitamm, Tonu

    2009-01-01

    system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins......The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon...

  13. Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

    DEFF Research Database (Denmark)

    Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J

    1989-01-01

    Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine...... represented a primary stimulus in all subjects. First, basal 2',5'A activity increased severalfold in response to yellow fever vaccination. In IDDM subjects, this increase was significantly lower (P = .025). Second, the 2',5'A activity increased proportionately to the higher basal 2',5'A activity in IDDM...

  14. Biochemical and genetic characterization of a carbamyl phosphate synthetase mutant of Escherichia coli K12.

    Science.gov (United States)

    Bolivar, F; Galván, M; Martuscelli, J

    1976-05-01

    An unusual Escherichia coli K12 mutant for carbamyl phosphate synthetase is described. The mutation was generated by bacteriophage MUI insertion and left a 5% residual activity of the enzyme using either ammonia or glutamine as donors. The mutation is recessive to the wild-type allele and maps at or near the pyrA gene, but the mutant requires only arginine and not uracil for growth. By a second block in the pyrB gene it was possible to shift the accumulated carbamyl phosphate to arginine biosynthesis. The Km values and the levels of ornithine activation and inhibition by UMP were normal in the mutant enzyme.

  15. PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carter, Andrew T.; Beiche, Flora; Hove-Jensen, Bjarne

    1997-01-01

    In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS......) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has...

  16. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J

    2012-01-01

    -dependent methyltransferase 144, GTP cyclohydrolase 398, DNA topoisomerase VI subunit A 209, DNA topoisomerase VI subunit B 192, Type A Flavoprotein 911, NAD(P)H:rubredoxin oxidoreductase (Fatty acid metabolism) 120, NAD(P)H:rubredoxin oxidoreductase 120, cofactor-independent phosphoglycerate mutase 909, bis(5'-adenosyl......, transcriptional regulator 364, glutamine synthetase 120, N6-adenine-specific DNA methylase 194, ArsR family transcriptional regulator 113, 5'-methylthioadenosine phosphorylase II 280, DNA repair and recombination protein RadA 323, 30S ribosomal protein S6e 106, pyruvate ferredoxin oxidoreductase subunit beta 282...

  17. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

  18. A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

    Directory of Open Access Journals (Sweden)

    Paula Bustamante

    2016-06-01

    Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

  19. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-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...

  20. Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene.

    NARCIS (Netherlands)

    Nijtmans, L.G.J.; Henderson, N.S.; Attardi, G.; Holt, L.J.

    2001-01-01

    Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome.

  1. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...

  2. δ-(L-α-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    Lende, Ted R. van der; Kamp, Mart van de; Berg, Marco van den; Sjollema, Klaas; Bovenberg, Roel A.L.; Veenhuis, Marten; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-α-aminoadipate, L-cysteine, and L-valine into

  3. delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    van der Lende, T.R.; de Kamp, M.; den Berg, M.van; Sjollema, K.; Bovenberg, R.A.L.; Veenhuis, M; Konings, W.N; Driessen, A.J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteme, and

  4. Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1988-01-01

    A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40 degrees C, the strain was fully viable at this temperature as long as guanosine, uridine, histi...

  5. Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens)

    Science.gov (United States)

    Michelle J. Serapiglia; Rakesh Minocha; Subhash C. Minocha

    2008-01-01

    We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount...

  6. Evaluation of Methionine Content in a High-Fat and Choline-Deficient Diet on Body Weight Gain and the Development of Non-Alcoholic Steatohepatitis in Mice.

    Science.gov (United States)

    Chiba, Tsuyoshi; Suzuki, Sachina; Sato, Yoko; Itoh, Tatsuki; Umegaki, Keizo

    2016-01-01

    Non-alcoholic steatohepatitis (NASH) is a globally recognized liver disease. A methionine- and choline-deficient diet is used to induce NASH in mice; however, this diet also causes severe body weight loss. To resolve this issue, we examined the effects of methionine content in a high-fat and choline-deficient (HFCD) diet on body weight and the development of NASH in mice. C57BL/6J mice (male, 10 weeks of age) were fed an L-amino acid rodent (control) diet, high-fat (HF) diet, or HFCD diet containing various amounts of methionine (0.1-0.6% (w/w)) for 12 weeks. Plasma lipid levels, hepatic lipid content and inflammatory marker gene expression were measured, and a pathological analysis was conducted to evaluate NASH. The 0.1% methionine in HFCD diet suppressed body weight gain, which was lower than that with control diet. On the other hand, the 0.2% methionine in HFCD diet yielded similar body weight gains as the control diet, while more than 0.4% methionine showed the same body weight gains as the HF diet. Liver weights and hepatic lipid contents were the greatest with 0.1% methionine and decreased in a methionine dose-dependent manner. Pathological analysis, NAFLD activity scores and gene expression levels in the liver revealed that 0.1% and 0.2% methionine for 12 weeks induced NASH, whereas 0.4% and 0.6% methionine attenuated the induction of NASH by HFCD diet. However, the 0.2% methionine in HFCD diet did not induce insulin resistance, despite the body weight gain. The 0.2% methionine in HFCD diet for 12 weeks was able to induce NASH without weight loss.

  7. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    Energy Technology Data Exchange (ETDEWEB)

    Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  8. Pig performance increases with the addition of DL-methionine and L-lysine to ensiled cassava leaf protein diets.

    Science.gov (United States)

    Ly, Nguyen Thi Hoa; Ngoan, Le Duc; Verstegen, Martin Wilhelmus Antonius; Hendriks, Wouter Hendrikus

    2012-01-01

    Two studies were conducted to determine the impact of supplementation of diets containing ensiled cassava leaves as the main protein source with synthetic amino acids, DL-methionine alone or with L-lysine. In study 1, a total of 40 pigs in five units, all cross-breds between Large White and Mong Cai, with an average initial body weight of 20.5 kg were randomly assigned to four treatments consisting of a basal diet containing 45% of dry matter (DM) from ensiled cassava leaves (ECL) and ensiled cassava root supplemented with 0%, 0.05%, 0.1% and 0.15% DL-methionine (as DM). Results showed a significantly improved performance and protein gain by extra methionine. This reduced the feed cost by 2.6%, 7.2% and 7.5%, respectively. In study 2, there were three units and in each unit eight cross-bred (Large White × Mong Cai) pigs with an initial body weight of 20.1 kg were randomly assigned to the four treatments. The four diets were as follows: a basal diet containing 15% ECL (as DM) supplemented with different amounts of amino acids L-lysine and DL-methionine to the control diet. The results showed that diets with 15% of DM as ECL with supplementation of 0.2% lysine +0.1% DL-methionine and 0.1% lysine +0.05% DL-methionine at the 20-50 kg and above 50 kg, respectively, resulted in the best performance, protein gain and lowest costs for cross-bred (Large White × Mong Cai) pigs. Ensiled cassava leaves can be used as a protein supplement for feeding pigs provided the diets contain additional amounts of synthetic lysine and methionine.

  9. Metal-Catalyzed Oxidation of Protein Methionine Residues in Human Parathyroid Hormone (1-34): Formation of Homocysteine and a Novel Methionine-Dependent Hydrolysis Reaction

    Science.gov (United States)

    Mozziconacci, Olivier; Ji, Junyan A.; Wang, Y. John; Schöneich, Christian

    2013-01-01

    The oxidation of PTH(1-34) catalyzed by ferrous ethylenediaminetetraacetic acid (EDTA) is site-specific. The oxidation of PTH(1-34) is localized primarily to the residues Met[8] and His[9]. Beyond the transformation of Met[8] and His[9] into methionine sulfoxide and 2-oxo-histidine, respectively, we observed a hydrolytic cleavage between Met[8] and His[9]. This hydrolysis requires the presence of FeII and oxygen and can be prevented by diethylenetriaminepentaacetic acid (DTPA) and phosphate buffer. Conditions leading to this site-specific hydrolysis also promote the transformation of Met[8] into homocysteine, indicating that the hydrolysis and transformation of homocysteine may proceed through a common intermediate. PMID:23289936

  10. Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase.

    Directory of Open Access Journals (Sweden)

    Ljubica Caldovic

    Full Text Available The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS, carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1 catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C. Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app for acetyl coenzyme A increased while the Km(app for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under

  11. Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum

    DEFF Research Database (Denmark)

    Theilgaard, Hanne Birgitte; Kristiansen, K.N.; Henriksen, Claus Maxel

    1997-01-01

    delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)(2)SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The mole......delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)(2)SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography...

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

    DEFF Research Database (Denmark)

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman

    2004-01-01

    Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology......-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine....

  13. β-Keto and β-hydroxyphosphonate analogs of biotin-5’-AMP are inhibitors of holocarboxylase synthetase

    OpenAIRE

    Sittiwong, Wantanee; Cordonier, Elizabeth L.; Zempleni, Janos; Dussault, Patrick H.

    2014-01-01

    Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (β-ketoP) and hydroxyphosphonate (β-hydroxyP) analogs of biotin-5′-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7 μM and 203.7 μM. By comparison, an IC50 value of 7 μM was observed with the previously reported biotinol-5'-AMP. The Ki values, 3.4 μM and 17.3 μM, r...

  14. Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

    Science.gov (United States)

    Burke, Charles; Croteau, Rodney

    2002-02-01

    Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.

  15. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-03-01

    The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  17. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    Science.gov (United States)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  18. tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

    Science.gov (United States)

    Myers, Christopher A; Kuhla, Birte; Cusack, Stephen; Lambowitz, Alan M

    2002-03-05

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

  19. An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

    Science.gov (United States)

    Khan, Sameena; Garg, Ankur; Sharma, Arvind; Camacho, Noelia; Picchioni, Daria; Saint-Léger, Adélaïde; de Pouplana, Lluís Ribas; Yogavel, Manickam; Sharma, Amit

    2013-01-01

    Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations. PMID:23776638

  20. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme

    Directory of Open Access Journals (Sweden)

    Ana Rita Seabra

    2015-07-01

    Full Text Available Glutamine Synthetase (GS catalyses the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of glutamine synthetase isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  1. Mammalian folylpoly-γ-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

    International Nuclear Information System (INIS)

    Cichowicz, D.J.; Shane, B.

    1987-01-01

    Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of M/sub r/ 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K + was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the K/sub m/ value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied

  2. Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene.

    Science.gov (United States)

    Meussen, Bas J; Weusthuis, Ruud A; Sanders, Johan P M; Graaff, Leo H de

    2012-02-01

    Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid.

  3. Effectiveness and mode of action of phosphonate inhibitors of plant glutamine synthetase.

    Science.gov (United States)

    Occhipinti, Andrea; Berlicki, Łukasz; Giberti, Samuele; Dziedzioła, Gabriela; Kafarski, Paweł; Forlani, Giuseppe

    2010-01-01

    Aiming at the rational design of new herbicides, the availability of the three-dimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry. Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterisation showed a non-competitive mechanism against glutamate and an uncompetitive mechanism against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site. Results made it possible to define the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity.

  4. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    Science.gov (United States)

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Marta Spodenkiewicz

    2016-10-01

    Full Text Available Glutamine synthetase (GS is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  6. Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.

    Science.gov (United States)

    Ben-Salem, Salma; Gleeson, Joseph G; Al-Shamsi, Aisha M; Islam, Barira; Hertecant, Jozef; Ali, Bassam R; Al-Gazali, Lihadh

    2015-06-01

    Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 6.3.5.4), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.

  7. Mutations in the glutaminyl-tRNA synthetase gene cause early-onset epileptic encephalopathy.

    Science.gov (United States)

    Kodera, Hirofumi; Osaka, Hitoshi; Iai, Mizue; Aida, Noriko; Yamashita, Akio; Tsurusaki, Yoshinori; Nakashima, Mitsuko; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

    2015-02-01

    Aminoacylation is the process of attaching amino acids to their cognate tRNA, and thus is essential for the translation of mRNA into protein. This direct interaction of tRNA with amino acids is catalyzed by aminoacyl-tRNA synthetases. Using whole-exome sequencing, we identified compound heterozygous mutations [c.169T>C (p.Tyr57His) and c.1485dup (p.Lys496*)] in QARS, which encodes glutaminyl-tRNA synthetase, in two siblings with early-onset epileptic encephalopathy (EOEE). Recessive mutations in QARS, including the loss-of-function missense mutation p.Tyr57His, have been reported to cause intractable seizures with progressive microcephaly. The p.Lys496* mutation is novel and causes truncation of the QARS protein, leading to a deletion of part of the catalytic domain and the entire anticodon-binding domain. Transient expression of the p.Lys496* mutant in neuroblastoma 2A cells revealed diminished and aberrantly aggregated expression, indicating the loss-of-function nature of this mutant. Together with the previous report, our data suggest that abnormal aminoacylation is one of the underlying pathologies of EOEE.

  8. Effect of methionine supplementation in chicken feed on the quality and shelf life of fresh poultry meat.

    Science.gov (United States)

    Albrecht, Antonia; Herbert, Ulrike; Miskel, Dennis; Heinemann, Celine; Braun, Carina; Dohlen, Sophia; Zeitz, Johanna O; Eder, Klaus; Saremi, Behnam; Kreyenschmidt, Judith

    2017-08-01

    The aim of this study was to investigate the influence of different methionine sources and concentrations on the quality and spoilage process of broiler meat. The trial was comprised of 7 treatment groups: one basal group (suboptimal in Methionine+Cysteine; i.e., 0.89, 0.74, 0.69% in DM SID Met+Cys in starter, grower, and finisher diets, respectively) and 3 doses (0.10, 0.25, and 0.40%) of either DL-Methionine (DLM) or DL-2-hydroxy-4-methylthio butanoic acid (DL-HMTBA) on an equimolar basis of the DLM-supplemented groups. The broilers were fed the diets for 35 d, then slaughtered and processed. The filets were aerobically packed and stored under temperature controlled conditions at 4°C. Meat quality investigations were comprised of microbial investigations (total viable count and Pseudomonas spp.), pH and drip loss measurements of the filets. The shelf life of the meat samples was determined based on sensory parameters. After slaughtering, all supplemented meat samples showed a high quality, whereby no differences between the 2 methionine sources could be detected for the microbial load, pH, and drip loss. In comparison to the control group, the supplemented samples showed a higher sensory quality, characterized by a fresh smell and fresh red color. Methionine supplementation had a significant influence on meat quality parameters during storage. The microbial load, pH and drip loss of the chicken filets were positively correlated to the methionine concentration. Additionally, the microbial load at the end of storage was positively correlated to pH and drip loss values. Nevertheless, the microbial parameters were in a normal range and the positive correlation to methionine concentration did not affect the sensory shelf life. The mean sensory shelf life of the broiler filets varied between 7 to 9 d. During storage, no difference in the development of sensory parameters was observed between the supplemented groups, while the spoilage process of the basal group

  9. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, K

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...

  10. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed ...

  11. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... sequencing of sucrose synthase gene fragment from sor- ghum using primers designed at their conserved exons. MATERIALS AND METHODS. Multiple sequence alignment. Sucrose synthase gene sequences of various cereals like rice, maize, and barley were accessed from NCBI Genbank database.

  12. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

  13. Immune function and hematology of male cotton rats (Sigmodon hispidus) in response to food supplementation and methionine

    Science.gov (United States)

    Webb, R.E.; Leslie, David M.; Lochmiller, R.L.; Masters, R.E.

    2003-01-01

    We examined effects of supplementation of food quantity and quality (=enhanced methionine) on hematologic and immunologic parameters of wild, but enclosed, adult male cotton rats (Sigmodon hispidus) in north-central Oklahoma. Sheet metal enclosures were stocked with a high density of wild-caught cotton rats (160 animals/ha) and randomly assigned a treatment of no supplementation, mixed-ration supplementation or methionine-enhanced supplementation. Aside from small increases in counts of red blood cells and hematocrit levels, most indices of erythrocytic characteristics were not affected by supplementation with the mixed-ration or enhanced methionine. In contrast, platelet counts were highest in mixed-ration and methionine treatments and counts of total white blood cells were highest with methionine supplementation, albeit relative proportions of different leukocytes did not differ among treatments. Immunologically, neither delayed-type hypersensitivity response nor hemolytic-complement activity differed among treatments. Supplementation of food quantity and quality did not broadly affect hematologic parameters and immune function of male cotton rats, but enhanced platelet and leukocyte counts may confer advantages to overall health. Clarification of the role of such effects on population limitation or regulation requires additional research.

  14. Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

    Science.gov (United States)

    Clevenger, Kenneth D; Ye, Rosa; Bok, Jin Woo; Thomas, Paul M; Islam, Md Nurul; Miley, Galen P; Robey, Matthew T; Chen, Cynthia; Yang, KaHoua; Swyers, Michael; Wu, Edward; Gao, Peng; Wu, Chengcang C; Keller, Nancy P; Kelleher, Neil L

    2018-03-20

    The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-C T and the internal C-domains as BenZ-C 1 and BenZ-C 2 . Unexpectedly, we also uncovered evidence suggesting BenY-C T or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal C T domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

  15. Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide.

    Science.gov (United States)

    Liu, Yuchen; Dos Santos, Patricia C; Zhu, Xiang; Orlando, Ron; Dean, Dennis R; Söll, Dieter; Yuan, Jing

    2012-02-17

    Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

  16. Cisplatin interaction with cysteine and methionine in aqueous solution: computational DFT/PCM study.

    Science.gov (United States)

    Zimmermann, Tomás; Chval, Zdenĕk; Burda, Jaroslav V

    2009-03-12

    In this paper we explore cisplatin interactions with sulfur-containing amino acids in a polarizable continuum model. Two cisplatin hydrated complexes were considered as reactants (chloro complex, cis-[Pt(NH3)2Cl(H2O)]+; hydroxo complex, cis-[Pt(NH3)2(OH)(H2O)]+). We considered the following reaction mechanism: first step, substitution of the aqua ligand by amino acid; second step, dissociative chelate formation. For the optimized complex (at the B3LYP/6-31+G(d)/COSMO level), the energy profile was determined using the B3LYP/6-311++G(2df,2pd) level and two different PCM models-COSMO and UAKS/DPCM methods which were adapted for use on transition metal complexes. The results show thermodynamic preference for bonding by cysteine sulfur followed by the amino group nitrogen, methionine thioether sulfur, and carboxyl-group oxygen. Methionine slightly prefers the Pt-N(Met) coordination in the chloro complex, but in the hydroxo complex it prefers the Pt-S(Met) coordination. A similar trend follows from the bonding energies: BE(Pt-S(Cys)) = 80.8 kcal/mol and BE(Pt-N(Met)) = 76 kcal/mol. According to the experimental observations, the most stable structures found are kappa2(S,N) chelates. In the case of methionine, the same thermodynamic stability is predicted also for the kappa2(N,O) chelate. This differs from the gas-phase results, where kappa2(S,N) and even kappa2(S,O) were found to be more stable than kappa2(N,O) complex.

  17. Regulation of longevity and oxidative stress by nutritional interventions: role of methionine restriction.

    Science.gov (United States)

    Sanchez-Roman, Ines; Barja, Gustavo

    2013-10-01

    Comparative studies indicate that long-lived mammals have low rates of mitochondrial reactive oxygen species production (mtROSp) and oxidative damage in their mitochondrial DNA (mtDNA). Dietary restriction (DR), around 40%, extends the mean and maximum life span of a wide range of species and lowers mtROSp and oxidative damage to mtDNA, which supports the mitochondrial free radical theory of aging (MFRTA). Regarding the dietary factor responsible for the life extension effect of DR, neither carbohydrate nor lipid restriction seems to modify maximum longevity. However protein restriction (PR) and methionine restriction (at least 80% MetR) increase maximum lifespan in rats and mice. Interestingly, only 7weeks of 40% PR (at least in liver) or 40% MetR (in all the studied organs, heart, brain, liver or kidney) is enough to decrease mtROSp and oxidative damage to mtDNA in rats, whereas neither carbohydrate nor lipid restriction changes these parameters. In addition, old rats also conserve the capacity to respond to 7weeks of 40% MetR with these beneficial changes. Most importantly, 40% MetR, differing from what happens during both 40% DR and 80% MetR, does not decrease growth rate and body size of rats. All the available studies suggest that the decrease in methionine ingestion that occurs during DR is responsible for part of the aging-delaying effect of this intervention likely through the decrease of mtROSp and ensuing DNA damage that it exerts. We conclude that lowering mtROS generation is a conserved mechanism, shared by long-lived species and dietary, protein, and methionine restricted animals, that decreases damage to macromolecules situated near the complex I mtROS generator, especially mtDNA. This would decrease the accumulation rate of somatic mutations in mtDNA and maybe finally also in nuclear DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Key enzymes involved in methionine catabolism by cheese lactic acid bacteria.

    Science.gov (United States)

    Hanniffy, S B; Peláez, C; Martínez-Bartolomé, M A; Requena, T; Martínez-Cuesta, M C

    2009-11-15

    Cheese microbiota and their enzymatic conversion of l-methionine to volatile sulphur compounds (VSCs) play an important role in aroma formation during cheese ripening. Here, lactic acid bacteria (LAB) strains isolated from raw goats' milk cheeses were screened for the major enzymes critical to the formation of VSCs from l-methionine. A large natural biodiversity in enzyme capabilities and high inter- and intra-species variability was found among the LAB isolates investigated. From those isolates tested, lactococci displayed higher C-S lyase specificities towards the sulphur-containing compounds examined than did Lactobacillus and Leuconostoc, in some cases generating higher levels of VSCs than B. linens, known to be an efficient producer of methanethiol (MTL) and related VSCs. Moreover, these differences in C-S lyase activities (determined spectrophotometrically by measuring the formation of free thiol groups) were shown to correspond with the enzymatic potential of the isolates as determined by visualization of enzymatic activities. This technique could therefore prove valuable for the detection and preliminary characterization of C-S lyase activities among LAB isolates. Lactococci were also found to possess higher aminotransferase activities than lactobacilli and leuconostocs, while glutamate dehydrogenase activities were observed to be highest among Leuconostoc and Lactobacillus spp. Meanwhile, alpha-keto acid decarboxylase activities were highly variable and were measurable in only a limited number of isolates, mainly lactobacilli. From these data, combining indigenous isolates showing high VSCs-producing capabilities with those that facilitate the completion of the metabolic pathway responsible for degrading l-methionine into volatile compounds may provide an efficient approach to enhance cheese aroma development.

  19. Methionine sulfoxides on PrPSc: a prion-specific covalent signature.

    Science.gov (United States)

    Canello, Tamar; Engelstein, Roni; Moshel, Ofra; Xanthopoulos, Konstantinos; Juanes, María E; Langeveld, Jan; Sklaviadis, Theodoros; Gasset, Maria; Gabizon, Ruth

    2008-08-26

    Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrP (Sc), an aberrant form of the membrane protein PrP (C). In the absence of an established form-specific covalent difference, the infectious properties of PrP (Sc) were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27-30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of alphaPrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrP (Sc) are present as methionine sulfoxides this modification could not be found on brain PrP (C) as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrP (Sc) differs both within and between species, adding another diversity factor to the structure of PrP (Sc) molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrP (Sc) and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.

  20. On the Correlation between EPR and Positron Annihilation Measurements on gamma-Irradiated Acetyl Methionine

    DEFF Research Database (Denmark)

    Eldrup, Morten Mostgaard; Lund-Thomsen, E.; Mogensen, O. E.

    1972-01-01

    The dose dependence of the relative EPR signal intensity and positron lifetime spectrum was measured for γ‐irradiated acetyl methionine in the dose range from 0 to 30 Mrad. Angular correlation measurements were performed for the doses 0 and 30 Mrad. The result of the irradiation was the creation...... of EPR centers and inhibition of positronium formation. For one sample, irradiated with a dose of 30 Mrad, EPR and positron lifetime spectra were followed over a period of 50 days after the irradiation. The inhibiting effect and the EPR signal intensity decreased with time. No simple correlation could...

  1. Effects of sucrose on rFVIIa aggregation and methionine oxidation

    DEFF Research Database (Denmark)

    Soenderkaer, Susanne; Carpenter, John F; van de Weert, Marco

    2004-01-01

    The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability......) spectroscopy and circular dichroism (CD) spectroscopy were used to study protein conformation. Stability studies showed that increasing sucrose concentrations reduced the loss of monomeric rFVIIa, and decreased formation of dimeric/oligomeric and polymeric rFVIIa. Preferential exclusion of the sugar from...

  2. 11C-methionine uptake in the brain of phenylketonuric children

    International Nuclear Information System (INIS)

    Comar, D.; Chopinet, A.; Maziere, M.; Berger, G.; Todd-Pokropek, A.

    The investigation covered 9 children aged between 7 and 77 months. The brain uptake for each examination before and after correction (for each child correction due to pericerebral activity, calculated by a method described) and the ratio between the corrected uptake rates of two examinations are reported. The results show clearly the strong resistance of the blood brain barrier to the passage of methionine in non-dieting phenylketonuric children. Moreover analysis of the brain radioactivity variation with time during the two examinations suggests a partial inhibition of brain protein synthesis, especially when the blood phenylalanine content is high [fr

  3. Catalytic Promiscuity of the Radical S-adenosyl-L-methionine Enzyme NosL.

    Science.gov (United States)

    Ding, Wei; Ji, Xinjian; Li, Yongzhen; Zhang, Qi

    2016-01-01

    Catalytic promiscuity plays a key role in enzyme evolution and the acquisition of novel biological functions. Because of the high reactivity of radical species, in our view enzymes involving radical-mediated mechanisms could intrinsically be more prone to catalytic promiscuity. This mini-review summarizes the recent advances in the study of NosL, a radical S-adenosyl-L-methionine (SAM)-dependent L-tryptophan (L-Trp) lyase. We demonstrate here the interesting chemistry and remarkable catalytic promiscuity of NosL, and attempt to highlight the high evolvability of radical SAM enzymes and the potential to engineer these enzymes for novel and improved activities.

  4. Identification of methionine-processed HPr in the equine pathogen Streptococcus equi.

    Science.gov (United States)

    Sutcliffe, I C; Trigg, J; Harrington, D

    2000-10-01

    Using preparative electrophoresis, a low molecular weight protein has been partially purified from a cell extract of the equine pathogen Streptococcus equi susp. equi. N-terminal sequence analysis and Western blotting revealed the protein to be HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Interestingly, the only form of the HPr protein detected in S. equi was one with the amino-terminal methionine removed, a modification that has previously been associated with surface localization of streptococcal HPr proteins.

  5. Synthesis of cysteine from methionine in normal adult subjects: effect of route of alimentation.

    Science.gov (United States)

    Stegink, L D; Den Besten, L

    1972-11-03

    Parenteral alimentation solutions free of cysteine, probably an essential amino acid for premature infants, were administered continuously to eight healthy men through catheters in the superior vena cava and through nasogastric tubes. When the preparation was administered parenterally, the plasma cystine concentration dropped markedly. When feeding was switched to the oral route, the concentration rose immediately, but returned to baseline only when a cystine-containing diet was fed. These studies indicate that the synthesis of cysteine from methionine is limited, even in the adult subject, when cystine-free diets are administered parenterally.

  6. Kinetics of reversible reductive carbonylation of heme in human cystathionine β-synthase.

    Science.gov (United States)

    Carballal, Sebastián; Cuevasanta, Ernesto; Marmisolle, Inés; Kabil, Omer; Gherasim, Carmen; Ballou, David P; Banerjee, Ruma; Alvarez, Beatriz

    2013-07-02

    Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2(•-)) that exists in rapid equilibrium with S2O4(2-). Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10(-3) s(-1) (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 10(2) M(-1) s(-1). Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed a hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H2S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H2S, and superoxide signaling pathways.

  7. The Methionine Transamination Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5 Acetyltransferase and the PGC-1α Transcriptional Coactivator.

    Science.gov (United States)

    Tavares, Clint D J; Sharabi, Kfir; Dominy, John E; Lee, Yoonjin; Isasa, Marta; Orozco, Jose M; Jedrychowski, Mark P; Kamenecka, Theodore M; Griffin, Patrick R; Gygi, Steven P; Puigserver, Pere

    2016-05-13

    Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Insight into the reaction mechanism of lipoyl synthase: a QM/MM study.

    Science.gov (United States)

    Dong, Geng; Cao, Lili; Ryde, Ulf

    2018-03-01

    Lipoyl synthase (LipA) catalyses the final step of the biosynthesis of the lipoyl cofactor by insertion of two sulfur atoms at the C6 and C8 atoms of the protein-bound octanoyl substrate. In this reaction, two [4Fe4S] clusters and two molecules of S-adenosyl-L-methionine are used. One of the two FeS clusters is responsible for the generation of a powerful oxidant, the 5'-deoxyadenosyl radical (5'-dA • ). The other (the auxiliary cluster) is the source of both sulfur atoms that are inserted into the substrate. In this paper, the spin state of the FeS clusters and the reaction mechanism is investigated by the combined quantum mechanical and molecular mechanics approach. The calculations show that the ground state of the two FeS clusters, both in the [4Fe4S] 2+ oxidation state, is a singlet state with antiferromagnetically coupled high-spin Fe ions and that there is quite a large variation of the energies of the various broken-symmetry states, up to 40 kJ/mol. For the two S-insertion reactions, the highest energy barrier is found for the hydrogen-atom abstraction from the octanoyl substrate by 5'-dA • . The formation of 5'-dA • is very facile for LipA, with an energy barrier of 6 kJ/mol for the first S-insertion reaction and without any barrier for the second S-insertion reaction. In addition, the first S ion attack on the C6 radical of octanoyl was found to take place directly by the transfer of the H6 from the substrate to 5'-dA • , whereas for the second S-insertion reaction, a C8 radical intermediate was formed with a rate-limiting barrier of 71 kJ/mol.

  9. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  10. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

    Science.gov (United States)

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. PMID:10611289

  11. Hepatic metabolism of 11C-methionine and secretion of 11C-protein measured by PET in pigs

    DEFF Research Database (Denmark)

    Horsager, Jacob; Lausten, Susanne Bach; Bender, Dirk

    2017-01-01

    -proteins in arterial plasma was measured during the experiment. There were no statistically significant differences between the laparotomy group and the pneumoperitoneum group in any of the calculated parameters. Average mean hepatic systemic metabolic clearance Kmet was 0.212 mL plasma/mL liver tissue/min, secretion...... allocated to either laparotomy or pneumoperitoneum. 24 hours after surgery a 70-min dynamic PET scanning of the liver with arterial blood sampling was performed immediately after intravenous injection of 11C-methionine. Time course of arterial plasma 11C-methionine concentration was used as input function...... and that of liver tissue 11C-concentration as output function in an extended Patlak analysis that accounted for irreversible metabolism of 11C-methionine (hepatic systemic metabolic clearance Kmet) and secretion of 11C-protein + 11C-metabolites into blood (rate constant kloss). Appearance of 11C...

  12. Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

    Science.gov (United States)

    Parker, J; Fishman, S E

    1979-01-01

    The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA. PMID:374370

  13. Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Bernard, Stéphanie M.; Møller, Anders Laurell Blom; Dionisio, Giuseppe

    2008-01-01

    We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2...

  14. Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

    Science.gov (United States)

    Parker, J; Fishman, S E

    1979-04-01

    The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA.

  15. Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical, biochemical, and molecular genetic aspects

    NARCIS (Netherlands)

    Santer, R.; Muhle, H.; Suormala, T.; Baumgartner, E. R.; Duran, M.; Yang, X.; Aoki, Y.; Suzuki, Y.; Stephani, U.

    2003-01-01

    We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the

  16. Mitochondrial aminoacyl-tRNA synthetase single-nucleotide polymorphisms that lead to defects in refolding but not aminoacylation

    DEFF Research Database (Denmark)

    Banerjee, Rajat; Reynolds, Noah M; Yadavalli, Srujana S

    2011-01-01

    that mutations in nuclear-encoded components of the mitochondrial translation machinery, such as aminoacyl-tRNA synthetases (aaRSs), can also lead to disease. In some cases, mutations can be directly linked to losses in enzymatic activity; however, for many, their effect is unknown. To investigate how aa...

  17. Novel applications of plant polyketide synthases.

    Science.gov (United States)

    Abe, Ikuro

    2012-04-01

    The structurally and mechanistically simple type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkably diverse structures and biological activities. By exploiting the enzymes, we combined precursor-directed biosynthesis with nitrogen-containing substrates and structure-based enzyme engineering and generated unnatural, novel polyketide-alkaloid scaffolds with promising biological activities. The nucleophilic nitrogen atom and the engineered enzymes thus facilitated the formation of additional CC and CN bonds during the enzymatic transformations. The methodology will contribute to the further production of chemically and structurally divergent, unnatural natural products, as well as the rational design of novel biocatalysts with unprecedented catalytic functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  19. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    Science.gov (United States)

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  20. Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases

    Directory of Open Access Journals (Sweden)

    Tukalo M. A.

    2013-07-01

    Full Text Available In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA rather than usual 4 or 5 nucleotides (type 1 tRNA. In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong