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Sample records for methionine adenosyltransferase 1a

  1. Transsulfuration in an adult with hepatic methionine adenosyltransferase deficiency.

    PubMed Central

    Gahl, W A; Bernardini, I; Finkelstein, J D; Tangerman, A; Martin, J J; Blom, H J; Mullen, K D; Mudd, S H

    1988-01-01

    We investigated sulfur and methyl group metabolism in a 31-yr-old man with partial hepatic methionine adenosyltransferase (MAT) deficiency. The patient's cultured fibroblasts and erythrocytes had normal MAT activity. Hepatic S-adenosylmethionine (SAM) was slightly decreased. This clinically normal individual lives with a 20-30-fold elevation of plasma methionine (0.72 mM). He excretes in his urine methionine and L-methionine-d-sulfoxide (2.7 mmol/d), a mixed disulfide of methanethiol and a thiol bound to an unidentified group X, which we abbreviate CH3S-SX (2.1 mmol/d), and smaller quantities of 4-methylthio-2-oxobutyrate and 3-methylthiopropionate. His breath contains 17-fold normal concentrations of dimethylsulfide. He converts only 6-7 mmol/d of methionine sulfur to inorganic sulfate. This abnormally low rate is due not to a decreased flux through the primarily defective enzyme, MAT, since SAM is produced at an essentially normal rate of 18 mmol/d, but rather to a rate of homocysteine methylation which is abnormally high in the face of the very elevated methionine concentrations demonstrated in this patient. These findings support the view that SAM (which is marginally low in this patient) is an important regulator that helps to determine the partitioning of homocysteine between degradation via cystathionine and conservation by reformation of methionine. In addition, these studies demonstrate that the methionine transamination pathway operates in the presence of an elevated body load of that amino acid in human beings, but is not sufficient to maintain methionine levels in a normal range. PMID:3339126

  2. Refolding and characterization of methionine adenosyltransferase from Euglena gracilis.

    PubMed

    Garrido, Francisco; Estrela, Sylvie; Alves, Claudia; Sánchez-Pérez, Gabino F; Sillero, Antonio; Pajares, María A

    2011-09-01

    Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 μmol/min/mg and K(m) values of approximately 85 and 260 μM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed β-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.

  3. The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

    PubMed Central

    Garrido, Francisco; Reytor, Edel; Portillo, Francisco; Pajares, María A.

    2016-01-01

    Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases. PMID:27548429

  4. Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III☆

    PubMed Central

    Schröder, Paul C.; Fernández-Irigoyen, Joaquín; Bigaud, Emilie; Serna, Antonio; Renández-Alcoceba, Rubén; Lu, Shelly C.; Mato, José M.; Prieto, Jesús; Corrales, Fernando J.

    2015-01-01

    Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Proteomics: The clinical link. PMID:22270009

  5. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes

    PubMed Central

    Murray, Ben; Antonyuk, Svetlana V.; Marina, Alberto; Lu, Shelly C.; Mato, Jose M.; Hasnain, S. Samar; Rojas, Adriana L.

    2016-01-01

    The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a “structural movie” of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE. PMID:26858410

  6. Understanding Molecular Recognition of Promiscuity of Thermophilic Methionine Adenosyltransferase, sMAT from Sulfolobus solfataricus

    PubMed Central

    Wang, Fengbin; Singh, Shanteri; Zhang, Jianjun; Huber, Tyler D.; Helmich, Kate E.; Sunkara, Manjula; Hurley, Katherine A.; Goff, Randal D.; Bingman, Craig A.; Morris, Andrew J.; Thorson, Jon S.; Phillips, George N.

    2014-01-01

    Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoMet) bound, and unbound forms. Notably, among these structures, the AdoEth-bound form offers the first MAT structure containing a non-native product and cumulatively, these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in E. coli and human. As a thermostable MAT structure from archaea, the structures herein also provide as a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed ‘alkylrandomization’ and/or the study of methylation in the context of biological processes. PMID:24649856

  7. Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer

    SciTech Connect

    Tomasi, Maria Lauda; Ryoo, Minjung; Skay, Anna; Tomasi, Ivan; Giordano, Pasquale; Mato, José M.; Lu, Shelly C.

    2013-07-15

    Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70–75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100 pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell. -- Highlights: • MAT2A knockdown depletes putrescine and leads to apoptosis. • Putrescine attenuates MAT2A knockdown-induced apoptosis and growth

  8. Impact of glutathione supplementation of parenteral nutrition on hepatic methionine adenosyltransferase activity

    PubMed Central

    Elremaly, Wesam; Mohamed, Ibrahim; Rouleau, Thérèse; Lavoie, Jean-Claude

    2015-01-01

    Background The oxidation of the methionine adenosyltransferase (MAT) by the combined impact of peroxides contaminating parenteral nutrition (PN) and oxidized redox potential of glutathione is suspected to explain its inhibition observed in animals. A modification of MAT activity is suspected to be at origin of the PN-associated liver disease as observed in newborns. We hypothesized that the correction of redox potential of glutathione by adding glutathione in PN protects the MAT activity. Aim To investigate whether the addition of glutathione to PN can reverse the inhibition of MAT observed in animal on PN. Methods Three days old guinea pigs received through a jugular vein catheter 2 series of solutions. First with methionine supplement, (1) Sham (no infusion); (2) PN: amino acids, dextrose, lipids and vitamins; (3) PN-GSSG: PN+10 μM GSSG. Second without methionine, (4) D: dextrose; (5) D+180 μM ascorbylperoxide; (6) D+350 μM H2O2. Four days later, liver was sampled for determination of redox potential of glutathione and MAT activity in the presence or absence of 1 mM DTT. Data were compared by ANOVA, p<0.05. Results MAT activity was 45±4% lower in animal infused with PN and 23±7% with peroxides generated in PN. The inhibition by peroxides was associated with oxidized redox potential and was reversible by DTT. Correction of redox potential (PN+GSSG) or DTT was without effect on the inhibition of MAT by PN. The slope of the linear relation between MAT activity and redox potential was two fold lower in animal infused with PN than in others groups. Conclusion The present study suggests that prevention of peroxide generation in PN and/or correction of the redox potential by adding glutathione in PN are not sufficient, at least in newborn guinea pigs, to restore normal MAT activity. PMID:26722840

  9. Methionine adenosyltransferase:adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression.

    PubMed

    Kim, Jong-So; Coon, Steven L; Blackshaw, Seth; Cepko, Constance L; Møller, Morten; Mukda, Sujira; Zhao, Wan-Qian; Charlton, Clivel G; Klein, David C

    2005-01-07

    (S)-adenosylmethionine (SAM) is a critical element of melatonin synthesis as the methyl donor in the last step of the pathway, the O-methylation of N-acetyl 5-hydroxytryptamine by hydroxyindole-O-methyltransferase. The activity of the enzyme that synthesizes SAM, methionine adenosyltransferase (MAT), increases 2.5-fold at night in the pineal gland. In this study, we found that pineal MAT2A mRNA and the protein it encodes, MAT II, also increase at night, suggesting that the increase in MAT activity is caused by an increase in MAT II gene products. The night levels of MAT2A mRNA in the pineal gland were severalfold higher than in other neural and non-neural tissues examined, consistent with the requirement for SAM in melatonin synthesis. Related studies indicate that the nocturnal increase in MAT2A mRNA is caused by activation of a well described neural pathway that mediates photoneural-circadian regulation of the pineal gland. MAT2A mRNA and MAT II protein were increased in organ culture by treatment with norepinephrine (NE), the sympathetic neurotransmitter that stimulates the pineal gland at night. NE is known to markedly elevate pineal cAMP, and here it was found that cAMP agonists elevate MAT2A mRNA levels by increasing MAT2A mRNA synthesis and that drugs that block cAMP activation of cAMP dependent protein kinase block effects of NE. Therefore, the NE-cAMP dependent increase in pineal MAT activity seems to reflect an increase in MAT II protein, which occurs in response to cAMP-->protein kinase-dependent increased MAT2A expression. The existence of this MAT regulatory system underscores the importance that MAT plays in melatonin biogenesis. These studies also point to the possibility that SAM production in other tissues might be regulated through cAMP.

  10. Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites.

    PubMed Central

    Gasset, María; Alfonso, Carlos; Neira, José L; Rivas, Germán; Pajares, María A

    2002-01-01

    The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established. PMID:11772402

  11. NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

    PubMed

    González, Beatriz; Garrido, Francisco; Ortega, Rebeca; Martínez-Júlvez, Marta; Revilla-Guarinos, Ainhoa; Pérez-Pertejo, Yolanda; Velázquez-Campoy, Adrián; Sanz-Aparicio, Julia; Pajares, María A

    2012-01-01

    Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

  12. NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

    PubMed Central

    Ortega, Rebeca; Martínez-Júlvez, Marta; Revilla-Guarinos, Ainhoa; Pérez-Pertejo, Yolanda; Velázquez-Campoy, Adrián; Sanz-Aparicio, Julia; Pajares, María A.

    2012-01-01

    Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells. PMID:23189196

  13. ROLE OF METHIONINE ADENOSYLTRANSFERASE α2 AND β PHOSPHORYLATION AND STABILIZATION IN HUMAN HEPATIC STELLATE CELL TRANS-DIFFERENTIATION

    PubMed Central

    Ramani, Komal; Donoyan, Shant; Tomasi, Maria Lauda; Park, Sunhee

    2016-01-01

    Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members [ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (B-Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation. PMID:25294683

  14. Inhibition of hepatic methionine adenosyltransferase by peroxides contaminating parenteral nutrition leads to a lower level of glutathione in newborn Guinea pigs.

    PubMed

    Elremaly, Wesam; Rouleau, Thérèse; Lavoie, Jean-Claude

    2012-12-15

    Premature newborn infants on total parenteral nutrition (TPN) are at risk of oxidative stress because of peroxides contaminating TPN and low glutathione level. Low cysteine availability limits glutathione synthesis. In this population, the main source of cysteine derives from the hepatic conversion of methionine. The first enzyme of this conversion, methionine adenosyltransferase (MAT), contains redox-sensitive cysteinyl residues. We hypothesize that inhibition of MAT by peroxides contaminating TPN leads to a lower availability of cysteine for glutathione synthesis. At 3 days of life, animals were fitted with a jugular catheter for intravenous infusion. Four groups were compared by ANOVA (P<0.05): (1) Control, without surgery, fed regular chow; (2) Sham, fitted with an obstructed catheter, fed orally regular chow; (3) TPN, fed exclusively TPN (dextrose, amino acids, fat, vitamins) containing 350 μM peroxides; (4) H2O2, fed regular chow orally and infused with 350 μM H2O2. Four days later, MAT activity and glutathione in liver and blood were lower in TPN and H2O2 groups. The redox potential was more oxidized in blood and liver of the TPN group. In conclusion, peroxides generated in TPN inhibit methionine adenosyltransferase activity with, among consequences, a low level of glutathione and a more oxidized redox potential.

  15. MAT1A variants modulate the effect of dietary fatty acids on plasma homocysteine concentrations and DNA damage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary n-3 polyunsaturated fatty acids (PUFA) are associated with decreased plasma homocysteine (Hcy), an important biomarker for cardiovascular disease. Methionine adenosyltransferase (MAT1A) is an enzyme involved in formation of form S-adenosylmethionine during methionine metabolism. The objectiv...

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

    PubMed

    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%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA

  17. Methionine and methionine sulfoxide treatment induces M1/classical macrophage polarization and modulates oxidative stress and purinergic signaling parameters.

    PubMed

    Dos Santos, Lien M; da Silva, Tatiane M; Azambuja, Juliana H; Ramos, Priscila T; Oliveira, Pathise S; da Silveira, Elita F; Pedra, Nathalia S; Galdino, Kennia; do Couto, Carlus A T; Soares, Mayara S P; Tavares, Rejane G; Spanevello, Roselia M; Stefanello, Francieli M; Braganhol, Elizandra

    2017-01-01

    Methionine is an essential amino acid involved in critical metabolic process, and regulation of methionine flux through metabolism is important to supply this amino acid for cell needs. Elevation in plasma methionine commonly occurs due to mutations in methionine-metabolizing enzymes, such as methionine adenosyltransferase. Hypermethioninemic patients exhibit clinical manifestations, including neuronal and liver disorders involving inflammation and tissue injury, which pathophysiology is not completely established. Here, we hypothesize that alterations in macrophage inflammatory response may contribute to deleterious effects of hypermethioninemia. To this end, macrophage primary cultures were exposed to methionine (1 mM) and/or its metabolite methionine sulfoxide (0.5 mM), and M1/proinflammatory or M2/anti-inflammatory macrophage polarization was evaluated. In addition, inflammation-related pathways including oxidative stress parameters, as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities; reactive oxygen species (ROS) production, and purinergic signaling, as ATP/ADP/AMPase activities, were investigated. Methionine and/or methionine sulfoxide induced M1/classical macrophage activation, which is related to proinflammatory responses characterized by increased iNOS activity and TNF-α release. Further experiments showed that treatments promoted alterations on redox state of macrophages by differentially modulated SOD and CAT activities and ROS levels. Finally, methionine and/or methionine sulfoxide treatment also altered the extracellular nucleotide metabolism, promoting an increase of ATPase/ADPase activities in macrophages. In conclusion, these findings contribute to better understand the participation of proinflammatory responses in cell injury observed in hypermethioninemic patients.

  18. Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress

    PubMed Central

    Allu, Praveen Kumar; Marada, Adinarayana; Boggula, Yerranna; Karri, Srinivasu; Krishnamoorthy, Thanuja; Sepuri, Naresh Babu V.

    2015-01-01

    Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway. PMID:25428986

  19. Choline and methionine differentially alter methyl carbon metabolism in bovine neonatal hepatocytes

    PubMed Central

    Chandler, Tawny L.

    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

  20. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  1. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild. PMID:22521563

  2. L-Methionine Production.

    PubMed

    Shim, Jihyun; Shin, Yonguk; Lee, Imsang; Kim, So Young

    2016-11-22

    L-Methionine has been used in various industrial applications such as the production of feed and food additives and has been used as a raw material for medical supplies and drugs. It functions not only as an essential amino acid but also as a physiological effector, for example, by inhibiting fat accumulation and enhancing immune response. Producing methionine from fermentation is beneficial in that microorganisms can produce L-methionine selectively using eco-sustainable processes. Nevertheless, the fermentative method has not been used on an industrial scale because it is not competitive economically compared with chemical synthesis methods. Presented are efforts to develop suitable strains, engineered enzymes, and alternative process of producing L-methionine that overcomes problems of conventional fermentation methods. One of the alternative processes is a two-step process in which the L-methionine precursor is produced by fermentation and then converted to L-methionine by enzymes. Directed efforts toward strain development and enhanced enzyme engineering will advance industrial production of L-methionine based on fermentation.

  3. Photooxidation of Methionine

    ERIC Educational Resources Information Center

    Lewis, Catherine; Scouten, William H.

    1976-01-01

    Describes an experiment in which the photooxidation of methionine using free methylene blue as the sensitizer is applied to the isolated amino acid or to the methionyl residues of a complex polypeptide. (MLH)

  4. Methionine Biosynthesis in Lemna

    PubMed Central

    Thompson, Gregory A.; Datko, Anne H.; Mudd, S. Harvey; Giovanelli, John

    1982-01-01

    Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine γ-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of

  5. PqqE from Methylobacterium extorquens AM1: a radical S-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen

    PubMed Central

    Saichana, Natsaran; Tanizawa, Katsuyuki; Pechoušek, Jiří; Novák, Petr; Yakushi, Toshiharu; Toyama, Hirohide; Frébortová, Jitka

    2016-01-01

    Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S]2+ cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S]2+ and [2Fe-2S]2+ clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions. PMID:26188050

  6. N-Terminal Methionine Processing.

    PubMed

    Wingfield, Paul T

    2017-04-03

    Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-methionine. In general, if the side chains of the penultimate residues have a radius of gyration of 1.29 Å or less, methionine is cleaved. © 2017 by John Wiley & Sons, Inc.

  7. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants.

    PubMed

    Sánchez-Aguayo, Inmaculada; Rodríguez-Galán, José Manuel; García, Remedios; Torreblanca, José; Pardo, José Manuel

    2004-12-01

    S-Adenosyl-L-methionine synthase (SAM; ATP: L-methionine adenosyltransferase, EC 2.5.1.6) catalyzes the biosynthesis of S-adenosyl-L-methionine (AdoMet), a universal methyl-group donor. This enzyme is induced by salinity stress in tomato (Lycopersicon esculentum Mill.). To elucidate the role of SAM and AdoMet in the adaptation of plants to a saline environment, the expression pattern and histological distribution of SAM was investigated in control and salt-stressed tomato plants. Immunohistochemical analysis showed that SAM proteins were expressed in all cell types and plant organs, albeit with preferential accumulation in lignified tissues. Lignin deposition was estimated by histochemical tests and the extent of tissue lignification in response to salinity was quantified by image analysis. The average number of lignified cells in vascular bundles was significantly greater in plants under salt stress, with a maximal expansion of the lignified area found in the root vasculature. Accordingly, the greatest abundance of SAM gene transcripts and proteins occurred in roots. These results indicate that increased SAM activity correlated with a greater deposition of lignin in the vascular tissues of plants under salinity stress. A model is proposed in which an increased number of lignified tracheary elements in tomato roots under salt stress may enhance the cell-to-cell pathway for water transport, which would impart greater selectivity and reduced ion uptake, and compensate for diminished bulk flow of water and solutes along the apoplastic pathway.

  8. Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon, methionine, and transsulfuration pathways in neonatal Holstein calves.

    PubMed

    Jacometo, C B; Zhou, Z; Luchini, D; Corrêa, M N; Loor, J J

    2017-02-01

    An important mechanism of nutritional "programming" induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from -21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 μM) at -10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-β-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ∼50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless

  9. Effects of chronic dietary selenomethionine exposure on repeat swimming performance, aerobic metabolism and methionine catabolism in adult zebrafish (Danio rerio).

    PubMed

    Thomas, Jith K; Wiseman, Steve; Giesy, John P; Janz, David M

    2013-04-15

    swimming resulted in lesser concentrations of glycogen in the body, exposure to SeMet in the diet had no significant effect on glycogen content. Exposure to SeMet significantly down-regulated mRNA abundance of protein tyrosine phosphatase 1B (PTP 1B) in muscle, and β-hydroxyacyl coenzyme A dehydrogenase (HOAD), sterol regulatory element binding protein 1 (SREBP 1) and methionine adenosyltransferase 1 alpha (MAT 1A) in liver of adult zebrafish. Overall the results of this study suggest chronic exposure of adult zebrafish to SeMet in the diet can cause both cellular and organismal effects that could affect fitness and survivability of fish.

  10. The eutT gene of Salmonella enterica Encodes an oxygen-labile, metal-containing ATP:corrinoid adenosyltransferase enzyme.

    PubMed

    Buan, Nicole R; Suh, Sang-Jin; Escalante-Semerena, Jorge C

    2004-09-01

    The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP:co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT+ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe2+ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe2+. We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.

  11. Methionine requirements in healthy adolescents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate methionine requirements in healthy adolescents, we conducted an experiment in 28 children age 15.6+2.1 years, wt. 57.8+9.7 kg, using the intravenous indicator amino acid oxidation and balance (IV-IAAOB) technique. Children were randomly assigned to 7 different levels of methionine int...

  12. Acute Liver Injury Induces Nucleocytoplasmic Redistribution of Hepatic Methionine Metabolism Enzymes

    PubMed Central

    Delgado, Miguel; Garrido, Francisco; Pérez-Miguelsanz, Juliana; Pacheco, María; Partearroyo, Teresa; Pérez-Sala, Dolores

    2014-01-01

    Abstract Aims: The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results: Using the rat model of d-galactosamine intoxication, severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities, and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine treatment induced hepatic nuclear accumulation of methionine adenosyltransferase (MAT) α1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear MAT activity. Acetaminophen (APAP) intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and APAP that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased glutathione reduced form/glutathione oxidized form ratios and was prevented by N-acetylcysteine (NAC) and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by NAC in galactosamine-treated cells. Innovation: Cytoplasmic and nuclear changes in proteins that regulate the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion: Altogether these results demonstrate galactosamine- and APAP-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of

  13. Crystallization and preliminary X-ray crystallographic studies of a PduO-type ATP:cob(I)alamin adenosyltransferase from Bacillus cereus

    SciTech Connect

    Park, Ae Kyung; Moon, Jin Ho; Lee, Sung Haeng; Chi, Young Min

    2008-07-01

    Orthorhombic crystals of a PduO-type ATP:cob(I)alamin adenosyltransferase from B. cereus were obtained both as an apoenzyme and in the presence of Mg{sup 2+} and ATP. Cobalamin adenosyltransferases transfer a 5′-deoxyadenosyl moiety from ATP and covalently attach it to the cobalt(I) ion of the corrin ring of cobalamin to generate adenosylcobalamin. The PduO-type adenosyltransferase from Bacillus cereus was overexpressed in Escherichia coli, purified and crystallized as the apoenzyme as well as in complex with Mg{sup 2+} and ATP (MgATP). Diffraction data were collected to 1.9 Å resolution for the native crystals and 2.0 Å resolution for the complexed crystals. Both crystals belonged to the orthorhombic space group C222{sub 1}; the native crystals have unit-cell parameters a = 64.93, b = 137.08, c = 158.55 Å. The asymmetric unit contained one trimer, with a corresponding V{sub M} of 2.69 Å{sup 3} Da{sup −1}.

  14. 63 FR 41290 - Synthetic Methionine From Japan

    Federal Register 2010, 2011, 2012, 2013, 2014

    1998-08-03

    ... COMMISSION Synthetic Methionine From Japan AGENCY: United States International Trade Commission. ACTION: Institution of a five-year review concerning the antidumping duty order on synthetic methionine from Japan... antidumping duty order on synthetic methionine from Japan would be likely to lead to continuation...

  15. Methionine requirements for the preimplantation bovine embryo.

    PubMed

    BONILLA, Luciano; LUCHINI, Daniel; DEVILLARD, Estelle; HANSEN, Peter J

    2010-10-01

    The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium - bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 µmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 µmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 µmol/l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 µmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine. In conclusion, the methionine requirement for preimplantation development is between 14 and 21 µmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of

  16. 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.

    PubMed

    Jiang, Hua; Hurt, K Joseph; Breen, Kelsey; Stabler, Sally P; Allen, Robert H; Orlicky, David J; Maclean, Kenneth N

    2015-08-14

    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.

  17. 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

    PubMed Central

    Jiang, Hua; Hurt, K. Joseph; Breen, Kelsey; Stabler, Sally P.; Allen, Robert H.; Orlicky, David J.; Maclean, Kenneth N.

    2015-01-01

    ABSTRACT 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. PMID:26276101

  18. Methionine sulfoximine intensifies cancer anorexia.

    PubMed

    Chance, W T; Zhang, F S; Fischer, J E

    1991-05-01

    Consistent anorexia was first observed 33 days after inoculating Fischer 344 rats with methylcholanthrene-induced sarcoma. Daily treatment of a similar group of rats with the glutamine synthetase inhibitor, methionine sulfoximine, elicited significant reductions of feeding by day 29 at a dose that had no effect on nontumor-bearing rats. Blood concentrations of ammonia were elevated in both groups of tumor-bearing rats and brain ammonia level was increased in the methionine sulfoximine-treated tumor-bearing rats. Forebrain concentrations of tyrosine, tryptophan, DOPAC and 5-HIAA were elevated in both groups of tumor-bearing rats. Since ammonia is detoxified through the glutamine synthetase reaction, these results suggest that blood and brain ammonia concentrations are more important than the neurochemical consequences of ammonia detoxification for the etiology of cancer anorexia.

  19. Methionine restriction and lifespan control

    PubMed Central

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N.

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending lifespan. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with/without adequate nutrition (e.g. particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend lifespan in various model organisms. We discuss beneficial effects of methionine-restricted (MR) diet, the molecular pathways involved, and the use of this regimen in longevity interventions. PMID:26663138

  20. The physiological role of reversible methionine oxidation.

    PubMed

    Drazic, Adrian; Winter, Jeannette

    2014-08-01

    Sulfur-containing amino acids such as cysteine and methionine are particularly vulnerable to oxidation. Oxidation of cysteine and methionine in their free amino acid form renders them unavailable for metabolic processes while their oxidation in the protein-bound state is a common post-translational modification in all organisms and usually alters the function of the protein. In the majority of cases, oxidation causes inactivation of proteins. Yet, an increasing number of examples have been described where reversible cysteine oxidation is part of a sophisticated mechanism to control protein function based on the redox state of the protein. While for methionine the dogma is still that its oxidation inhibits protein function, reversible methionine oxidation is now being recognized as a powerful means of triggering protein activity. This mode of regulation involves oxidation of methionine to methionine sulfoxide leading to activated protein function, and inactivation is accomplished by reduction of methionine sulfoxide back to methionine catalyzed by methionine sulfoxide reductases. Given the similarity to thiol-based redox-regulation of protein function, methionine oxidation is now established as a novel mode of redox-regulation of protein function. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

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

    PubMed

    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.

  2. Influence of copper supplementation on the relationship between dietary methionine and free plasma methionine.

    PubMed

    Ekperigin, H E; Vohra, P

    1981-09-01

    One-day-old broiler chicks were adapted to a basal, isolated soyprotein-cornstarch diet containing 20% protein, 0.59% methionine and 3,300 kcal metabolizable energy (ME)/kg. They were then fed experimental diets consisting of three levels (0, 500, 1,000 ppm) of copper added to each of the basal diet plus four levels (0, 0.4, 0.8, or 1.5%) of L-methionine for 1 or 4 weeks. Growth was retarded after 1 week by all levels of supplementary methionine, and by 500 or 1,000 ppm excess copper. The plasma concentrations of free methionine, serine, alpha-aminobutyric acid and cystathionine were increased by excess dietary methionine. Excess copper prevented the increase in plasma methionine. After 4 weeks, the plasma methionine concentration and rate of growth of chicks fed 0.4% excess L-methionine did not differ significantly from basal values. The growth retardation caused by 500 ppm excess copper was alleviated by 0.4% supplemental methionine, and the elevations in plasma methionine and liver and spleen iron concentrations observed in chicks fed 1.5% excess methionine were reduced by 1,000 ppm excess copper. The patterns of the relationship between dietary methionine and liver or spleen iron, in the presence or absence of supplementary copper, were similar to those between dietary and free plasma methionine.

  3. Efficiency of methionine retention in ducks.

    PubMed

    Adeola, Olayiwola

    2007-03-01

    The accretion of methionine and protein as a function of methionine intake was assessed in growing ducks between 22 and 42 d post-hatching. Four graded doses of dl-methionine at 0, 0 x 5, 1 x 0 or 1 x 5 g/kg diet were added to a methionine-limiting basal diet and fed to four replicate groups of four ducks each. The growth and efficiency of food use for growth increased linearly (P<0 x 05) as a function of methionine intake. The accretion of body protein increased (P<0 x 001) from 87 x 5 to 182 x 2 g, and that of methionine from 1616 to 3125 mg, over the 21 d period as dietary methionine increased. The accretion rate of methionine in the body (y, mg/d) as a function of methionine intake (x, mg/d) of ducks fed diets containing supplemental methionine at 0, 0 x 5, 1 x 0 or 1 x 5 g/kg diet from day 22 to day 42 post-hatching gave the regression equation: y=-148 x 86 (se 32 x 558)+0 x 312 (se 0 x 0384)X, r2=0 x 8253. For protein accretion rate in the body (y, mg/d) as a function of methionine intake (x, mg/d), the regression equation was: y= -9782 (se 2204)+19 x 505 (se 2 x 5994)x, r2=0 x 8009. There was a linear relationship between methionine (y, mg/d) and protein (x, mg/d) accretion in ducks that was described by the equation y=12 x 757 (se 7 x 4019)+0 x 01 525 (se 0 x 00 107)x, r2=0 x 9355. The results of these studies suggest a constant utilisation of methionine over the range 2 x 4-3 x 9 g digestible methionine/kg diet, with an efficiency of 31 %. Furthermore, the results suggest a quantitative relationship of 15 mg methionine for every gram of protein accretion.

  4. 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).

    PubMed

    Rolland, Marine; Skov, Peter V; Larsen, Bodil K; Holm, Jørgen; Gómez-Requeni, Pedro; Dalsgaard, Johanne

    2016-08-01

    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 as a signalling factor in different metabolic pathways. The study investigated the effect of increasing dietary methionine intake on the intermediary metabolism in the liver of juvenile rainbow trout. For this purpose, five diets were formulated with increasing methionine levels from 0.60 to 1.29% dry matter. 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 significantly affected by the increase in dietary methionine. Changes in gene expression reflected to some extent the decrease in ammonia excretion (P=0.022) and in the hepatosomatic index (HSI; P<0.001) when dietary methionine increased. Postprandial plasma methionine concentrations correlated positively with the dietary level (P<0.001) at the different sampling points. The study shows that the expression of several genes related to the hepatic intermediary metabolism in rainbow trout responded in a dose-dependent manner to increasing levels of dietary methionine.

  5. Methionine metabolism in Yucatan miniature swine.

    PubMed

    McBreairty, Laura E

    2016-06-01

    Methionine is an essential amino acid which when not incorporated into protein, can be converted to S-adenosylmethionine, the universal methyl donor in over 200 transmethylation reactions, which include creatine and phosphatidylcholine (PC) synthesis, as well as deoxyribonucleic acid (DNA) methylation. Following transmethylation, homocysteine is formed, which can be converted to cysteine via transsulfuration or remethylated to methionine by receiving a methyl group from folate or betaine. Changes to methyl group availability in utero can lead to permanent changes in epigenetic patterns of DNA methylation, which has been implicated in "fetal programming", a phenomenon associated with poor nutrition during fetal development that results in low birth weight and disease in later life. It has been shown that programming can also occur in the neonate. Our global objective was to understand how the variability of nutrients involved in methionine metabolism can affect methionine and methyl group availability. We hypothesize that nutrients that converge on methionine metabolism can affect methionine availability for its various functions. In this thesis, we used intrauterine growth restricted (IUGR) piglets to investigate whether a global nutritional insult in utero can lead to a perturbed methionine metabolism. Our results demonstrate that IUGR piglets have a lower capacity to dispose of homocysteine via both transsulfuration and remethylation pathways, as well as a lower incorporation of methyl groups into PC. The second objective of this thesis was to determine whether variation in methionine supply and demand can affect methionine availability. We demonstrated that stimulating either acute or chronic creatine synthesis leads to lower methyl incorporation into protein and PC in pigs. Furthermore, when methionine is limiting, supplementation with either folate or betaine leads to higher methionine availability for protein synthesis. Finally, because creatine is

  6. Metabolism of 5-methylthioribose to methionine

    SciTech Connect

    Miyazaki, J.H.; Yang, S.F.

    1987-06-01

    During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine is released as 5'-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, (/sup 14/C)MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, L-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O/sub 2/ in the conversion of MTR to methionine is discussed.

  7. Modulation of Potassium Channel Function by Methionine Oxidation and Reduction

    NASA Astrophysics Data System (ADS)

    Ciorba, Matthew A.; Heinemann, Stefan H.; Weissbach, Herbert; Brot, Nathan; Hoshi, Toshinori

    1997-09-01

    Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.

  8. Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins

    PubMed Central

    2012-01-01

    Background Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative. Results We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity. Conclusion Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation. PMID:23088625

  9. Dissimilation of Methionine by Achromobacter starkeyi1

    PubMed Central

    Ruiz-Herrera, José; Starkey, Robert L.

    1970-01-01

    Methionine was decomposed by some bacteria which were isolated from soil. The sulfur of the methionine was liberated as methanethiol, and part of this became oxidized to dimethyl disulfide. Detailed studies with one of these cultures, Achromobacter starkeyi, indicated that the first step in methionine decomposition was its oxidadative deamination to α-keto-γ-methyl mercaptobutyrate by a constitutive amino acid oxidase. The following steps were carried out by inducible enzymes, the synthesis of which was inhibited by chloramphenicol. α-Keto-γ-methyl mercaptobutyrate was split producing methanethiol and α-keto butyrate which was oxidized to propionate. The metabolism of propionate was similar to that described for animal tissues; the propionate was carboxylated to succinate via methyl malonyl coenzyme A, and the succinate was metabolized through the Krebs cycle. PMID:16559105

  10. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Zinc methionine sulfate. 172.399 Section 172.399... CONSUMPTION Special Dietary and Nutritional Additives § 172.399 Zinc methionine sulfate. Zinc methionine... conditions: (a) The additive is the product of the reaction between equimolar amounts of zinc sulfate and...

  11. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  12. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  13. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  14. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  15. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  16. Structural Characterization of a Human-Type Corrinoid Adenosyltransferase Confirms That Coenzyme B[subscript 12] Is Synthesized through a Four-Coordinate Intermediate

    SciTech Connect

    St. Maurice, Martin; Mera, Paola; Park, Kiyoung; Brunold, Thomas C.; Escalante-Semerena, Jorge C.; Rayment, Ivan

    2008-11-18

    ATP:cob(I)alamin adenosyltransferases (ACAs) catalyze the transfer of the 5{prime}-deoxyadenosyl moiety from ATP to the upper axial ligand position of cobalamin in the synthesis of coenzyme B{sub 12}. For the ACA-catalyzed reaction to proceed, cob(II)alamin must be reduced to cob(I)alamin in the enzyme active site. This reduction is facilitated through the generation of a four-coordinate cob(II)alamin intermediate on the enzyme. We have determined the high-resolution crystal structure of a human-type ACA from Lactobacillus reuteri with a four-coordinate cob(II)alamin bound in the enzyme active site and with the product, adenosylcobalamin, partially occupied in the active site. The assembled structures represent snapshots of the steps in the ACA-catalyzed formation of the cobalt-carbon bond of coenzyme B{sub 12}. The structures define the corrinoid binding site and provide visual evidence for a base-off, four-coordinate cob(II)alamin intermediate. The complete structural description of ACA-mediated catalysis reveals the molecular features of four-coordinate cob(II)alamin stabilization and provides additional insights into the molecular basis for dysfunction in human patients suffering from methylmalonic aciduria.

  17. Local flexibility facilitates oxidization of buried methionine residues.

    PubMed

    Xu, Kuiran; Uversky, Vladimir N; Xue, Bin

    2012-06-01

    In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.

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

    PubMed

    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

  19. Factors influencing methionine toxicity in young bobwhite quail

    USGS Publications Warehouse

    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.

  20. Characterization of isolated yeast growth response to methionine analogs.

    PubMed

    Saengkerdsub, Suwat; Lingbeck, Jody M; Wilkinson, Heather H; O'Bryan, Corliss A; Crandall, Philip G; Muthaiyan, Arunachalam; Biswas, Debabrata; Ricke, Steven C

    2013-01-01

    Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g(-1) dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content.

  1. Methionine restriction beyond life-span extension.

    PubMed

    Ables, Gene P; Hens, Julie R; Nichenametla, Sailendra N

    2016-01-01

    Dietary methionine restriction (MR) extends life span across species via various intracellular regulatory mechanisms. In rodents, MR induces resistance against adiposity, improves hepatic glucose metabolism, preserves cardiac function, and reduces body size, all of which can affect the onset of age-related diseases. Recent studies have shown that MR-affected biomarkers, such as fibroblast growth factor 21, adiponectin, leptin, cystathionine β synthase, and insulin-like growth factor 1, can potentially alter physiology. The beneficial effects of MR could be explained in part by its ability to reduce mitochondrial oxidative stress. Studies have revealed that MR can reduce reactive oxygen species that damage cells and promote cancer progression. It has been demonstrated that either MR or the targeting of specific genes in the methionine cycle could induce cell apoptosis while decreasing proliferation in several cancer models. The complete mechanism underlying the actions of MR on the cell cycle during cancer has not been fully elucidated. Epigenetic mechanisms, such as methylation and noncoding RNAs, are also possible downstream effectors of MR; future studies should help to elucidate some of these mechanisms. Despite evidence that changes in dietary methionine can affect epigenetics, it remains unknown whether epigenetics is a mechanism in MR. This review summarizes research on MR and its involvement in metabolism, cancer, and epigenetics.

  2. the Eutt enzyme of Salmonella enterica is a unique ATP:Cob(I)alamin adenosyltransferase metalloprotein that requires ferrous ions for maximal activity.

    PubMed

    Moore, Theodore C; Mera, Paola E; Escalante-Semerena, Jorge C

    2014-02-01

    ATP:co(I)rrinoid adenosyltransferase (ACAT) enzymes convert vitamin B12 to coenzyme B12. EutT is the least understood ACAT. We report the purification of EutT to homogeneity and show that, in vitro, free dihydroflavins drive the adenosylation of cob(II)alamin bound to EutT. Results of chromatography analyses indicate that EutT is dimeric in solution, and unlike other ACATs, EutT catalyzes the reaction with sigmoidal kinetics indicative of positive cooperativity for cob(II)alamin. Maximal EutT activity was obtained after metalation with ferrous ions. EutT/Fe(II) protein lost all activity upon exposure to air and H2O2, consistent with previously reported results indicating that EutT was an oxygen-labile metalloprotein containing a redox-active metal. Results of in vivo and in vitro analyses of single-amino-acid variants affecting a HX11CCXXC(83) motif conserved in EutT proteins showed that residues His67, Cys80, and Cys83 were required for EutT function in vivo, while Cys79 was not. Unlike that of other variants, the activity of the EutT(C80A) variant was undetectable in vitro, suggesting that Cys80 was critical to EutT function. Results of circular dichroism studies indicate that the presence or absence of a metal ion does not affect protein folding. EutT can now be purified in the presence of oxygen and reactivated with ferrous ions for maximal activity.

  3. The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

    PubMed Central

    Moore, Theodore C.; Mera, Paola E.

    2014-01-01

    ATP:co(I)rrinoid adenosyltransferase (ACAT) enzymes convert vitamin B12 to coenzyme B12. EutT is the least understood ACAT. We report the purification of EutT to homogeneity and show that, in vitro, free dihydroflavins drive the adenosylation of cob(II)alamin bound to EutT. Results of chromatography analyses indicate that EutT is dimeric in solution, and unlike other ACATs, EutT catalyzes the reaction with sigmoidal kinetics indicative of positive cooperativity for cob(II)alamin. Maximal EutT activity was obtained after metalation with ferrous ions. EutT/Fe(II) protein lost all activity upon exposure to air and H2O2, consistent with previously reported results indicating that EutT was an oxygen-labile metalloprotein containing a redox-active metal. Results of in vivo and in vitro analyses of single-amino-acid variants affecting a HX11CCXXC83 motif conserved in EutT proteins showed that residues His67, Cys80, and Cys83 were required for EutT function in vivo, while Cys79 was not. Unlike that of other variants, the activity of the EutTC80A variant was undetectable in vitro, suggesting that Cys80 was critical to EutT function. Results of circular dichroism studies indicate that the presence or absence of a metal ion does not affect protein folding. EutT can now be purified in the presence of oxygen and reactivated with ferrous ions for maximal activity. PMID:24336938

  4. Comparison of the effects of seleno-l-methionine, seleno-dl-methionine, and selenized yeast on reproduction of mallards

    USGS Publications Warehouse

    Heinz, G.H.; Hoffman, D.J.

    1996-01-01

    The toxicities of seleno-L-methionine, seleno-DL-methionine, and selenized yeast were compared. Ten pairs of mallards were fed a control diet and 15 pairs were fed diets containing 10 ppm selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. Hatching of fertile eggs was significantly lower for females fed 10 ppm selenium as seleno-DL-methionine (7.6%) and seleno-L-methionine (6.4%) than for controls (41.3%). Survival of ducklings was lower when their parents had been fed 10 ppm selenium as seleno-L-methionine (20.0%) than for controls (98.4%). The number of 6-day-old ducklings produced per female was significantly lower for mallards fed 10 ppm selenium as seleno-DL-methionine (0.47) or selenized yeast (2.67) than for controls (6.10), and was significantly lower for mallards fed seleno-L-methionine (0.13) than for mallards fed selenized yeast. The eighth eggs of females fed the DL or L forms of selenomethionine contained means of 9.2 and 8.9 ppm selenium, wet weight; these means were higher than the mean (6.6 ppm) for females fed selenized yeast. Among embryos that died at 7 days of age or older, the percentage of embryos that were deformed was 1.3% for controls, 24.6% for seleno-DL-methionine, 28.2% for seleno-L-methionine, and 11.0% for selenized yeast. The results suggested that seleno-DL-methionine and seleno-L-methionine were of similar toxicity and were both more toxic than selenium from selenized yeast.

  5. Stimulation of Ethylene Production in Apple Tissue Slices by Methionine

    PubMed Central

    Lieberman, Morris; Kunishi, Alice; Mapson, L. W.; Wardale, D. A.

    1966-01-01

    Methionine can induce more than a 100% increase in ethylene production by apple tissue slices. The increased amount of ethylene derives from carbons 3 and 4 of methionine. Only post-climacteric fruit tissues are stimulated by methionine, and stimulation is optimum after 8 months' storage. Copper chelators such as sodium diethyl dithiocarbamate and cuprizone very markedly inhibit ethylene production by tissue slices. Carbon monoxide does not effect ethylene production by the slices. These data suggest that the mechanism for the conversion of methionine to ethylene, in apple tissues, is similar to the previously described model system for producing ethylene from methionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systems in tissues derives from methionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase. PMID:16656267

  6. Comparative dynamics of methionine side-chain in FMOC-methionine and in amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Vugmeyster, Liliya; Ostrovsky, Dmitry

    2017-04-01

    We compared the dynamics of key methionine methyl groups in the water-accessible hydrophobic cavity of amyloid fibrils and Fluorenylmethyloxycarbonyl-Methionine (FMOC-Met), which renders general hydrophobicity to the environment without the complexity of the protein. Met35 in the hydrated cavity was recently found to undergo a dynamical cross-over from the dominance of methyl rotations at low temperatures to the dominance of diffusive motion of methyl axis at high temperatures. Current results indicate that in FMOC-Met this cross-over is suppressed, similar to what was observed for the dry fibrils, indicating that hydration of the cavity is driving the onset of the dynamical transition.

  7. Methionine-to-Cysteine Recycling in Klebsiella aerogenes

    PubMed Central

    Seiflein, Thomas A.; Lawrence, Jeffrey G.

    2001-01-01

    In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5′-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella. PMID:11114934

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

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    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.

  9. L-methionine degradation potentialities of cheese-ripening microorganisms.

    PubMed

    Bonnarme, P; Lapadatescu, C; Yvon, M; Spinnler, H E

    2001-11-01

    Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase

  10. Methionine as a Precursor of Ethylene—Commentary

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  12. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Abstracts Service Registry No. 65-82-7) is the derivative of the amino acid methionine formed by addition of... percent L- and DL-methionine (expressed as the free amino acid) by weight of the total protein of the...) The amounts of additive and each amino acid contained in any mixture. (3) Adequate directions for...

  13. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  14. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...-Acetyl-L-methionine (Chemical Abstracts Service Registry No. 65-82-7) is the derivative of the amino acid... provide a total of 3.1 percent L- and DL-methionine (expressed as the free amino acid) by weight of the... contained therein. (2) The amounts of additive and each amino acid contained in any mixture. (3)...

  15. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  16. High Methionine Diet Poses Cardiac Threat: A Molecular Insight.

    PubMed

    Chaturvedi, Pankaj; Kamat, Pradip K; Kalani, Anuradha; Familtseva, Anastasia; Tyagi, Suresh C

    2016-07-01

    High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: (1) by augmenting oxidative stress; (2) by inflammatory manifestations; and (3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice: (1) WT; (2) WT + methionine; (3) CBS(+/-) ; (4) CBS(+/-) +methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS(+/-) and CBS(+/-) +methionine. Higher oxidative stress was indicated by high levels of SOD-1 in methionine fed mouse hearts whereas IL-1β, IL-6, TNFα, and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion, our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling, and decreased cardiac function.

  17. The distribution of methionine-enkephalin and leucine-enkephalin in the brain and peripheral tissues

    PubMed Central

    Hughes, J.; Kosterlitz, H.W.; Smith, T.W.

    1977-01-01

    1 A method is described for the rapid extraction of opioid peptides from the brain and other tissues. The method is based on acid extraction of tissues followed by adsorption of the extract onto Amberlite XAD-2 resin. Elution with methanol separates the enkephalins and α-endorphin from β-endorphin. 2 Over 90% of the opioid peptide activity isolated from brain and gut of several species by our method was due to methionine- and leucine-enkephalin. In contrast, the major opioid peptide activity recovered from the pituitary was due to peptides of much greater mol. wt. than the enkephalins. 3 An opioid peptide with properties unlike those of the known endorphins or enkephalins was present in brain extracts. This peptide, termed ε-endorphin, has an apparent mol. wt. of 700 to 1200; it constituted between 5 to 10% of the total opioid activity in our extracts. 4 A differential assay of methionine- and leucine-enkephalin was made either by destroying methionine-enkephalin activity with cyanogen bromide or by separating the peptides by thin layer chromatography. 5 The ratio of methionine-enkephalin to leucine-enkephalin varied greatly in different brain regions. The highest proportions of leucine-enkephalin were found in the cerebral cortex and hippocampus. 6 Formaldehyde perfusion and fixation of the brain in vivo had no significant effect on the brain content of enkephalin, indicating that proteolytic breakdown is not a major problem in the extraction of these peptides. 7 It is suggested that the enkephalins may have a neurotransmitter role in both brain and peripheral tissues and that methionine- and leucine-enkephalin may subserve separate neuronal functions. PMID:597668

  18. Detection and Measurement of Methionine Oxidation in Proteins.

    PubMed

    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.

  19. Removal of N-terminal methionine from haemoglobin nascent peptides by a membrane-bound rat liver methionine aminopeptidase.

    PubMed Central

    Termignoni, C; Freitas, J O; Guimarães, J A

    1986-01-01

    A membrane-bound aminopeptidase able to remove methionine from haemoglobin nascent peptides is described. The enzyme also hydrolyses methionine from methionyl-lysyl-bradykinin but not lysine from lysyl-bradykinin. The tripeptide Met-Ala-Ser is poorly hydrolysed. This aminopeptidase also splits amino acid 2-naphthylamides, being, however, less specific with respect to these synthetic substrates. PMID:3087345

  20. Bioavailability of different dietary supplemental methionine sources in animals.

    PubMed

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

    2015-06-01

    Dietary methionine is indispensable for animal maintenance, growth and development. L-methionine (L-Met), and its synthetic forms DL-methionine (DL-Met) and 2-hydroxy-4 (methylthio) butanoic acid (HMTBA) are common supplemental methionine sources in animal diets. There are different characteristics for cellular absorption, transport, metabolism and bio-efficiency between these three dietary methionine sources. Moreover, there are differences in their utilization among various species such as chickens, pigs and ruminants. As a methionine precursor, HMTBA is efficacious in the promotion of growth in animals. It is absorbed mainly by monocarboxylate transporter 1 (MCT1), coupled with the activity of the Na(+)/H(+) exchanger (NHE3), while DL-Met uptake occurs via multiple carrier-mediated systems. Liver, kidney and small intestine can metabolize D-Met and HMTBA to L-Met through oxidation and transamination. In ruminants, the non-hepatic tissues act as major sites of HMTBA conversion, which are different from that in chickens and pigs. HMTBA also has additional benefits in anti-oxidation. Understanding the characteristics of uptake and metabolism of different methionine sources will greatly benefit the industry and bioscience research.

  1. Protein-borne methionine residues as structural antioxidants in mitochondria.

    PubMed

    Schindeldecker, Mario; Moosmann, Bernd

    2015-07-01

    Methionine is an oxidant-labile amino acid whose major oxidation products, methionine sulfoxides, can be readily repaired by various NADPH-dependent methionine sulfoxide reductases. Formally, the methionine oxidation-reduction circuit could act as a cellular antioxidant system, by providing a safe sink for oxidants that might cause much more damage if reacting otherwise. This concept is supported by focal experimental evidence; however, the global importance, scope and biochemical role of protein-borne methionine as an inbuilt macromolecular antioxidant have remained incompletely defined. In analyzing proteomic methionine usage on different levels of comparison, we find that protein methionine (i) is primarily an antioxidant of mitochondria, especially of the inner mitochondrial membrane, (ii) responds strongly to respiratory demands on an evolutionary timescale, (iii) acts locally, by selectively protecting its carrier protein, and (iv) might be utilized as a molecular predictor of aerobic metabolic rate in animals, to complement traditional markers like the presence of a respiratory pigment. Our data support the idea that proteins in need of a long lifespan or acting in dangerous environments may acquire massive structural alterations aimed at increasing their resistance to oxidation. Counterintuitively though, they sometimes do so by accumulating particularly labile rather than particularly stable building blocks, illustrating that the technical concept of cathodic protection is also employed by the animate nature.

  2. Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidases by Bengamide Derivatives

    SciTech Connect

    Lu, Jing-Ping; Yuan, Xiu-Hua; Yuan, Hai; Wang, Wen-Long; Wan, Baojie; Franzblau, Scott G.; Ye, Qi-Zhuang

    2012-05-29

    Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.

  3. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional... reaction between equimolar amounts of zinc sulfate and DL-methionine in purified water. (b) The...

  4. In vitro Characterization of Uptake Mechanism of L-[methyl-3H]-methionine in Hepatocellular Carcinoma

    PubMed Central

    Kuang, Yu; Wang, Fangjing; Corn, David J.; Tian, Haibin; Lee, Zhenghong

    2015-01-01

    Purpose Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-11C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-11C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-11C]-Met in HCC cells. Procedures A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-3H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively. Results Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na+ dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-3H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of

  5. Growth rate of Enterobacteriaceae at elevated temperatures: limitation by methionine.

    PubMed

    Ron, E Z

    1975-10-01

    The effect of elevated temperatures on growth rate was studied in five strains of Enterobacteriaceae. In all the strains tested a shift to the elevated temperature resulted in an immediate decrease in growth rate which was due to limitation in the availability of endogenous methionine. The first biosynthetic enzyme of the methionine pathway-homoserine transsuccinylase-was studied in extracts of Aerobacter aerogenes, Salmonella typhimurium, and Escherichia coli and was shown to be temperature sensitive in all of them.

  6. Loss of conformational stability in calmodulin upon methionine oxidation.

    PubMed Central

    Gao, J; Yin, D H; Yao, Y; Sun, H; Qin, Z; Schöneich, C; Williams, T D; Squier, T C

    1998-01-01

    We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in

  7. [NMR screening of potential inhibitors of Citrobacter freundii methionine].

    PubMed

    Batuev, E A; Lizunov, A Iu; Morozova, E A; Klochkov, V V; Anufrieva, N V; Demidkina, T V; Pol'shakov, V I

    2014-01-01

    Methionine γ-lyase [EC 4.4.1.11] participates in a methionine catabolism at a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. Lack of this enzyme at mammals allows consider it as a perspective target for rational antibacterial drug design. Currently in medical practice there are no the preparations based on an inhibition of methionine γ-lyase activity. We present results of the search of potential inhibitors of the enzyme using the NMR screening techniques based on identification of compounds, which able to bind specifically to their biological target. Study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds, capable to interact with enzyme. Identification of binding was carried out by means of saturation transfer difference (STD) spectroscopy and WaterLOGSY technique. At the final stage the experimental assessment of inhibiting ability of the selected compounds in the reaction of γ-elimination of L-methionine catalyzed by methionine γ-lyase was carried out. Binding constants of two leading compounds were determined using the WaterLOGSY method. The research expands structural group of potential inhibitors of methionine γ-lyase and allows approach to the design of the inhibitors with higher efficacy.

  8. Methionine salvage pathway in relation to ethylene biosynthesis

    SciTech Connect

    Miyazaki, J.H.

    1987-01-01

    The recycling of methionine during ethylene biosynthesis (the methionine cycle) was studied. During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine (SAM) is released at 5'-methylthioadenosine (MTA), which is recycled to methionine via 5'-methylthioribose (MTS). In mungbean hypocotyls and cell-free extracts of avocado fruit, (/sup 14/C)MTR was converted to labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB) as intermediates. Radioactive tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract required an amino group donor. Among several potential donors tested, L-glutamine was the most efficient. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, with little evolution of other /sup 14/C-labeled one-carbon compounds, indicating that the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR.

  9. Modeling the oxidation of methionine residues by peroxides in proteins.

    PubMed

    Chennamsetty, Naresh; Quan, Yong; Nashine, Vishal; Sadineni, Vikram; Lyngberg, Olav; Krystek, Stanley

    2015-04-01

    We report the use of molecular modeling to predict the oxidation propensity of methionine residues in proteins. Oxidation of methionine to the sulfoxide form is one of the major degradation pathways for therapeutic proteins. Oxidation can occur during production, formulation, or storage of pharmaceuticals and it often reduces or eliminates biological activity. We use a molecular model based on atomistic simulations called 2-shell water coordination number to predict the oxidation rates for several model proteins and therapeutic candidates. In addition, we implement models that are based on static and simulation average of the solvent-accessible area (SAA) for either the side chain or the sulfur atom in the methionine residue. We then compare the results from the different models against the experimentally measured relative rates of methionine oxidation. We find that both the 2-shell model and the simulation-averaged SAA models are accurate in predicting the oxidation propensity of methionine residues for the proteins tested. We also find the appropriate parameter ranges where the models are most accurate. These models have significant predictive power and can be used to enable further protein engineering or to guide formulation approaches in stabilizing the unstable methionine residues.

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

    PubMed

    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.

  11. Toxicity of seleno-l-methionine, seleno-dl-methionine, high selenium wheat, and selenized yeast to mallard ducklings

    USGS Publications Warehouse

    Heinz, G.H.; Hoffman, D.J.; LeCaptain, L.J.

    1996-01-01

    The toxicity of four chemical forms of selenium (seleno-L-methionine, seleno-DL-methionine, selenized yeast, and high selenium wheat) was compared in day-old mallard ducklings (Anas platyrhynchos). In the first experiment, in which the basal diet was 75% wheat, survival after 2 weeks was lower for ducklings fed 30 ?g/g selenium as seleno-L-methionine (36%) than for ducklings fed 30 ?g/g selenium as seleno-DL-methionine (100%) or 30 ?g/g selenium from high selenium yeast (88%). In a second experiment, in which the basal diet was a commercial duck feed, survival after 2 weeks was 100% in ducklings fed 30 ?g/g selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. The greater toxicity of the L form of selenomethionine was probably related to the palatability or nutritional nature of the wheat-based diet used in experiment 1, but the exact reason for the difference between the DL and L forms is unknown. Biologically incorporated selenium, derived from high selenium wheat was no more toxic than selenium derived from the two purified forms of selenomethionine, and the selenium in selenized yeast was not as toxic as that in the two forms of selenomethionine.

  12. Metabolic characteristics and importance of the universal methionine salvage pathway recycling methionine from 5'-methylthioadenosine.

    PubMed

    Albers, Eva

    2009-12-01

    The methionine salvage pathway, also called the 5'-methylthioadenosine (MTA) cycle, recycles the sulfur of MTA, which is a by-product in the biosyntheses of polyamine and the plant hormone ethylene. MTA is first converted to 5'-methylthioribose-1-phosphate either by MTA phosphorylase or the combined action of MTA nucleosidase and 5'-methylthioribose kinase. Subsequently, five additional enzymatic steps, catalyzed by four or five proteins, will form 4-methylthio-2-oxobutyrate, the deaminated form of methionine. The final transamination is achieved by transaminases active in the amino acid biosynthesis. This pathway is present with some variations in all types of organisms and seems to be designed for a quick removal of MTA achieved by high affinities of the first enzymes. During evolution some enzymes have attained additional functions, like a proposed role in nuclear mRNA processing by the aci-reductone dioxygenase. For others the function seems to be lost due to conditions in specific ecological niches, such as, presence of sulfur and/or absence of oxygen resulting in that, for example, Escherichia coli is lacking a functional pathway. The pathway is regulated as response to sulfur availability and take part in the regulation of polyamine synthesis. Some of the enzymes in the pathway show separate specificities in different organisms and some others are unique for groups of bacteria and parasites. Thus, promising targets for antimicrobial agents have been identified. Other medical topics to which this pathway has connections are cancer, apoptosis, and inflammatory response.

  13. Transcriptional analysis of L-methionine catabolism in Brevibacterium linens ATCC9175.

    PubMed

    Cholet, Orianne; Hénaut, Alain; Bonnarme, Pascal

    2007-04-01

    The expression of genes possibly involved in L-methionine and lactate catabolic pathways were performed in Brevibacterium linens (ATCC9175) in the presence or absence of added L-methionine. The expression of 27 genes of 39 selected genes differed significantly in L-methionine-enriched cultures. The expression of the gene encoding L-methionine gamma-lyase (MGL) is high in L-methionine-enriched cultures and is accompanied by a dramatic increase in volatile sulfur compounds (VSC) biosynthesis. Several genes encoding alpha-ketoacid dehydrogenase and one gene encoding an acetolactate synthase were also up-regulated by L-methionine, and are probably involved in the catabolism of alpha-ketobutyrate, the primary degradation product of L-methionine to methanethiol. Gene expression profiles together with biochemical data were used to propose catabolic pathways for L-methionine in B. linens and their possible regulation by L-methionine.

  14. Dissimilation of Methionine by a Demethiolase of Aspergillus Species1

    PubMed Central

    Ruiz-Herrera, José; Starkey, Robert L.

    1969-01-01

    Enzyme preparations obtained from the mycelium of Aspergillus species broke down methionine by co-dissimilation. The deaminase and demethiolase activities of crude extracts were increased 100-fold by precipitation with (NH4)2SO4 and column chromatography on diethylaminoethyl cellulose. The enzyme acted on d-methionine but not on l-methionine. The enzyme was labile: it was inactivated by oxygen and ascorbic acid but ethylenediaminetetraacetic acid and mercaptoethanol preserved its activity. Enzyme activity decreased even at 4 and −30 C and was lost rapidly above 45 C. It was most rapid at 35 C and at pH 8.0 to 9.0. For the following reasons, it was concluded that deamination and demethiolation of methionine were effected by the same enzyme: both activities increased equally at each stage of purification; ammonia, methanethiol, and α-keto butyric acid were formed in amounts equivalent to the amount of methionine dissimilated; the Km and optimal pH for formation of both keto acid and methanethiol were the same; both activities remained in the same fractions that were separated by electrophoresis and the activities were equivalent. The purified enzyme demethiolated α-keto methionine and α-hydroxy methionine and split the sulfur linkage of ethionine but did not cleave cystathionine. Few amino acids were deaminated. The enzyme was sensitive to some carbonyl and sulfhydryl reagents and was relatively insensitive to heavy metals other than Hg++. The Km was 1.3 × 10−3 to 1.5 × 10−3m at pH 7.0. No requirement for cofactors was noted, and attempts to dissociate the enzyme, including dialysis with hydroxylamine, were unsuccessful. PMID:5370277

  15. Methionine Sulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium

    PubMed Central

    Rouf, Syed Fazle; Kitowski, Vera; Böhm, Oliver M.; Rhen, Mikael; Jäger, Timo; Bange, Franz-Christoph

    2011-01-01

    Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H2O2, and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H2O2, as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium. PMID:22073230

  16. Oxidation increases the strength of the methionine-aromatic interaction

    PubMed Central

    Lewis, Andrew K.; Dunleavy, Katie; Senkow, Tiffany L.; Her, Cheng; Horn, Benjamin T.; Jersett, Mark A.; Mahling, Ryan; McCarthy, Megan R.; Perell, Gabriella T.; Valley, Christopher C.; Karim, Christine B.; Gao, Jiali; Pomerantz, William C. K.; Thomas, David D.; Cembran, Alessandro; Hinderliter, Anne; Sachs, Jonathan N.

    2016-01-01

    Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we show that oxidation increases the strength of the methionine-aromatic interaction motif—a driving force for protein folding and protein-protein interaction—by 0.5 – 1.4 kcal/mol. We find that non-hydrogen bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups are enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR experiments on model peptides demonstrate that oxidation of methionine stabilizes the interaction by 0.5–0.6 kcal/mol. We confirm the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on 1) calmodulin structure and dynamics and 2) lymphotoxin-α/TNFR1 binding. Thus, the methionine-aromatic motif is a determinant of protein structural and functional sensitivity to oxidative stress. PMID:27547920

  17. CHARACTERIZATION OF THE METHIONINE SULFOXIDE REDUCTASES OF SCHISTOSOMA MANSONI

    PubMed Central

    Oke, Tolulope T.; Moskovitz, Jackob; Williams, David L.

    2013-01-01

    Schistosomiasis, also known as Bilharzia, is an infectious disease caused by several species of Schistosoma. Twenty million individuals suffer severe symptoms and 200,000 people die annually from the disease. The host responds to the presence of S. mansoni by producing reactive oxygen species that cause oxidative stress. We hypothesized that schistosomes produce antioxidants in response to oxidative stress. A known antioxidant protein is methionine sulfoxide reductase (Msr). Methionine residues can be oxidized to methionine sulfoxide in the presence of oxidizing agents, which is readily reversed by the action of the Msr system. Two S. mansoni MsrB genes (MsrB1 and MsrB2) were cloned and the recombinant proteins expressed in bacteria and purified. The S. mansoni MsrB proteins contained the common conserved catalytic and zinc coordinating cysteines. Analysis of the proteins showed that both proteins promote the reduction of both free methionine sulfoxide (Met[O]) and dabsyl-Met(O) to free methionine (Met) and dabsyl-Met, respectively, while exhibiting differences in their specific activities towards these substrates. Using real-time PCR, both proteins were found to be expressed in all stages of the parasite’s life cycle with the highest level of expression of both proteins in the egg stage. This is the first description of MsrB proteins from a parasite. PMID:19604033

  18. Protein methionine oxidation augments reperfusion injury in acute ischemic stroke

    PubMed Central

    Gu, Sean X.; Blokhin, Ilya O.; Wilson, Katina M.; Dhanesha, Nirav; Doddapattar, Prakash; Grumbach, Isabella M.; Chauhan, Anil K.; Lentz, Steven R.

    2016-01-01

    Reperfusion injury can exacerbate tissue damage in ischemic stroke, but little is known about the mechanisms linking ROS to stroke severity. Here, we tested the hypothesis that protein methionine oxidation potentiates NF-κB activation and contributes to cerebral ischemia/reperfusion injury. We found that overexpression of methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that reverses protein methionine oxidation, attenuated ROS-augmented NF-κB activation in endothelial cells, in part, by protecting against the oxidation of methionine residues in the regulatory domain of calcium/calmodulin-dependent protein kinase II (CaMKII). In a murine model, MsrA deficiency resulted in increased NF-κB activation and neutrophil infiltration, larger infarct volumes, and more severe neurological impairment after transient cerebral ischemia/reperfusion injury. This phenotype was prevented by inhibition of NF-κB or CaMKII. MsrA-deficient mice also exhibited enhanced leukocyte rolling and upregulation of E-selectin, an endothelial NF-κB–dependent adhesion molecule known to contribute to neurovascular inflammation in ischemic stroke. Finally, bone marrow transplantation experiments demonstrated that the neuroprotective effect was mediated by MsrA expressed in nonhematopoietic cells. These findings suggest that protein methionine oxidation in nonmyeloid cells is a key mechanism of postischemic oxidative injury mediated by NF-κB activation, leading to neutrophil recruitment and neurovascular inflammation in acute ischemic stroke. PMID:27294204

  19. Production of methanethiol from methionine by Brevibacterium linens CNRZ 918.

    PubMed

    Ferchichi, M; Hemme, D; Nardi, M; Pamboukdjian, N

    1985-04-01

    The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.

  20. Methionine, homocysteine, one carbon metabolism and fetal growth.

    PubMed

    Kalhan, Satish C; Marczewski, Susan E

    2012-06-01

    Methionine and folate are the key components of one carbon metabolism, providing the methyl groups for numerous methyl transferase reactions via the ubiquitous methyl donor, s-adenosyl methionine. Methionine metabolism is responsive to nutrient intake, is regulated by several hormones and requires a number of vitamins (B12, pyridoxine, riboflavin) as co-factors. The critical relationship between perturbations in the mother's methionine metabolism and its impact on fetal growth and development is now becoming evident. The relation of folate intake to fetal teratogenesis has been known for some time. Studies in human pregnancy show a continuous decrease in plasma homocysteine, and an increase in plasma choline concentrations with advancing gestation. A higher rate of transsulfuration of methionine in early gestation and of transmethylation in the 3rd trimester was seen in healthy pregnant women. How these processes are impacted by nutritional, hormonal and other influences in human pregnancy and their effect on fetal growth has not been examined. Isocaloric protein restriction in pregnant rats, resulted in fetal growth restriction and metabolic reprogramming. Isocaloric protein restriction in the non-pregnant rat, resulted in differential expression of a number of genes in the liver, a 50% increase in whole body serine biosynthesis and high rate of transmethylation, suggesting high methylation demands. These responses were associated with a significant decrease in intracellular taurine levels in the liver suggesting a role of cellular osmolarity in the observed metabolic responses. These unique changes in methionine and one carbon metabolism in response to physiological, nutritional and hormonal influences make these processes critical for cellular and organ function and growth.

  1. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  2. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  3. Characterisation of Potential Antimicrobial Targets in Bacillus spp. I. Aminotransferases and Methionine Regeneration in Bacillus subtilis

    DTIC Science & Technology

    2002-07-01

    targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J. Berger and Marvin H. Knodel Defence R&D...Characterisation of potential antimicrobial targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J...examined in the gram-positive bacterium Bacillus subtilis. Homogenates of this bacterium were able to convert ketomethiobutyrate to methionine, utilising

  4. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  5. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  6. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  7. (11)C-Methionine uptake in secondary brain epilepsy.

    PubMed

    Lopci, E; Bello, L; Chiti, A

    2014-01-01

    Carbon-11 methionine ((11)C-Methionine) is a radio-labeled amino acid currently utilized in Positron Emission Tomography (PET) for imaging primary and metastatic brain tumors. Its clinical use relies mostly on oncologic applications, but the tracer has the potential to investigate other non-malignant conditions. So far, very limited evidence concerns the use of (11)C-Methionine in patients suffering from seizure; however, the tracer can find a proper utilization in this setting especially as a diagnostic complement to (18)F-Fluorodeoxyglucose ((18)F-FDG). Herein we report the case of a 57-year-old patient presenting with epileptic crises secondary to a brain metastasis from bladder carcinoma, who was investigated in our institution with (11)C-Methionine PET. The scan documented the disease recurrence in the left parietal lobe associated with a diffused tracer uptake in the surrounding cerebral circumvolutions, derived from the comitial status. After surgical removal of the metastatic lesion, the patient experienced a complete recovery of symptoms and no further onset of secondary seizure.

  8. Whole body methionine kinetics, transmethylation, transulfuration and remethylation during pregnancy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is evidence from a study of pregnant American women that methionine transmethylation (TM) and remethylation (RM) rates increase and transulfuration (TS) decreases as pregnancy progresses from trimester 1 to 3. To determine whether pregnant Indian women can make this adaptation successfully, me...

  9. The role of methionine metabolism in inflammatory bowel disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methionine (Met) cycle activity is critical for normal cell functions. Met metabolites S-adenosylmethionine (SAM) and methylthioadenosine (MTA) are anti-inflammatory, yet their role in inflammatory bowel disease (IBD) is poorly understood. We hypothesize that active IBD leads to changes in Met metab...

  10. Oxidized methionine is not a prion-specific covalent modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The oxidation of methionine residues in the '-helical region of PrPC has been proposed to be important for prion formation. This proposal has been supported by structural studies, model systems and antibody-based experimental evidence. We developed a sensitive mass spectrometry-based method to stu...

  11. Methionine splanchnic uptake is increased in critically ill children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During critical illness the splanchnic area is profoundly affected. There is no information on splanchnic uptake of amino acids in vivo, in critically ill children. Methionine splanchnic uptake in critically ill children will differ from estimates in healthy adults. We studied 24 critically ill chil...

  12. Technical note: Methionine, a precursor of methane in living plants

    NASA Astrophysics Data System (ADS)

    Lenhart, K.; Althoff, F.; Greule, M.; Keppler, F.

    2014-11-01

    When terrestrial plants were identified as producers of the greenhouse gas methane, much discussion and debate ensued, not only about their contribution to the global methane budget, but also with regard to the validity of the observation itself. Although the phenomenon has now become more accepted for both living and dead plants, the mechanism of methane formation in living plants remains to be elucidated and its precursor compounds identified. We made use of stable isotope techniques to verify in vivo formation of methane and, in order to identify the carbon precursor, 13C-positionally labelled organic compounds were employed. Here we show that the amino acid L-methionine acts as a methane precursor in living plants. Employing 13C-labelled methionine clearly identified the sulphur-bound methyl group of methionine as a carbon precursor of methane released from lavender (Lavandula angustifolia). Furthermore, when lavender plants were stressed physically, methane release rates and the stable carbon isotope values of the emitted methane greatly increased. Our results provide additional support that plants possess a mechanism for methane production and suggest that methionine might play an important role in the formation of methane in living plants, particularly under stress conditions.

  13. Technical Note: Methionine, a precursor of methane in living plants

    NASA Astrophysics Data System (ADS)

    Lenhart, K.; Althoff, F.; Greule, M.; Keppler, F.

    2015-03-01

    When terrestrial plants were identified as producers of the greenhouse gas methane, much discussion and debate ensued not only about their contribution to the global methane budget but also with regard to the validity of the observation itself. Although the phenomenon has now become more accepted for both living and dead plants, the mechanism of methane formation in living plants remains to be elucidated and its precursor compounds to be identified. We made use of stable isotope techniques to verify the in vivo formation of methane, and, in order to identify the carbon precursor, 13C positionally labeled organic compounds were employed. Here we show that the amino acid L-methionine acts as a methane precursor in living plants. Employing 13C-labeled methionine clearly identified the sulfur-bound methyl group of methionine as a carbon precursor of methane released from lavender (Lavandula angustifolia). Furthermore, when lavender plants were stressed physically, methane release rates and the stable carbon isotope values of the emitted methane greatly increased. Our results provide additional support that plants possess a mechanism for methane production and suggest that methionine might play an important role in the formation of methane in living plants, particularly under stress conditions.

  14. Amino acid nutrition beyond methionine and lysine for milk protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

  15. Oxidative Metabolism of Seleno-L-Methionine to L-Methionine Selenoxide by Flavin- Containing Monooxygenases

    PubMed Central

    Krause, Renee J.; Glocke, Steven C.; Sicuri, Anna Rita; Ripp, Sharon L.; Elfarra, Adnan A

    2008-01-01

    The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-L-methionine (SeMet) to L-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. Formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean ±S.D.; n=4) of 0.91 ± 0.29 mM and a Vmax value of 44 ± 8.0 nmol MetSeO/mg protein/min. Inclusion of 1-benzylimidazole, superoxide dismutase or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions. PMID:17173378

  16. Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.

    PubMed

    Preynat, A; Lapierre, H; Thivierge, M C; Palin, M F; Matte, J J; Desrochers, A; Girard, C L

    2009-02-01

    The present experiment was undertaken to determine the effects of dietary supplements of rumen-protected methionine and intramuscular injections of folic acid and vitamin B(12), given 3 wk before to 16 wk after calving, on glucose and methionine metabolism of lactating dairy cows. Twenty-four multiparous Holstein cows were assigned to 6 blocks of 4 cows each according to their previous milk production. Within each block, 2 cows were fed a diet estimated to supply methionine as 1.83% metabolizable protein, equivalent to 76% of methionine requirement, whereas the 2 other cows were fed the same diet supplemented daily with 18 g of rumen-protected methionine. Within each diet, the cows were administrated either no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid plus 10 mg of vitamin B(12.) To investigate metabolic changes at 12 wk of lactation, glucose and methionine kinetics were measured by isotope dilution using infusions of 3[U-(13)C]glucose, [(13)C]NaHCO(3) and 3[1-(13)C,(2)H(3)] methionine. Milk and plasma concentrations of folic acid and vitamin B(12) increased with vitamin injections. Supplementary B-vitamins increased milk production from 34.7 to 38.9 +/- 1.0 kg/d and increased milk lactose, protein, and total solids yields. Whole-body glucose flux tended to increase with vitamin supplementation with a similar quantitative magnitude as the milk lactose yield increase. Vitamin supplementation increased methionine utilization for protein synthesis through increased protein turnover when methionine was deficient and through decreased methionine oxidation when rumen-protected methionine was fed. Vitamin supplementation decreased plasma concentrations of homocysteine independently of rumen-protected methionine feeding, although no effect of vitamin supplementation was measured on methionine remethylation, but this could be due to the limitation of the technique used. Therefore, the effects of these B-vitamins on lactation performance

  17. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase

    SciTech Connect

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; Carlson, Hans K.; Wetmore, Kelly M.; Price, Morgan N.; Lavarone, Anthony T.; Deutschbauer, Adam M.; Arkin, Adam P.; Coates, John D.

    2015-05-12

    Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. In addition, bacteria are often

  18. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase

    DOE PAGES

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; ...

    2015-05-12

    Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and amore » putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. In addition, bacteria are often stressed in the environment by reactive chlorine species (RCS) of

  19. Potential for Development of an Escherichia coli—Based Biosensor for Assessing Bioavailable Methionine: A Review

    PubMed Central

    Chalova, Vesela I.; Froelich, Clifford A.; Ricke, Steven C.

    2010-01-01

    Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs. PMID:22319312

  20. Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver.

    PubMed

    Cabrero, C; Puerta, J; Alemany, S

    1987-12-30

    Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-Mr S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-Mr S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-Mr S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.

  1. Identification of activators of methionine sulfoxide reductases A and B

    PubMed Central

    Cudic, Predrag; Joshi, Neelambari; Sagher, Daphna; Williams, Brandon T.; Stawikowski, Maciej J.; Weissbach, Herbert

    2016-01-01

    The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in D. melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in C. elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB. PMID:26718410

  2. Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation

    PubMed Central

    Kuhns, Lisa G.; Mahawar, Manish; Sharp, Joshua S.; Benoit, Stéphane; Maier, Robert J.

    2014-01-01

    The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions. PMID:23181726

  3. Comparative genomics of transcriptional regulation of methionine metabolism in proteobacteria

    SciTech Connect

    Leyn, Semen A.; Suvorova, Inna A.; Kholina, Tatiana D.; Sherstneva, Sofia S.; Novichkov, Pavel S.; Gelfand, Mikhail S.; Rodionov, Dmitry A.; Kuipers, Oscar P.

    2014-11-20

    Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ~200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific and genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.

  4. Comparative genomics of transcriptional regulation of methionine metabolism in proteobacteria

    DOE PAGES

    Leyn, Semen A.; Suvorova, Inna A.; Kholina, Tatiana D.; ...

    2014-11-20

    Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ~200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific andmore » genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.« less

  5. Amino acid distribution and metabolism in pituitary adenomas using positron emission tomography with D-(/sup 11/C)methionine and L-(/sup 11/C)methionine

    SciTech Connect

    Bergstroem, M.M.; Muhr, C.; Lundberg, P.O.; Bergstroem, K.L.; Lundqvist, H.; Antoni, G.; Fasth, K.J.; Langstroem B3

    1987-05-01

    Four patients with hormonally inactive pituitary adenomas were examined with positron emission tomography (PET) after injection, during different examinations, of L-(methyl-/sup 11/C)methionine and D-(methyl-/sup 11/C)methionine, respectively. After the rapid distribution phase, the enantiomer L-(/sup 11/C)methionine, which is metabolically active, showed a considerable continuous irreversible trapping attributed to amino acid metabolism. The stereoisomer D-(/sup 11/C)methionine, which does not participate in protein synthesis, showed a rapid distribution within the whole adenoma tissue, with a distribution space on the order of 100%. A minimal irreversible trapping was observed which could be explained by technical factors. It is concluded that PET using the two enantiomers allows a separation of passive distribution and metabolism, and that L-(/sup 11/C)methionine can be used for in vivo quantitative studies of amino acid metabolism of pituitary adenomas.

  6. The polymorphisms in methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and the risk of colorectal cancer.

    PubMed

    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.

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

    PubMed Central

    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

  8. Polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and thymidylate synthase (TYMS) in multiple myeloma risk.

    PubMed

    Lima, Carmen S P; Ortega, Manoela M; Ozelo, Margareth C; Araujo, Renato C; De Souza, Cármino A; Lorand-Metze, Irene; Annichino-Bizzacchi, Joyce M; Costa, Fernando F

    2008-03-01

    We tested whether the polymorphisms of the methylenetetrahydrofolate reductase gene, MTHFR C677T and A1298C, the methionine synthase gene, MTR A2756G, the methionine synthase reductase gene, MTRR A66G, and the thymidylate synthase gene, TYMS 2R-->3R, involved in folate and methionine metabolism, altered the risk for multiple myeloma (MM). Genomic DNA from 123MM patients and 188 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. The frequency of the MTR 2756 AG plus GG genotype was higher in patients than in controls (39.8% versus 23.4%, P=0.001). Individual carriers of the variant allele G had a 2.31 (95% CI: 1.38-3.87)-fold increased risk for MM compared with others. In contrast, similar frequencies of the MTHFR, the MTRR and the TYMS genotypes were seen in patients and controls. These results suggest, for the first time, a role for the MTR A2756G polymorphism in MM risk in our country, but should be confirmed by large-scale epidemiological studies with patients and controls age matched.

  9. Microbial Decomposition of Methionine and Identity of the Resulting Sulfur Products1

    PubMed Central

    Segal, William; Starkey, Robert L.

    1969-01-01

    Various bacteria, actinomycetes, and filamentous fungi decomposed methionine, but only certain aerobic bacteria isolated from soil decomposed it in the absence of other organic substrates. These bacteria could grow on methionine as the only organic substrate and source of nitrogen and sulfur. Methionine was first deaminated and then demethiolated with production of methanethiol, part of which was oxidized to dimethyl disulfide. The amount of methanethiol that was oxidized varied with different cultures. A bacterial culture initially unable to grow on methionine developed capacity to do this in a medium which contained methionine and other growth substrates. The two sulfur products, methanethiol and dimethyl disulfide, are volatile and escaped from the media, resulting in a decrease in the sulfur content proportional to the amount of methionine decomposed. PMID:5788717

  10. Effect of Methionine Restriction on Bone Density and NK Cell Activity

    PubMed Central

    Dong, Jingming

    2016-01-01

    Methionine restriction (MR) is proven to increase the lifespan; and it also affects the bone density and the innate immune system. The aim of this study is to explore the effect of methionine restriction on bone density and natural killer (NK) cells. C57BL/6J mice were subjected to either basal diet (BD, containing 0.80% methionine) or methionine-restricted diet (containing 0.14% methionine). Mice with MR diet displayed reduced bone mass and decrease in the cytotoxicity of NK from the spleen, compared to BD animals. Also, mice with MR diet had an inferior body weight (P < 0.05) and higher plasma levels of adiponectin and FGF21 (P < 0.05) but lower concentrations of leptin and IGF-1 (P < 0.05). Overall, the investigation shows that methionine affects bone density and NK cell cytotoxicity. PMID:27882323

  11. Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway.

    PubMed

    Campbell, Kate; Vowinckel, Jakob; Keller, Markus A; Ralser, Markus

    2016-04-01

    Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway.

  12. Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

    NASA Astrophysics Data System (ADS)

    Pilo, Alice L.; McLuckey, Scott A.

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M + H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to `label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  13. Uptake of carbon-11-methionine and fluorodeoxyglucose in non-Hodgkin's lymphoma: A PET study

    SciTech Connect

    Leskinen-Kallio, S.; Ruotsalainen, U.; Nagren, K.T.; Teraes, M.J.; Joensuu, H. )

    1991-06-01

    Uptake of L-(methyl-11C)methionine (11C-methionine) and (18F)-2-fluoro-2-deoxy-D-glucose (FDG) was studied with PET in 14 patients with non-Hodgkin's lymphomas. The low molecular weight fraction of venous plasma separated by fast gel filtration was used as the input function for 11C-methionine studies, and tracer accumulation was analyzed according to Patlak and Gjedde. The average uptake rate of 11C-methionine was 0.0775 {plus minus} 0.0245 min-1 (s.d.) and of FDG 0.0355 {plus minus} 0.0293 min-1, 11C-methionine uptake rate being significantly higher than that of FDG (p less than 0.01). Carbon-11-methionine accumulated strongly in all but one of the lymphomas. FDG accumulated clearly in lymphomas of high-grade malignancy, whereas two intermediate- and three low-grade malignant lymphomas had a poor uptake rate. The tumor/plasma ratio of both 11C-methionine and FDG increased faster in high and intermediate-grade lymphomas than in low-grade lymphomas, but there was considerable overlap between the histologic grades. Carbon-11-methionine seems to be preferable in detecting tumors, while FDG was superior to 11C-methionine in distinguishing the high-grade malignant lymphomas from the other grades.

  14. Oxidation of methionine residues in polypeptide ions via gas-phase ion/ion chemistry.

    PubMed

    Pilo, Alice L; McLuckey, Scott A

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O](+)), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O](+) product is observed at a much greater abundance than the proton transfer product (viz., [M + H](+)). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to 'label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  15. Diversity of L-methionine catabolism pathways in cheese-ripening bacteria.

    PubMed

    Bonnarme, P; Psoni, L; Spinnler, H E

    2000-12-01

    Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified. L-Methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested. No L-methionine deaminase activity could be detected in any of the ripening bacteria and L-methionine aminotransferase was detected in CFEs of Brevibacterium linens, Micrococcus luteus, and Corynebacterium glutamicum. The results suggest that several pathways for L-methionine catabolism probably coexist in these ripening bacteria.

  16. Response of growing goslings to dietary supplementation with methionine and betaine.

    PubMed

    Yang, Z; Wang, Z Y; Yang, H M; Zhao, F Z; Kong, L L

    2016-12-01

    An experiment with a 2 × 3 factorial design with two concentrations of dietary betaine (0 and 600 mg/kg) and three dietary concentrations of methionine (0, 600 and 1200 mg/kg) was conducted using goslings to estimate growth, nutrient utilisation and digestibility of amino acids from 21 to 70 d of age. Three hundred geese were randomised at 18 d of age into 6 groups with 5 replicates per treatment and 10 geese per replicate. Increasing dietary concentrations of methionine gave a linear increase in body weight and average daily gain. The coefficient of crude fat retention increased as dietary methionine increased and there was a significant non-linear response to increasing dietary methionine. Similarly, increasing supplemental methionine gave linear increases in the digestibility of methionine and cysteine. The results of this study indicated that optimal dietary supplementation of methionine could increase growth performance and methionine and cysteine utilisation in growing goslings. Betaine supplementation had no apparent sparing effect on methionine needs for growth performance, but did improve the apparent cysteine digestibility.

  17. Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway

    PubMed Central

    Campbell, Kate; Vowinckel, Jakob; Keller, Markus A.

    2016-01-01

    Abstract Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway. Antioxid. Redox Signal. 24, 543–547. PMID:26596469

  18. Methionine kinetics in adult men: effects of dietary betaine on L-(2H3-methyl-1-13C)methionine

    SciTech Connect

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

    1991-08-01

    The effects of a daily 3-g supplement of betaine on kinetic aspects of L-(2H3-methyl-1-13C)methionine (MET) metabolism in healthy young adult men were explored. Four groups of four subjects each were given a control diet, based on an L-amino acid mixture supplying 29.5 and 21.9 mg.kg-1.d-1 of L-methionine and L-cystine for 4 d before the tracer study, conducted on day 5 during the fed state. Two groups received the control diet and two groups received the betaine supplement. Tracer was given intravenously (iv) or orally. The transmethylation rate of MET (TM), homocysteine remethylation (RM), and oxidation of methionine were estimated from plasma methionine labeling and 13C enrichment of expired air. RM tended to increase (P = 0.14) but the TM and methionine oxidation were significantly (P less than 0.05) higher after betaine supplementation when estimated with the oral tracer. No differences were detected with the intravenous tracer. Methionine concentration in plasma obtained from blood taken from subjects in the fed state was higher (P less than 0.01) with betaine supplementation. These results suggest that excess methyl-group intake may increase the dietary requirement for methionine.

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

    SciTech Connect

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

    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.

  20. Conversion of Methionine to Thiols by Lactococci, Lactobacilli, and Brevibacteria†

    PubMed Central

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and α-ketoglutarate was used as the amino group acceptor. In Lactococcus lactis subsp. cremoris S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of l-methionine γ-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix. PMID:9726877

  1. Retinol status and expression of retinol-related proteins in methionine-choline deficient rats.

    PubMed

    Miyazaki, Hiroshi; Takitani, Kimitaka; Koh, Maki; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2014-01-01

    Retinol and its derivative, retinoic acid, have pleiotropic functions including vision, immunity, hematopoiesis, reproduction, cell differentiation/growth, and development. Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases in developed countries and encompasses a broad spectrum of forms, ranging from steatosis to steatohepatitis, which develops further to cirrhosis. Retinol status has an important role in liver homeostasis. The purpose of this study was to evaluate the retinol status and expression of retinol-related proteins, including enzymes and binding proteins, in methionine-choline deficient (MCD) rats as a model of NAFLD. We examined retinol levels in the plasma and liver and gene expression for β-carotene 15,15'-monooxygenase (BCMO), lecithIn: retinol acyltransferase (LRAT), aldehyde dehydrogenase 1A1 (ALDH1A1), ALDH1A2, and cellular retinol binding protein (CRBP)-I in MCD rats. The plasma retinol levels in MCD rats were lower than those in the controls, whereas hepatic retinol levels in MCD rats were higher. BCMO expression in the intestine and liver in MCD rats was lower, whereas that in the testes and the kidneys was higher than in control rats. Expression of LRAT, CRBP-I, ALDH1A1, and ALDH1A2 in the liver of MCD rats was also higher. Altered expression of retinol-related proteins may affect retinol status in NAFLD.

  2. Pyridinylquinazolines Selectively Inhibit Human Methionine Aminopeptidase-1 in Cells

    PubMed Central

    Zhang, Feiran; Bhat, Shridhar; Gabelli, Sandra B.; Chen, Xiaochun; Miller, Michelle S.; Nacev, Benjamin A.; Cheng, Yim Ling; Meyers, David J.; Tenney, Karen; Shim, Joong Sup; Crews, Phillip; Amzel, L. Mario; Ma, Dawei; Liu, Jun O.

    2013-01-01

    Methionine aminopeptidases (MetAPs) which remove the initiator methionine from nascent peptides are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1–4). But all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1–4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells. PMID:23634668

  3. Metabolic Fate of Cysteine and Methionine in Rumen Digesta

    PubMed Central

    Nader, C. J.; Walker, D. J.

    1970-01-01

    Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both 35S and 14C, it was observed that a large proportion of the 35S appeared in the sulfide pool and of the 14C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the 14C to 35S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1% and for methionine, at most 11% of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of 35S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources. PMID:5485079

  4. Pyridinylquinazolines selectively inhibit human methionine aminopeptidase-1 in cells.

    PubMed

    Zhang, Feiran; Bhat, Shridhar; Gabelli, Sandra B; Chen, Xiaochun; Miller, Michelle S; Nacev, Benjamin A; Cheng, Yim Ling; Meyers, David J; Tenney, Karen; Shim, Joong Sup; Crews, Phillip; Amzel, L Mario; Ma, Dawei; Liu, Jun O

    2013-05-23

    Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.

  5. Interactive effects of selenium, methionine, and dietary protein on survival, growth, and physiology in mallard ducklings

    USGS Publications Warehouse

    Hoffman, D.J.; Sanderson, C.J.; LeCaptain, L.J.; Cromartie, E.; Pendleton, G.W.

    1992-01-01

    Concentrations of over 100 ppm (100 mg/kg) selenium (Se) have been found in aquatic food chains associated with irrigation drainwater. Both quantity and composition of dietary protein for wild ducklings may vary in selenium-contaminated environments. Day-old mallard (Anas platyrhynchos) ducklings received one of the following diets containing 22% protein: unsupplemented (controls), 15 ppm Se (as selenomethionine), 60 ppm Se, methionine supplemented, 15 ppm Se with methionine supplement, or 60 ppm Se with methionine supplement. In a second concurrent experiment the above sequence was repeated with a protein-restricted (11%) but isocaloric diet. In a third concurrent experiment all ducklings received 44% protein with 0, 15, or 60 ppm Se added. After 4 weeks, blood and tissue samples were collected for biochemical and histological examination. With 22% protein and 60 ppm Se in the diet, duckling survival and growth was reduced and histopathological lesions of the liver occurred. Antagonistic interactive effects occurred between supplementary methionine and Se, including complete to partial alleviation of the following Se effects by methionine: mortality, hepatic lesions, and altered glutathione and thiol status. With 11% protein, growth of controls was less than that with 22% protein, Se (60 ppm) caused 100% mortality, and methionine supplementation, although protective afforded less protection than it did with 22% protein. With 44% protein, ducklings experienced physiological stress, and Se was more toxic than with methionine-supplemented 22% protein. These findings suggest the potential for antagonistic effects of Se, methionine, and protein on duckling survival and physiology.

  6. Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

    SciTech Connect

    Kiene, R.P.; Visscher, P.T.

    1987-10-01

    Anoxic salt marsh sediments were amended with DL-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 ..mu..M) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO/sub 2/ and only small amounts of CH/sub 4/. At higher concentrations of methionine, the proportion of the methyl-sulfur groups converted to CH/sub 4/ increased.

  7. Ontogeny of methionine utilization and splanchnic uptake in critically ill children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine the rates of methionine splanchnic uptake and utilization in critically ill pediatric patients, we used two kinetic models: the plasma methionine enrichment,and the "intracellular" homocysteine enrichment. Twenty-four patients, eight infants, eight children, and eight adolescents, were ...

  8. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    EPA Science Inventory

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver Cytosol
    Shan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas
    S-adenosyl-L-methionine (AdoMet): ar...

  9. From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span.

    PubMed

    McIsaac, R Scott; Lewis, Kaitlyn N; Gibney, Patrick A; Buffenstein, Rochelle

    2016-01-01

    Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long-lived naked mole-rat, suggesting that it naturally presents with a life-extending phenotype akin to that observed in methionine-restricted animals. Similarly, long-lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine-restriction effects on life-span extension, however, remain unknown, as do their potential connections with caloric restriction, another well-established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span-from yeast to humans-and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span.

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

    SciTech Connect

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

    1989-07-01

    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, {sup 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 {sup 75}Se in erythrocyte proteins were determined. In comparison to the rats fed the basal diet without added methionine, significantly more {sup 75}Se-selenocysteine was found in liver and muscle, more {sup 75}Se was found in erythrocyte GSHPx and less {sup 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. Methionine residues around phosphorylation sites are preferentially oxidized in vivo under stress conditions

    PubMed Central

    Veredas, Francisco J.; Cantón, Francisco R.; Aledo, J. Carlos

    2017-01-01

    Protein phosphorylation is one of the most prevalent and well-understood protein modifications. Oxidation of protein-bound methionine, which has been traditionally perceived as an inevitable damage derived from oxidative stress, is now emerging as another modification capable of regulating protein activity during stress conditions. However, the mechanism coupling oxidative signals to changes in protein function remains unknown. An appealing hypothesis is that methionine oxidation might serve as a rheostat to control phosphorylation. To investigate this potential crosstalk between phosphorylation and methionine oxidation, we have addressed the co-occurrence of these two types of modifications within the human proteome. Here, we show that nearly all (98%) proteins containing oxidized methionine were also phosphoproteins. Furthermore, phosphorylation sites were much closer to oxidized methionines when compared to non-oxidized methionines. This proximity between modification sites cannot be accounted for by their co-localization within unstructured clusters because it was faithfully reproduced in a smaller sample of structured proteins. We also provide evidence that the oxidation of methionine located within phosphorylation motifs is a highly selective process among stress-related proteins, which supports the hypothesis of crosstalk between methionine oxidation and phosphorylation as part of the cellular defence against oxidative stress. PMID:28079140

  12. Methionine residues around phosphorylation sites are preferentially oxidized in vivo under stress conditions.

    PubMed

    Veredas, Francisco J; Cantón, Francisco R; Aledo, J Carlos

    2017-01-12

    Protein phosphorylation is one of the most prevalent and well-understood protein modifications. Oxidation of protein-bound methionine, which has been traditionally perceived as an inevitable damage derived from oxidative stress, is now emerging as another modification capable of regulating protein activity during stress conditions. However, the mechanism coupling oxidative signals to changes in protein function remains unknown. An appealing hypothesis is that methionine oxidation might serve as a rheostat to control phosphorylation. To investigate this potential crosstalk between phosphorylation and methionine oxidation, we have addressed the co-occurrence of these two types of modifications within the human proteome. Here, we show that nearly all (98%) proteins containing oxidized methionine were also phosphoproteins. Furthermore, phosphorylation sites were much closer to oxidized methionines when compared to non-oxidized methionines. This proximity between modification sites cannot be accounted for by their co-localization within unstructured clusters because it was faithfully reproduced in a smaller sample of structured proteins. We also provide evidence that the oxidation of methionine located within phosphorylation motifs is a highly selective process among stress-related proteins, which supports the hypothesis of crosstalk between methionine oxidation and phosphorylation as part of the cellular defence against oxidative stress.

  13. Thioredoxin-dependent Redox Regulation of Cellular Signaling and Stress Response through Reversible Oxidation of Methionines

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2011-06-01

    Generation of reactive oxygen species (ROS) is a common feature of many forms of stress to which plants are exposed. Successful adaptation to changing environmental conditions requires sensitive sensors of ROS such as protein-bound methionines that are converted to their corresponding methionine sulfoxides, which in turn can influence cellular signaling pathways. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress response in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the sensitivity of individual methionines within plant and animal calmodulin to ROS, the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes.

  14. Methionine deficiency reduces autophagy and accelerates death in intestinal epithelial cells infected with enterotoxigenic Escherichia coli.

    PubMed

    Tang, Yulong; Tan, Bie; Xiong, Xia; Li, Fengna; Ren, Wenkai; Kong, Xiangfeng; Qiu, Wei; Hardwidge, Philip R; Yin, Yulong

    2015-10-01

    Infections by enterotoxigenic Escherichia coli (ETEC) result in large economic losses to the swine industry worldwide. Dietary supplementation with amino acids has been considered as a potential mechanism to improve host defenses against infection. The goal of this study was to determine whether methionine deprivation alters ETEC interactions with porcine intestinal epithelial cells. IPEC-1 cells were cultured in media with or without L-methionine. Methionine deprivation resulted in enhanced ETEC adhesion and increased both the cytotoxicity and apoptotic responses of IPEC-1 cells infected with ETEC. Methionine deprivation inhibited IPEC-1 cell autophagic responses, suggesting that the increased cytotoxicity of ETEC to methionine-deprived IPEC-1 cells might be due to defects in autophagy.

  15. Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family.

    PubMed

    Trötschel, Christian; Follmann, Martin; Nettekoven, Jeannine A; Mohrbach, Tobias; Forrest, Lucy R; Burkovski, Andreas; Marin, Kay; Krämer, Reinhard

    2008-12-02

    The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.

  16. The role of methionine in the intracellular accumulation and function of folates

    SciTech Connect

    Scott, J.M.; McKenna, B.; McGing, P.; Molloy, A.; Dinn, J.; Weir, D.G.

    1983-01-01

    It is suggested that mammalian cells have evolved to respond to methionine deficiency since in such circumstances vital methylation reactions are put at risk, due to decreased levels of S-adenosyl-methionine. Decreased cellular homocysteine, as a result of decreased methionine, would also restrict cell division by decreased conversion of plasma 5-CH3-H/sub 4/PteGlu into intracellular polyglutamates. Cobalamin deficiency, either nutritional or due to exposure to the Co(I)cobalamin inactivating agent nitrous oxide, prevents the demethylation of 5-CH3-H/sub 4/PteGlu, which even in the presence of adequate amounts of homocysteine and methionine prevents rapidly proliferating cells from converting enough of the plasma 5-CH3-H/sub 4/ PteGlu into folylpolyglutamate forms to permit normal DNA biosynthesis and cell replication. This, together with the trapping of the cellular folate cofactors in the 5-CH3-H/sub 4/PteGlu form, results in megaloblastic changes occurring in tissues such as the marrow. The vital role of the methylation reactions was demonstrated by exposing monkeys to nitrous oxide which inactivated their methionine synthetase. The resultant ataxia and severe demyelination was prevented and diminished by methionine supplementation. When methionine synthetase was similarly inactivated in mice it was shown that while 5-CH3-H/sub 4/PteGlu enters mammalian cells, it is not converted into a polyglutamyl form and subsequently leaves the cell unmetabolised. In similar experiments in rats methionine was found to have only a small effect in restoring folylpolyglutamate biosynthesis. It was found that a decrease in the deoxythymidine salvage pathway by methionine has led others to the mistaken conclusion that methionine has an 'anti-folate' effect in bone marrow, i.e. that it decreases folate availability for thymidylate synthetase.

  17. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    PubMed

    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.

  18. Na-Stimulated Transport of l-Methionine in Brevibacterium linens CNRZ 918.

    PubMed

    Ferchichi, M; Hemme, D; Nardi, M

    1987-09-01

    The transport of l-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (K(m) = 55 muM) found is constitutive for l-methionine, stereospecific, and pH and temperature dependent. Entry of l-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that l-methionine and alpha-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor d-methionine was an inhibitor, whereas powerful inhibition was shown by l-cysteine, s-methyl-l-cysteine, dl-selenomethionine and dl-homocysteine. Sodium plays important and varied roles in l-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the K(m), (ii) it increases the specific activity (on a biomass basis) of the l-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. l-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that l-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na gradient is involved in the mechanism of energy coupling of l-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium

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

    PubMed Central

    Wen, Z. G.; Tang, J.; Xie, M.; Yang, P. L.; Hou, S. S.

    2016-01-01

    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 ducks. PMID

  20. Can betaine partially replace or enhance the effect of methionine by improving broiler growth and carcase characteristics?

    PubMed

    McDevitt, R M; Mack, S; Wallis, I R

    2000-09-01

    1. Growth rates and carcase characteristics were measured in male broiler chickens fed on a control diet deficient in methionine (c. 2.8 g/kg methionine) or a series of diets containing graded levels of betaine or DL-methionine or both additives. 2. We aimed to answer 2 main questions. First, can betaine replace part of the methionine in a broiler ration? Secondly is there a synergism between methionine and betaine? 3. Birds given the control diet or that supplemented only with betaine ate less, grew more slowly, had higher food convension ratio (FCR) and varied more in mass at 42 d than birds fed diets with DL-methionine. Adding 1.2 g/kg DL-methionine to the control ration produced the heaviest birds at 42 d (2500 g) with the 2nd heaviest breast muscle (366 g). 4. After correcting for treatment differences in body mass (analysis of convariance), birds fed on the control diet and the diet supplemented with betaine only, had relatively lighter breast muscles but relatively heavier abdominal fat pads than those of birds given diets supplemented with DL-methionine. However, adding betaine to diets containing added methionine further improved the relative breast muscle yield. 5. After correcting for differences in body mass between treatments, birds fed on diets containing most methionine had lighter viscera than birds fed diets deficient in methionine. This demonstrated gut plasticity, suggesting that the viscera enlarged to sequester methionine from low-methionine diets. 6. Our data refute the hypothesis that betaine can substitute for methionine in broilers fed diets that are marginally deficient in methionine plus cystine. However, betaine may improve carcase composition, especially breast meat yield.

  1. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    DOE PAGES

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; ...

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess lowmore » cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.« less

  2. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    SciTech Connect

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

  3. Reinventing Cell Penetrating Peptides Using Glycosylated Methionine Sulfonium Ion Sequences

    PubMed Central

    2015-01-01

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs. PMID:27162954

  4. Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase

    SciTech Connect

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-09-07

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

  5. Catalysis and inhibition of Mycobacterium tuberculosis methionine aminopeptidase

    PubMed Central

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-01-01

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II) and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells, because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity towards the Fe(II)-form, the Mn(II)-form, or the Co(II)- and Ni(II)-forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues. PMID:20038112

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

    PubMed

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

    2015-11-24

    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.

  7. Methionine enhances the contractile activity of human colon circular smooth muscle in vitro.

    PubMed

    Choe, Eun Kyung; Moon, Jung Sun; Park, Kyu Joo

    2012-07-01

    Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 µM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 µM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and N(w)-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.

  8. Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.

    PubMed

    Zhu, Qingfu; El-Mergawy, Rabab G; Heinemann, Stefan H; Schönherr, Roland; Jáč, Pavel; Scriba, Gerhard K E

    2013-09-01

    A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

  9. Growth hormone signaling is necessary for lifespan extension by dietary methionine.

    PubMed

    Brown-Borg, Holly M; Rakoczy, Sharlene G; Wonderlich, Joseph A; Rojanathammanee, Lalida; Kopchick, John J; Armstrong, Vanessa; Raasakka, Debbie

    2014-12-01

    Growth hormone significantly impacts lifespan in mammals. Mouse longevity is extended when growth hormone (GH) signaling is interrupted but markedly shortened with high-plasma hormone levels. Methionine metabolism is enhanced in growth hormone deficiency, for example, in the Ames dwarf, but suppressed in GH transgenic mice. Methionine intake affects also lifespan, and thus, GH mutant mice and respective wild-type littermates were fed 0.16%, 0.43%, or 1.3% methionine to evaluate the interaction between hormone status and methionine. All wild-type and GH transgenic mice lived longer when fed 0.16% methionine but not when fed higher levels. In contrast, animals without growth hormone signaling due to hormone deficiency or resistance did not respond to altered levels of methionine in terms of lifespan, body weight, or food consumption. Taken together, our results suggest that the presence of growth hormone is necessary to sense dietary methionine changes, thus strongly linking growth and lifespan to amino acid availability.

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

    PubMed Central

    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

  11. Effects of methyl-deficient diets on methionine and homocysteine metabolism in the pregnant rat.

    PubMed

    Wilson, Fiona A; Holtrop, Grietje; Calder, A Graham; Anderson, Susan E; Lobley, Gerald E; Rees, William D

    2012-06-15

    Although the importance of methyl metabolism in fetal development is well recognized, there is limited information on the dynamics of methionine flow through maternal and fetal tissues and on how this is related to circulating total homocysteine concentrations. Rates of homocysteine remethylation in maternal and fetal tissues on days 11, 19, and 21 of gestation were measured in pregnant rats fed diets with limiting or surplus amounts of folic acid and choline at two levels of methionine and then infused with L-[1-(13)C,(2)H(3)-methyl]methionine. The rate of homocysteine remethylation was highest in maternal liver and declined as gestation progressed. Diets deficient in folic acid and choline reduced the production of methionine from homocysteine in maternal liver only in the animals fed a methionine-limited diet. Throughout gestation, the pancreas exported homocysteine for methylation within other tissues. Little or no methionine cycle activity was detected in the placenta at days 19 and 21 of gestation, but, during this period, fetal tissues, especially the liver, synthesized methionine from homocysteine. Greater enrichment of homocysteine in maternal plasma than placenta, even in animals fed the most-deficient diets, shows that the placenta did not contribute homocysteine to maternal plasma. Methionine synthesis from homocysteine in fetal tissues was maintained or increased when the dams were fed folate- and choline-deficient methionine-restricted diets. This study shows that methyl-deficient diets decrease the remethylation of homocysteine within maternal tissues but that these rates are protected to some extent within fetal tissues.

  12. Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A.

    PubMed

    Brock, Jonathan W C; Ames, Jennifer M; Thorpe, Suzanne R; Baynes, John W

    2007-01-15

    Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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

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

    PubMed

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

    2016-09-23

    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

  15. The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats.

    PubMed

    Amaral, Cátia Lira Do; Bueno, Rafaela de Barros E Lima; Burim, Regislaine Valéria; Queiroz, Regina Helena Costa; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

    2011-05-18

    Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet's effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet's effects on genomic stability and DNA methylation.

  16. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  17. 13C-methionine breath tests for mitochondrial liver function assessment.

    PubMed

    Candelli, M; Miele, L; Armuzzi, A; Nista, E C; Pignataro, G; Fini, L; Cazzato, I A; Zocco, M A; Bartolozzi, F; Gasbarrini, G; Grieco, A; Gasbarrini, A

    2008-01-01

    13C-methionine breath test has been proposed as a non-invasive tool for the assessment of human hepatic mithocondrial function. Two methionine breath labeled with 13C in differents point of his molecular structure have been used for breath test analisys. Aim of this study was to compare two differently 13C-labeled methionines in the evaluation of mitochondrial oxidation in basal conditions and after an acute oxidative stress. 15 healthy male subjects (mean age 30.5 +/- 3.1) received [methyl-13C]-methionine dissolved in water. Breath samples were taken at baseline and and 10, 20, 30, 45, 60, 75, 90, 105 and 120 minutes after the ingestion of the labeled substrate. Forthy-eight hours later, subjects underwent the same test 30 minutes after ethanol ingestion (0,3 g/kg of body weight). Seven-day later, subjects underwent breath test using (L-methionine-1-13COOH) as substrate, in basal condition and after ethanol ingestion. At basal condition, the cumulative percentage of 13CO2 recovered in breath during the test period (%cum-dose) was higher using L-methionine-1-13COOH than [methyl-13C]-methionine (10.25 +/- 1.0 vs 4.07 +/- 0.8; p < 0.01). After ethanol ingestion, % cum dose was significantly decreased at 60 and 120 minutes with both methionines (120 min: 10.25 +/- 1.0 vs 5.03% +/- 1.8; < 0.01 and 4.07 +/- 0.8 vs 2.16% +/- 0.9; p < 0.01, respectively). However, %cum-dose during L-methionine-1-13C-breath test was significantly lower than that observed during methyl-13C-methionine breath test (120 minutes: 5.03% +/- 1.8 vs 2.16% +/- 0.9; p < 0.01). In conclusion, breath test based on L-methionine-1-13COOH seems to show a greater reliability when compared to [methyl-13C]-methionine to assess mitochondrial function because a larger amount of labeled carbon that reaches the Krebs' cicle.

  18. The reaction of methionine with hydroxyl radical: reactive intermediates and methanethiol production.

    PubMed

    Spasojević, Ivan; Bogdanović Pristov, Jelena; Vujisić, Ljubodrag; Spasić, Mihajlo

    2012-06-01

    The mechanisms of reaction of methionine with hydroxyl radical are not fully understood. Here, we unequivocally show using electron paramagnetic resonance spin-trapping spectroscopy and GC-FID and GC-MS, the presence of specific carbon-, nitrogen- and sulfur-centered radicals as intermediates of this reaction, as well as the liberation of methanethiol as a gaseous end product. Taking into account the many roles that methionine has in eco- and biosystems, our results may elucidate redox chemistry of this amino acid and processes that methionine is involved in.

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

    PubMed

    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.

  20. In Silico Analysis of Sequence-Structure-Function Relationship of the Escherichia coli Methionine Synthase.

    PubMed

    Kumar, Shiv; Bhagabati, Puja; Sachan, Reena; Kaushik, Aman Chandra; Dwivedi, Vivek Dhar

    2015-12-01

    The molecular evolution of various metabolic pathways in the organisms can be employed for scrutinizing the molecular aspects behind origin of life. In the present study, we chiefly concerned about the sequence-structure-function relationship between the Escherichia coli methionine synthase and their respective animal homologs by in silico approach. Using homology prediction technique, it was observed that only 79 animal species showed similarity with the E. coli methionine synthase. Also, multiple sequence alignment depicted only 25 conserved patterns between the E. coli methionine synthase and their respective animal homologs. Based on that, Pfam analysis identified the protein families of 22 conserved patterns among the attained 25 conserved patterns. Furthermore, the 3D structure was generated by HHpred and evaluated by corresponding Ramachandran plot specifying 93% of the ϕ and ψ residues angles in the most ideal regions. Hence, the designed structure was established as a good quality model for the full length of E. coli methionine synthase.

  1. Methionine and choline regulate the metabolic phenotype of a ketogenic diet.

    PubMed

    Pissios, Pavlos; Hong, Shangyu; Kennedy, Adam Richard; Prasad, Deepthi; Liu, Fen-Fen; Maratos-Flier, Eleftheria

    2013-01-01

    Low-carbohydrate ketogenic diets are commonly used as weight loss alternatives to low-fat diets, however the physiological and molecular adaptations to these diets are not completely understood. It is assumed that the metabolic phenotype of the ketogenic diet (KD) is caused by the absence of carbohydrate and high fat content, however in rodents the protein content of KD affects weight gain and ketosis. In this study we examined the role of methionine and choline in mediating the metabolic effects of KD. We have found that choline was more effective than methionine in decreasing the liver steatosis of KD-fed mice. On the other hand, methionine supplementation was more effective than choline in restoring weight gain and normalizing the expression of several fatty acid and inflammatory genes in the liver of KD-fed mice. Our results indicate that choline and methionine restriction rather than carbohydrate restriction underlies many of the metabolic effects of KD.

  2. Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene.

    PubMed

    Kahraman, Huseyin; Aytan, Emel; Kurt, Ash Giray

    2011-09-01

    The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.

  3. Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens.

    PubMed

    Amarita, Felix; Yvon, Mireille; Nardi, Michele; Chambellon, Emilie; Delettre, Jerôme; Bonnarme, Pascal

    2004-12-01

    The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.

  4. Crystallization and preliminary X-ray analysis of l-methionine γ-lyase 1 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Karaki, Tsuyoshi; Shimizu, Akira; Kamei, Kaeko; Harada, Shigeharu; Nozaki, Tomoyoshi

    2008-08-01

    l-Methionine γ-lyase 1, a key enzyme in sulfur-containing amino-acid degradation, from the protozoan parasite E. histolytica was crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 Å, β = 101.82°. The crystals diffract to beyond 2.0 Å resolution. The presence of a tetramer in the asymmetric unit (4 × 42.4 kDa) gives a Matthews coefficient of 2.8 Å{sup 3} Da{sup −1} and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

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

    PubMed

    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 T1/2 of enzymes in mice varies from 73 to 123min. The AUC0-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 (<5μM) in plasma of mice for about 6h. The recovery of methionine concentration showed the advantageous efficiency of MGL from C. sporogenes: 95% 0.010-0.022 vs 0.023-0.061 for MGL C. freundii and 0.036-0.056 for MGL C. 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.

  6. Appropriate statistical methods to compare dose responses of methionine sources.

    PubMed

    Kratzer, D D; Littell, R C

    2006-05-01

    Two sources of methionine (Met) activity are frequently used in commercial feed formulation: DL-2-hydroxy-4-(methylthio) butanoic acid (HMTBA), most commonly available as an 88% solution with 12% water; and DL-methionine (DLM, 99% powder). Despite the fact that both compounds have been in commercial use for over 50 yr, controversy and confusion remain with respect to their relative bioefficacy (RBE). This paper presents a review of the use of a nonlinear common plateau asymptotic regression technique (NLCPAR) that has been used to compare the 2 Met sources with particular emphasis on the validity of the basic assumptions of that model. The thesis of this paper is that the controversy is due, at least in part, to the misapplication of this regression technique to estimate the RBE of HMTBA and DLM. The NLCPAR model is a bioassay with the key dependent assumptions that HMTBA is a dilution of DLM, and that each follows dose-response curves of the same form and approach a common plateau. Because both provide Met activity, it may be considered reasonable to accept these assumptions; however, specifically testing them demonstrated that the assumption of a common dose-response is not supported by data. The common plateau assumption was tested with an alternative approach of fitting nonlinear separate plateaus asymptotic regression (NLSPAR) to a set of 13 published broiler studies in which the NLCPAR model had been used to estimate RBE of HMTBA and DLM. The hypothesis of a common plateau was rejected (P < 0.01), meaning that the conclusion that HMTBA had lower bioefficacy than DLM based on the NLCPAR methodology was not valid. An example using published data demonstrated that the NLSPAR model was a significantly better fit than the NLCPAR model, and showed that HMTBA and DLM followed different dose responses. Consequently, there was no single value for RBE for the entire dose range; rather, the RBE of the 2 compounds varied with use level. The evidence presented here

  7. Impact of methionine oxidation on calmodulin structural dynamics

    SciTech Connect

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin; Moen, Rebecca J.; Olenek, Michael J.; Klein, Jennifer C.; Thomas, David D.

    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

  8. Phase II Clinical Trials: D-methionine to Reduce Noise-Induced Hearing Loss

    DTIC Science & Technology

    2012-03-01

    loss (NIHL) and tinnitus in our troops. Hypotheses: Primary Hypothesis: Administration of oral D-methionine prior to and during weapons...reduce or prevent noise-induced tinnitus . Primary outcome to test the primary hypothesis: Pure tone air-conduction thresholds. Primary outcome to...test the secondary hypothesis: Tinnitus questionnaires. Specific Aims: 1. To determine whether administering oral D-methionine (D-met) can

  9. Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide.

    PubMed

    Richardson, David E; Regino, Celeste A S; Yao, Huirong; Johnson, Jodie V

    2003-12-15

    Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO4-, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO2. The kinetic parameters for HCO4- oxidation of free methionine have been obtained (k1 = 0.48 +/- 0.08 M(-1)s(-1) by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood ( approximately 25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H2O2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of alpha1-proteinase inhibitor (alpha1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO4- oxidation of alpha1-PI (0.36 +/- 0.06 M(-1)s(-1)) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the alpha1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized alpha1-PI. These results strongly suggest that HCO4- should be considered a reactive oxygen species in aerobic metabolism.

  10. Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

    PubMed Central

    Suzuki, T; Takahashi, E

    1976-01-01

    1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed. PMID:1008848

  11. Decreased rates of methionine synthesis by methylene tetrahydrofolate reductase-deficient fibroblasts and lymphoblasts.

    PubMed

    Boss, G R; Erbe, R W

    1981-06-01

    Methionine synthesis from homocysteine was measured in intact human fibroblasts and lymphoblasts using a [14C]formate label. Seven fibroblast lines and two lymphoblast lines derived from patients with 5,10-methylene tetrahydrofolate reductase deficiency had rates of methionine synthesis that were from 4 to 43% of normal. When the patients were divided by clinical status into mildly (two patients), moderately (two patients), and severely (three patients) affected, methionine biosynthesis expressed as a percent of control values was 43 and 33%, 11 and 10%, and 7, 6, and 4%, respectively, in fibroblasts. Similar data for the two lymphoblast lines were 36 and 26% for a mildly and moderately affected patient, respectively. These data are to be contrasted with the measurement of residual enzyme activity in cell extracts which agrees less precisely with the clinical status of the patients. In the presence of normal methionine synthetase activity, the rate of synthesis of methionine from homocysteine is a function of the activity of the enzyme 5,10-methylene tetrahydrofolate reductase, and measurement of the methionine biosynthetic capacity of cells deficient in this enzyme accurately reflects the clinical status of the patient from whom the cells were derived.

  12. Comparative value of L-, and D-methionine supplementation of an oat-based diet for humans.

    PubMed

    Kies, C; Fox, H; Aprahamian, S

    1975-07-01

    Total sulfur-containing amino acids have been found to be the first limiting amino acid in several foods in comparison with human amino acid requirements. Addition of methionine in appropriate amounts to these foods might be expected to improve protein value. Economically, DL-methionine would be preferable to L-methionine for this purpose. However, the comparative tuilization of L- DL-, and D-methionine is unclear. The objective of the current project was to compare the effectiveness of L-, DL-, and D-methionine supplementation of diets based on a food product known to be low in methionine value for human subjects. "Instant" oatmeal was fed to adult subjects to provide 4.0 g of nitrogen/day. In randomly arranged periods, these diets were supplemented with L-, DL-, or D-methionine at two levels (0.58 and 1.16 g of methionine/day). An unsupplemented diet was used in a control period. Diets were adequate in vitamins, minerals, and energy. Mean nitrogen balances of subjects while receiving the L-methionine supplements at the 0.58 and 1.16 g levels were minus0.10 and +0.06 g of nitrogen, respectively. At similar levels of DL-methionine supplementation, nitrogen balances were minus0.12 and minus0.15 g of nitrogen, respectively, and minus0.24 and minus0.18 g of nitrogen with D-methionine supplementation. The mean nitrogen balance when no supplement was used was minus0.22 g of nitrogen. Thus, D-methionine is seemingly poorly utilized by the human. Urinary methionine excretion data supported these results.

  13. Fragmentation network of doubly charged methionine: Interpretation using graph theory.

    PubMed

    Ha, D T; Yamazaki, K; Wang, Y; Alcamí, M; Maeda, S; Kono, H; Martín, F; Kukk, E

    2016-09-07

    The fragmentation of doubly charged gas-phase methionine (HO2CCH(NH2)CH2CH2SCH3) is systematically studied using the self-consistent charge density functional tight-binding molecular dynamics (MD) simulation method. We applied graph theory to analyze the large number of the calculated MD trajectories, which appears to be a highly effective and convenient means of extracting versatile information from the large data. The present theoretical results strongly concur with the earlier studied experimental ones. Essentially, the dication dissociates into acidic group CO2H and basic group C4NSH10. The former may carry a single or no charge and stays intact in most cases, whereas the latter may hold either a single or a double charge and tends to dissociate into smaller fragments. The decay of the basic group is observed to follow the Arrhenius law. The dissociation pathways to CO2H and C4NSH10 and subsequent fragmentations are also supported by ab initio calculations.

  14. Methionine Uptake and Required Radiation Dose to Control Glioblastoma

    SciTech Connect

    Iuchi, Toshihiko; Hatano, Kazuo; Uchino, Yoshio; Itami, Makiko; Hasegawa, Yuzo; Kawasaki, Koichiro; Sakaida, Tsukasa; Hara, Ryusuke

    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.

  15. Meta-analysis of lactation performance in dairy cows receiving supplemental dietary methionine sources or postruminal infusion of methionine.

    PubMed

    Zanton, G I; Bowman, G R; Vázquez-Añón, M; Rode, L M

    2014-11-01

    The objectives of our study were to evaluate the productive response to methionine supplementation in lactating dairy cows and to define a relationship between metabolizable Met (MP Met) intake and production. A database of 64 papers meeting the selection criteria was developed evaluating postruminally infused dl-methionine (9 papers with 18 control diets and 35 treatment comparisons), 2-hydroxy-4-methylthio butanoic acid (HMTBa) provided as either a liquid or Ca salt form (17 papers with 34 control diets and 46 treatment comparisons), Mepron (Evonik Industries, Essen, Germany; 18 papers with 35 control diets and 42 treatment comparisons), and Smartamine (Adisseo Inc., Antony, France; 20 papers with 30 control diets and 39 treatment comparisons). Dietary ingredients and their accompanying nutritional compositions as described in the reports were entered into the Cornell-Penn-Miner software to model the diets and to predict nutrients that were not reported in the original publication. Data were analyzed using a weighted analysis of response to supplementation compared with the intraexperiment control, as well as through a regression analysis to changing dietary MP Met. Data included in the analysis were from experiments published between 1970 and 2011 with cows supplemented with between 3.5 and 67.9 g of Met or its equivalent from HMTBa. Cows supplemented with Smartamine consumed more, whereas cows supplemented with Mepron consumed less DM compared with controls. Milk yield did not significantly respond to Met supplementation, although it tended to increase for cows supplemented with HMTBa and Mepron. Milk protein yield was increased due to supplementation from all sources or from infusion, and protein concentration was greater for all supplements or infusion of dl-Met, except for cows supplemented with HMTBa. Irrespective of Met source, milk protein yield increased 2.23 g of protein/g of MP Met until reaching the breakpoint. Milk fat yield was increased for Mepron

  16. Selenium-methionine and chromium-methionine supplementation of sheep around parturition: impacts on dam and offspring performance.

    PubMed

    Mousaie, Amir; Valizadeh, Reza; Chamsaz, Mahmoud

    2017-04-01

    To examine the effects of maternal energy restriction along with selenium-methionine (Se-Met) and chromium-methionine (Cr-Met) supplementation on performance of pregnant sheep and their offspring, the following treatments were allotted randomly to 40 multiparous Baluchi ewes (53.9 ± 1.15 kg of body weight [BW]) from 5 weeks prior to 5 weeks after parturition: (1) Control diet (60% and 100% of NRC energy requirements in pre- and post-partum, respectively); (2) Control diet plus 5 mg Se-Met/kg dry matter (DM); (3) Control diet plus 3 mg Cr-Met/kg DM and (4) Control diet plus 5 mg Se-Met and 3 mg Cr-Met/kg DM (Se-Cr-Met) of concentrate diet. The results indicated that Cr-Met alone or in combination with Se-Met increased average DM intake of ewes. In addition, Group Cr-Met had higher average BW than the Control (p < 0.05). Se-Met and/or Cr-Met supplementation led to decreased average serum cholesterol of the ewes (p < 0.05). Groups Cr-Met and Se-Cr-Met displayed decreased average serum malondialdehyde compared to the Control (p < 0.05). At 24 h post-partum, Group Se-Cr-Met had a greater serum Se content than the Control (p = 0.006). Compared with the Control, the Se concentration in milk was significantly increased from 30 to 138 µg/l and 197 µg/l in Groups Se-Met and Se-Cr-Met, respectively (p < 0.01), which proved that Se-Met supplementation can increase the Se concentration of ewe milk. Furthermore, feeding Cr-Met may attenuate BW loss post-partum and Se-Met and/or Cr-Met supplements may ameliorate oxidative stress condition in ewes around parturition.

  17. The Cus efflux system removes toxic ions via a methionine shuttle.

    PubMed

    Su, Chih-Chia; Long, Feng; Yu, Edward W

    2011-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.

  18. Control of protein life-span by N-terminal methionine excision

    PubMed Central

    Giglione, Carmela; Vallon, Olivier; Meinnel, Thierry

    2003-01-01

    Peptide deformylases (PDFs) have been discovered recently in eukaryotic genomes, and it appears that N-terminal methionine excision (NME) is a conserved pathway in all compartments where protein synthesis occurs. This work aimed at uncovering the function(s) of NME in a whole proteome, using the chloroplast-encoded proteins of both Arabidopsis thaliana and Chlamydomonas reinhardtii as model systems. Dis ruption of PDF1B in A.thaliana led to an albino phenotype, and an extreme sensitivity to the PDF- specific inhibitor actinonin. In contrast, a knockout line for PDF1A exhibited no apparent phenotype. Photosystem II activity in C.reinhardtii cells was substantially reduced by the presence of actinonin. Pulse–chase experiments revealed that PDF inhibi tion leads to destabilization of a crucial subset of chloroplast-encoded photosystem II components in C.reinhardtii. The same proteins were destabilized in pdf1b. Site-directed substitutions altering NME of the most sensitive target, subunit D2, resulted in similar effects. Thus, plastid NME is a critical mechanism specifically influencing the life-span of photosystem II polypeptides. A general role of NME in modulating the half-life of key subsets of proteins is suggested. PMID:12505980

  19. Divergent modulation of swine ileal microbiota by formic acid and methionine hydroxy analogue-free acid.

    PubMed

    Apajalahti, J; Rademacher, M; Htoo, J K; Redshaw, M; Kettunen, A

    2009-06-01

    Management of intestinal microbiota of monogastric animals has increased in importance since the ban of growth promoting antibiotics in many countries. Organic acids have been used as alternatives to antibiotics by many feed manufacturers. Regardless of the wide usage, the effect, dose response and mode of action of acids on intestinal microbes is poorly understood. In this study, we investigated the effects of dietary supplementation of three commonly used products, namely formic acid (FA) (90%), dl-methionine (DLM) (99%) and liquid methionine hydroxy analogue-free acid (88%), on ileal microbiota of pigs. Laboratory simulation system, mimicking swine ileum, was used to study the products at various concentrations and combinations. Furthermore, selected combinations were tested in a piglet trial to confirm the findings made in in vitro studies. FA turned out to have a dual effect on ileal microbiota. At concentrations below 0.5%, it significantly stimulated bacteria, but at higher inclusion rates it was highly inhibitory. This finding, which was consistent in in vitro and in vivo studies, implies that reducing the dose of FA does not lead to a diluted inhibitory effect, but in fact, an opposite, stimulatory effect on intestinal microbiota. It is highly important that feed compounders acknowledge this finding. Unlike FA, the inhibitory effect of methionine hydroxy analogue on ileal bacteria was linearly dose dependent and significant at inclusion levels above 0.2%, in vitro. Partial replacement of methionine hydroxy analogue by FA, or FA by methionine hydroxy analogue, led to an unpredictable outcome due to the dual effects of FA; e.g., a minor inclusion of added FA changed the inhibitory effect of methionine hydroxy analogue into microbial stimulation by FA. Inhibition of ileal microbiota by methionine hydroxy analogue was detected only in in vitro studies, suggesting that intact methionine hydroxy analogue may not have reached the ileum, in live animals. Therefore

  20. Cysteine dietary supplementation reverses the decrease in mitochondrial ROS production at complex I induced by methionine restriction.

    PubMed

    Gomez, A; Gomez, J; Lopez Torres, M; Naudi, A; Mota-Martorell, N; Pamplona, R; Barja, G

    2015-06-01

    It has been described that dietary cysteine reverses many of the beneficial changes induced by methionine restriction in aging rodents. In this investigation male Wistar rats were subjected to diets low in methionine, supplemented with cysteine, or simultaneously low in methionine and supplemented with cysteine. The results obtained in liver showed that cysteine supplementation reverses the decrease in mitochondrial ROS generation induced by methionine restriction at complex I. Methionine restriction also decreased various markers of oxidative and non-oxidative stress on mitochondrial proteins which were not reversed by cysteine. Instead, cysteine supplementation also lowered protein damage in association with decreases in mTOR activation. The results of the present study add the decrease in mitochondrial ROS production to the various beneficial changes induced by methionine restriction that are reversed by cysteine dietary supplementation.

  1. Recycling of 5'-methylthioadenosine-ribose carbon atoms into methionine in tomato tissue in relation to ethylene production.

    PubMed

    Wang, S Y; Adams, D O; Lieberman, M

    1982-07-01

    The ribose moiety of 5'-methylthioadenosine (MTA) is metabolized to form the four-carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-(14)C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C(2)H(4) and other unidentified compounds. However, when [U-(14)C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C(2)H(4), but not MTA. This suggests that C(2)H(4) formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O(2) dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue.

  2. Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato.

    PubMed

    Tu, H M; Godfrey, L W; Sun, S S

    1998-07-01

    A cDNA encoding the methionine-rich (19 mol% Met) protein in Brazil nut was placed under the regulation of CaMV 35S promoter and nopaline synthase terminator and introduced into the potato cultivar Russet Burbank via Agrobacterium-mediated transformation. To further enhance the Met content in the transgenic plants, chimeric genes containing four mutant constructs, BoxIa (with 5 additional Met), BoxIIa (2 additional Met), BoxIaIIa (7 additional Met), and BoxIIa2 (7 additional Met), were also generated by sequence modifications of the cDNA and transferred into potato. Analysis of the microtubers and leaves of the transgenic potato plants revealed, in general, with the exception of the BoxIIa2, the presence of mRNA transcripts of the expected size and the correctly processed Met-rich 9 kDa subunit polypeptides. The expression levels in the leaves among the various constructs and individual transgenic plants varied between <0.01% and 0.2% of total protein. The corresponding expression in the tubers was usually 2- to 4-fold lower than in leaves. In the case of BoxIIa2, which contains two tandem repeats of the BoxIIa mutant sequence, a larger (10.5-11 kDa) polypeptide was detected. These findings demonstrated that it is feasible to exploit the variable region of the Brazil Nut 2S protein for enhanced Met contents and perhaps for other desirable properties.

  3. 1- sup 13 C; methyl-2H3 methionine kinetics in humans: Methionine conservation and cystine sparing

    SciTech Connect

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

    1990-05-01

    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).

  4. Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXR{alpha}-null mice

    SciTech Connect

    Gyamfi, Maxwell Afari; Tanaka, Yuji; He Lin; Klaassen, Curtis D.; Wan, Y.-J.Y.

    2009-01-15

    Retinoid X receptor-{alpha} (RXR{alpha}) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXR{alpha} deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXR{alpha}-null (H-RXR{alpha}-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid {beta}-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXR{alpha}-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXR{alpha}-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXR{alpha}-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXR{alpha}-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXR{alpha}-null mice. In conclusion, these data suggest a critical role for RXR{alpha} in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.

  5. Metabolic engineering of Escherichia coli for microbial production of L-methionine.

    PubMed

    Huang, Jian-Feng; Liu, Zhi-Qiang; Jin, Li-Qun; Tang, Xiao-Ling; Shen, Zhen-Yang; Yin, Huan-Huan; Zheng, Yu-Guo

    2017-04-01

    L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16 mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli ΔmetJ ΔmetI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43.65% higher than that of E. coli W3110 ΔmetJ/pTrcA*H. Furthermore, deletion of lysA, which blocks the lysine biosynthesis pathway, led to a further 8.5-fold increase in L-methionine titer of E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H. Finally, addition of Na2 S2 O3 to the media led to an increase of fermentation titer of 11.45%. After optimization, constructed E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H was able to produce 9.75 g/L L-methionine with productivity of 0.20 g/L/h in a 5 L bioreactor. This novel metabolically tailored strain of E. coli provides an efficient platform for microbial production of L-methionine. Biotechnol. Bioeng. 2017;114: 843-851. © 2016 Wiley Periodicals, Inc.

  6. Functional Consequences of Methionine Oxidation of hERG Potassium Channels

    PubMed Central

    Su, Zhi; Limberis, James; Martin, Ruth L.; Xu, Rong; Kolbe, Katrin; Heinemann, Stefan H.; Hoshi, Toshinori; Cox, Bryan F.; Gintant, Gary A.

    2010-01-01

    Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in hERG1 (human ether à go-go related gene 1), which encodes cardiac IKr channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 µM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to −60 or −100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or −10 mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel. PMID:17624316

  7. Towards methionine overproduction in Corynebacterium glutamicum--methanethiol and dimethyldisulfide as reduced sulfur sources.

    PubMed

    Bolten, Christoph J; Schröder, Hartwig; Dickschat, Jeroen; Wittmann, Christoph

    2010-08-01

    In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

  8. In vivo metabolism of L-methionine in mice: evidence for stereoselective formation of methionine-d-sulfoxide and quantitation of other major metabolites.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2006-12-01

    Flavin-containing monooxygenases (FMOs) 1-4 oxidize methionine (Met) to methionine sulfoxide (MetO). FMO3, the primary isoform expressed in adult human liver, has the lowest Km and favors methionine-d-sulfoxide (Met-d-O) formation over methionine-l-sulfoxide. Because female mice, but not males, also express FMO3 in liver, levels of Met and its major metabolites were determined in male or female mice dosed with 400 mg/kg Met i.p. The results show that Met levels in male and female mouse liver or plasma increased significantly at both 15 and 30 min after the Met treatment; Met plasma and liver levels at 30 min were similar to or lower than the corresponding levels at 15 min. Liver and plasma MetO levels increased significantly in both sexes at 30 min, and Met-d-O was the major MetO diastereomer detected. Interestingly, less than 0.1% of the Met dose was excreted in the urine (0-24 h) as Met and Met-d-O. S-Adenosylmethionine (SAM) was the major metabolite detected in liver at 15 min. Liver SAM levels at 30 min were lower than the levels at 15 min, and the plasma SAM levels at both 15 and 30 min were much lower than the corresponding levels in the liver. Increases in liver and/or plasma S-adenosyl-l-homocysteine, 5'-deoxy-5'-(methylthio)adenosine, and N-acetyl-l-methionine were also detected. Taken together, these results suggest that mice extensively and rapidly used the Met dose. Although mice exhibited increases in tissue MetO levels, a major role for FMO3 in Met-d-O formation is not certain since the MetO increases were mostly similar in both males and females.

  9. Methionine increases BDNF DNA methylation and improves memory in epilepsy

    PubMed Central

    Parrish, R Ryley; Buckingham, Susan C; Mascia, Katherine L; Johnson, Jarvis J; Matyjasik, Michal M; Lockhart, Roxanne M; Lubin, Farah D

    2015-01-01

    Objective Temporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus. Methods Using the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model. Results We found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade. Interpretation Our findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE. PMID:25909085

  10. Definitive Endoderm Differentiation of Human Embryonic Stem Cells Combined with Selective Elimination of Undifferentiated Cells by Methionine Deprivation.

    PubMed

    Tsuyama, Tomonori; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Human embryonic stem cells (ESCs) show a characteristic feature in that they are highly dependent on methionine metabolism. Undifferentiated human ESCs cannot survive under condition that methionine is deprived from culture medium. We describe here a procedure for definitive endoderm differentiation from human ESCs, in which human ESCs are subject to 10 days' (d) differentiation combined with methionine deprivation between differentiation days (d) 8 to (d) 10. Methionine deprivation results in elimination of undifferentiated cells from the culture with no significant loss of definitive endoderm cells, as compared to those cultured under complete condition throughout the whole culture period.

  11. Impact of food supplementation and methionine on high densities of cotton rats: Support of the amino-acid-quality hypothesis?

    USGS Publications Warehouse

    Webb, R.E.; Leslie, David M.; Lochmiller, R.L.; Masters, R.E.

    2005-01-01

    Considerable research supports the tenet that quantity and quality of food limit vertebrate populations. We evaluated predictions that increased availabilities of food and the essential amino acid methionine were related to population limitation of the hispid cotton rat (Sigmodon hispidus). Effects of supplemental food and methionine on density, survival, and reproductive parameters of wild cotton rats were assessed in north-central Oklahoma in 1998-1999. Twelve enclosed groups of 16 adult cotton rats each (8 male, 8 female) were randomly assigned to either no supplementation (control), supplementation with a mixed ration that had methionine at slightly below maintenance levels (0.20%), or a methionine-enhanced mixed ration (1.20%). In general, densities of cotton rats were twice as high and were sustained longer with dietary supplementation, and methionine-supplemented populations maintained the highest densities. Treatment effects on survival depended on time of year, with higher survival in supplemented enclosures in October and November. Per capita recruitment was highest with methionine-enhanced food. Treatment effects on proportions of overall and female cotton rats in reproductive condition depended on sampling date, but males were most reproductively active with methionine supplementation. Methionine supplementation resulted in an earlier and longer reproductive season. Density-dependent and density-independent factors no doubt interplay to determine population dynamics of cotton rats, but our results suggest that methionine plays a role in the population dynamics of wild cotton rats, apparently by enhancing overall density, recruitment, and reproductive activity of males.

  12. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity

    PubMed Central

    Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M.; Bortolato, Marco

    2015-01-01

    Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous SNP that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood posttranslational mechanisms. One posttranslational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, while brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined. PMID:24735585

  13. Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters.

    PubMed

    Bozzi, Aaron T; Bane, Lukas B; Weihofen, Wilhelm A; McCabe, Anne L; Singharoy, Abhishek; Chipot, Christophe J; Schulten, Klaus; Gaudet, Rachelle

    2016-09-13

    Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.

  14. Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

    PubMed Central

    Bozzi, Aaron T.; Bane, Lukas B.; Weihofen, Wilhelm A.; McCabe, Anne L.; Singharoy, Abhishek; Chipot, Christophe J.; Schulten, Klaus

    2016-01-01

    Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur’s presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it—more effectively than any other amino acid—increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium. PMID:27573840

  15. Parasites suppress immune-enhancing effect of methionine in nestling great tits.

    PubMed

    Wegmann, Michèle; Voegeli, Beatrice; Richner, Heinz

    2015-01-01

    After birth, an organism needs to invest both in somatic growth and in the development of efficient immune functions to counter the effects of pathogens, and hence an investment trade-off is predicted. To explore this trade-off, we simultaneously exposed nestling great tits (Parus major) to a common ectoparasite, while stimulating immune function. Using a 2 × 2 experimental design, we first infested half of the nests with hen fleas (Ceratophyllus gallinae) on day 3 post-hatch and later, on day 9-13 post-hatch, and then supplemented half of the nestlings within each nest with an immuno-enhancing amino acid (methionine). We then assessed the non-specific immune response by measuring both the inflammatory response to a lipopolysaccharide (LPS) and assessing the levels of acute phase proteins (APP). In parasite-infested nestlings, methionine had a negative effect on body mass close to fledging. Methionine had an immune-enhancing effect in the absence of ectoparasites only. The inflammatory response to LPS was significantly lower in nestlings infested with fleas and was also lower in nestlings supplemented with methionine. These patterns of immune responses suggest an immunosuppressive effect of ectoparasites that could neutralise the immune-enhancing effect of methionine. Our study thus suggests that the trade-off between investment in life history traits and immune function is only partly dependent on available resources, but shows that parasites may influence this trade-off in a more complex way, by also inhibiting important physiological functions.

  16. Methionine supplementation influences melanin-based plumage colouration in Eurasian kestrel, Falco tinnunculus, nestlings.

    PubMed

    Parejo, Deseada; Silva, Nadia

    2009-11-01

    The extent to which the expression of melanin-based plumage colouration in birds is genetically or environmentally determined is controversial. Here, we performed a between-nest design supplementation with either the sulphur amino acid dl-methionine or with water to investigate the importance of the non-genetic component of melanin-based plumage colouration in the Eurasian kestrel, Falco tinnunculus. Methionine affects growth and immunity, thus we aimed to modify nestling growth and immunity before feather development. Then, we measured the effect of the experiment on colouration of two melanin-based plumage patches of nestling kestrels. We found that methionine slowed down nestling growth through treatment administration and that nestlings compensated by speeding up their growth later. We did not find any effects of methionine on nestling immunity (i.e. lymphocyte counts, natural antibody levels or complement-mediated immunity). Effects on growth seemed to be mirrored by changes in nestling colouration in the two sexes: methionine-nestlings showed less intense brown plumage on their backs compared with control nestlings. These results provide support for a non-genetic determination of a melanin-based plumage patch in the two sexes of nestling kestrels.

  17. Methionine sulfoxides in serum proteins as potential clinical biomarkers of oxidative stress

    PubMed Central

    Suzuki, Satoko; Kodera, Yoshio; Saito, Tatsuya; Fujimoto, Kazumi; Momozono, Akari; Hayashi, Akinori; Kamata, Yuji; Shichiri, Masayoshi

    2016-01-01

    Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that the mass spectra intensity ratio of oxidized to non-oxidized methionine residues in serum tryptic proteins can be accurately quantified using a single drop of human serum and give stable and reproducible results. Our data demonstrate that two methionine residues in serum albumin (Met-111 and Met-147) are highly oxidized to methionine sulfoxide in patients with diabetes and renal failure and in healthy smokers versus non-smoker controls. This label-free mass spectrometry approach to quantify redox changes in methionine residues should facilitate the identification of additional circulating biomarkers suitable for predicting the development or progression of human diseases. PMID:27929071

  18. Methionine deprivation suppresses triple-negative breast cancer metastasis in vitro and in vivo

    PubMed Central

    Jang, Young Jin; Son, Joe Eun; Kwon, Jung Yeon; Lim, Tae-gyu; Kim, Sunghoon; Park, Jung Han Yoon; Kim, Jong-Eun; Lee, Ki Won

    2016-01-01

    Nutrient deprivation strategies have been proposed as an adjuvant therapy for cancer cells due to their increased metabolic demand. We examined the specific inhibitory effects of amino acid deprivation on the metastatic phenotypes of the human triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs 578T, as well as the orthotopic 4T1 mouse TNBC tumor model. Among the 10 essential amino acids tested, methionine deprivation elicited the strongest inhibitory effects on the migration and invasion of these cancer cells. Methionine deprivation reduced the phosphorylation of focal adhesion kinase, as well as the activity and mRNA expression of matrix metalloproteinases MMP-2 and MMP-9, two major markers of metastasis, while increasing the mRNA expression of tissue inhibitor of metalloproteinase 1 in MDA-MB-231 cells. Furthermore, methionine restriction downregulated the metastasis-related factor urokinase plasminogen activatior and upregulated plasminogen activator inhibitor 1 mRNA expression. Animals on the methionine-deprived diet showed lower lung metastasis rates compared to mice on the control diet. Taken together, these results suggest that methionine restriction could provide a potential nutritional strategy for more effective cancer therapy. PMID:27579534

  19. Methionine and S-Adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis

    PubMed Central

    Zubiete-Franco, Imanol; García-Rodríguez, Juan Luis; Martínez-Uña, Maite; Martínez-Lopez, Nuria; Woodhoo, Ashwin; Juan, Virginia Gutiérrez-De; Beraza, Naiara; Lage-Medina, Sergio; Andrade, Fernando; Fernandez, Marta Llarena; Aldámiz-Echevarría, Luis; Fernández-Ramos, David; Falcon-Perez, Juan Manuel; Lopitz-Otsoa, Fernando; Fernandez-Tussy, Pablo; Barbier-Torres, Lucía; Luka, Zigmund; Wagner, Conrad; García-Monzón, Carmelo; Lu, Shelly C.; Aspichueta, Patricia; Mato, José María; Martínez-Chantar, María Luz; Varela-Rey, Marta

    2015-01-01

    Background & Aims Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe NAFLD. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism. Methods We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice. Results We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and Methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameloriated liver steatosis. Conclusions These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis. PMID:26394163

  20. Dietary crude protein has minimal effect on the activity of selected enzymes of methionine catabolism in kittens fed diets near-limiting in methionine.

    PubMed

    Strieker, M J; Morris, J G; Avery, E H; Freedland, R A; Rogers, Q R

    2008-04-01

    Previous experiments have shown that increasing the dietary crude protein (CP) of cats does not increase urea cycle enzymes or alanine amino transferase as occurs in rats. Also when an essential amino acid (EAA) is limiting in a diet for growing kittens, the kittens do not exhibit an amino acid imbalance when other EAAs are added to the diet. To study the metabolic basis for these observations which are different from that found in omnivores and herbivores, the hypothesis that increased dietary CP decreases methionine catabolism, so more is spared for growth, was tested. Fifteen male kittens were randomly assigned to one of three dietary treatments. Each diet contained 2.5 g l-methionine/kg diet and 200, 300 or 500 g CP/kg diet. The livers and kidneys were removed and assayed for methionine transaminase (MTA), cystathionase (CASE) and cystathionine synthase (CS). Free amino acid concentrations were determined in liver, kidney and plasma. The 300 and 500 g CP/kg groups had significantly greater kidney weights and body weight gains than the 200 g CP/kg group. Hepatic MTA activity was lower in the 300 than the 200 or 500 g CP/kg groups (p < 0.05). Renal MTA and CASE activities were 35% and 50% greater, respectively, for the 500 g CP/kg group than for the 200 g CP/kg diet group (p < 0.05). Renal CS activities for the 300 and 500 g CP/kg groups were 29% (p > 0.05) and 38% (p < 0.05) greater, respectively, than the 200 g CP/kg group. Cyst(e)ine concentrations were lower in the livers of the 500 g CP/kg group than the 200 g CP/kg group (p < 0.05). Cystathionine was lower in plasma and kidney from the 500 g CP/kg diet group than from the 200 g CP/kg diet group (p < 0.05). It was concluded that the metabolic basis for the increased growth of kittens fed diets marginally limiting in methionine, with increasing concentrations of dietary CP, was not mediated through decreased enzyme activity associated with the catabolism of methionine, but was the result of an increase in

  1. Impact of Methionine Oxidation on Calmodulin Structural Dynamics

    PubMed Central

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin; Moen, Rebecca J.; Olenek, Michael J.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    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 population ranging from 1.5 to 3 nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM’s functional interaction with its target proteins by perturbing this Ca-dependent structural shift. PMID:25478640

  2. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

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

    NASA Astrophysics Data System (ADS)

    Drössler, 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.

  4. Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters.

    PubMed

    Marie, Benjamin; Joubert, Caroline; Belliard, Corinne; Tayale, Alexandre; Zanella-Cléon, Isabelle; Marin, Frédéric; Gueguen, Yannick; Montagnani, Caroline

    2012-05-01

    Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO3 and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.

  5. Methionine-mediated gene expression and characterization of the CmhR regulon in Streptococcus pneumoniae

    PubMed Central

    Afzal, Muhammad; Shafeeq, Sulman

    2016-01-01

    This study investigated the transcriptomic response of Streptococcus pneumoniae D39 to methionine. Transcriptome comparison of the S. pneumoniae D39 wild-type grown in chemically defined medium with 0–10 mM methionine revealed the elevated expression of various genes/operons involved in methionine synthesis and transport (fhs, folD, gshT, metA, metB-csd, metEF, metQ, tcyB, spd-0150, spd-0431 and spd-0618). Furthermore, β-galactosidase assays and quantitative RT-PCR studies demonstrated that the transcriptional regulator, CmhR (SPD-0588), acts as a transcriptional activator of the fhs, folD, metB-csd, metEF, metQ and spd-0431 genes. A putative regulatory site of CmhR was identified in the promoter region of CmhR-regulated genes and this CmhR site was further confirmed by promoter mutational experiments. PMID:28348831

  6. Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase*

    PubMed Central

    Samson, Andre L.; Knaupp, Anja S.; Kass, Itamar; Kleifeld, Oded; Marijanovic, Emilia M.; Hughes, Victoria A.; Lupton, Chris J.; Buckle, Ashley M.; Bottomley, Stephen P.; Medcalf, Robert L.

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of “nucleocytoplasmic coagulation.” Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a “linchpin” whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression. PMID:25086035

  7. Betaine supplementation is less effective than methionine restriction in correcting phenotypes of CBS deficient mice.

    PubMed

    Gupta, Sapna; Wang, Liqun; Kruger, Warren D

    2016-01-01

    Cystathionine beta synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Betaine supplementation, which can lower tHcy by stimulating homocysteine remethylation to methionine, is often given to CBS deficient patients in combination with other treatments such as methionine restriction and supplemental B-vitamins. However, the effectiveness of betaine supplementation by itself in the treatment of CBS deficiency has not been well explored. Here, we have examined the effect of a betaine supplemented diet on the Tg-I278T Cbs (-/-) mouse model of CBS deficiency and compared its effectiveness to our previously published data using a methionine restricted diet. Tg-I278T Cbs (-/-) mice on betaine, from the time of weaning until for 240 days of age, had a 40 % decrease in mean tHcy level and a 137 % increase in serum methionine levels. Betaine-treated Tg-I278T Cbs (-/-) mice also exhibited increased levels of betaine-dependent homocysteine methyl transferase (BHMT), increased levels of the lipogenic enzyme stearoyl-coenzyme A desaturase (SCD-1), and increased lipid droplet accumulation in the liver. Betaine supplementation largely reversed the hair loss phenotype in Tg-I278T Cbs (-/-) animals, but was far less effective than methionine restriction in reversing the weight-loss, fat-loss, and osteoporosis phenotypes. Surprisingly, betaine supplementation had several negative effects in control Tg-I278T Cbs (+/-) mice including decreased weight gain, lean mass, and bone mineral density. Our findings indicate that while betaine supplementation does have some beneficial effects, it is not as effective as methionine restriction for reversing the phenotypes associated with severe CBS deficiency in mice.

  8. Metabolic adaptations to methionine restriction that benefit health and lifespan in rodents.

    PubMed

    Perrone, Carmen E; Malloy, Virginia L; Orentreich, David S; Orentreich, Norman

    2013-07-01

    Restriction of dietary methionine by 80% slows the progression of aged-related diseases and prolongs lifespan in rodents. A salient feature of the methionine restriction phenotype is the significant reduction of adipose tissue mass, which is associated with improvement of insulin sensitivity. These beneficial effects of MR involve a host of metabolic adaptations leading to increased mitochondrial biogenesis and function, elevated energy expenditure, changes of lipid and carbohydrate homeostasis, and decreased oxidative damage and inflammation. This review summarizes observations from MR studies and provides insight about potential mediators of tissue-specific responses associated with MR's favorable metabolic effects that contribute to health and lifespan extension.

  9. Isotopic Dilution GC/MS Method for Methionine Determination in Biological Media

    NASA Astrophysics Data System (ADS)

    Horj, Elena; Iordache, Andreea; Culea, Monica

    2011-10-01

    The isotopic dilution mass spectrometry technique is the method of choice for sensitive and accurate determination of analytes in biological samples. The aim of this work was to establish a sensitive analytical method for the determination of methionine in different biological media. Quantitation of methionine from the resultant tracer spectrum requires deconvolution of the enrichment of the isotopomers. Deconvolution of the ion abundance ratios to yield tracer-to-tracee ratio for the isotopomer was done using Brauman's least squares approach. Comparison with regression curve calculation method is presented. The method was applied for amino-acids determination in beef, pork and fish meat.

  10. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  11. Modified bean seed protein phaseolin did not accumulate stably in transgenic tobacco seeds after methionine enhancement mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three dimensional structure...

  12. [Phenotypic and technological influences of the Lupinus mutabilis (Tarwi) seed on its methionine availability and sulfur content].

    PubMed

    Oliveros, M; Schoeneberger, H; Gross, R; Reynoso, Z

    1983-09-01

    The present study was carried out to determine the content of available methionine and sulphur in seed cultivars of Lupinus mutabilis from different Andean regions, and to study the influence of processing on methionine and sulphur contents. An additional objective was to evaluate interrelationships among these chemical characteristics and protein quality, as measured by the protein efficiency ratio (PER) method. Results revealed a high variability in the content of available methionine and sulphur between the different ecotypes and varieties of Lupinus mutabilis. Fertilization with CaSO4 (200 kg/ha) did alter the content of available methionine and sulphur in Lupinus albus seeds. Traditional water-debittering of lupines did not affect the methionine content of the seeds, whereas oil-extraction and alcohol-debittering led to a decrease in available methionine (14 and 23% reduction, respectively). Production of a protein isolate further reduced the methionine content (54%). Regression analysis revealed a high correlation between available methionine and sulphur (r = 0.83), between sulphur and PER (r = 0.98) in the processed lupine samples, and lupine mixtures with other protein sources.

  13. Metabolism of excess methionine in the liver of intact rat: an in vivo /sup 2/H NMR study

    SciTech Connect

    London, R.E.; Gabel, S.A.; Funk, A.

    1987-11-03

    L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo /sup 2/H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-(methyl-/sup 2/H/sub 3/)methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small /sup 2/H resonances assigned to N/sup 1/-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.

  14. The oxidation of methionine-54 of epoetinum alfa does not affect molecular structure or stability, but does decrease biological activity.

    PubMed

    Labrenz, Steven R; Calmann, Melissa A; Heavner, George A; Tolman, Glen

    2008-01-01

    Erythropoietin therapy is used to treat severe anemia in renal failure and chemotherapy patients. One of these therapies based on recombinant human erythropoietin is marketed under the trade name of EPREX and utilizes epoetinum alfa as the active pharmaceutical ingredient. The effect of oxidation of methionine-54 on the structure and stability of the erythropoietin molecule has not been directly tested. We have observed partial and full chemical oxidation of methionine-54 to methionine-54 sulfoxide, accomplished using tert-Butylhydroperoxide and hydrogen peroxide, respectively. A blue shift in the fluorescence center of spectral mass wavelength was observed as a linear response to the level of methionine sulfoxide in the epoetinum alfa molecule, presumably arising from a local change in the environment near tryptophan-51, as supported by potassium iodide quenching studies. Circular dichroism studies demonstrated no change in the folded structure of the molecule with methionine oxidation. The thermal unfolding profiles of partial and completely oxidized epoetinum alfa overlap, with a T(m) of 49.5 degrees C across all levels of methionine sulfoxide content. When the protein was tested for activity, a decrease in biological activity was observed, correlating with methionine sulfoxide levels. An allosteric effect between Met54, Trp51, and residues involved in receptor binding is proposed. These results indicate that methionine oxidation has no effect on the folded structure and global thermodynamic stability of the recombinant human erythropoietin molecule. Oxidation can affect potency, but only at levels significantly in excess of those seen in EPREX.

  15. Bioavailability of D-methionine relative to L-methionine for nursery pigs using the slope-ratio assay

    PubMed Central

    Kong, Changsu; Ahn, Jong Young

    2016-01-01

    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. PMID:27651987

  16. Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.

    PubMed

    Leahy, D J; Erickson, H P; Aukhil, I; Joshi, P; Hendrickson, W A

    1994-05-01

    Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.

  17. Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit.

    PubMed

    Gonda, Itay; Lev, Shery; Bar, Einat; Sikron, Noga; Portnoy, Vitaly; Davidovich-Rikanati, Rachel; Burger, Joseph; Schaffer, Arthur A; Tadmor, Ya'akov; Giovannonni, James J; Huang, Mingyun; Fei, Zhangjun; Katzir, Nurit; Fait, Aaron; Lewinsohn, Efraim

    2013-05-01

    Sulfur-containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur-containing and other volatiles. L-methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C- and ²H-labeled L-methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an L-methionine aminotransferase and preserves the main carbon skeleton of L-methionine. The second route apparently involves the action of an L-methionine-γ-lyase activity, releasing methanethiol, a backbone for formation of thiol-derived aroma volatiles. Exogenous L-methionine also generated non-sulfur volatiles by further metabolism of α-ketobutyrate, a product of L-methionine-γ-lyase activity. α-Ketobutyrate was further metabolized into L-isoleucine and other important melon volatiles, including non-sulfur branched and straight-chain esters. Cell-free extracts derived from ripe melon fruit exhibited L-methionine-γ-lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing L-methionine-γ-lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co-segregated with the levels of sulfur-containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that L-methionine is a precursor of both sulfur and non-sulfur aroma volatiles in melon fruit.

  18. Carbon-11-methionine and PET in evaluation of treatment response of breast cancer.

    PubMed Central

    Huovinen, R.; Leskinen-Kallio, S.; Någren, K.; Lehikoinen, P.; Ruotsalainen, U.; Teräs, M.

    1993-01-01

    Uptake of L-methyl-11C-methionine (11C-methionine) in breast cancer metastases was studied with positron emission tomography (PET). Eight patients with soft tissue metastases were studied twice: before the onset of chemotherapy (4), hormonal therapy (3) or radiotherapy (1) and 3-14 weeks later. The radioactivity concentration of the low molecular weight fraction of venous plasma samples separated by fast gel filtration was used as input function. The input corrected uptake rate of 11C-methionine (Ki) in breast cancer metastases before the treatment ranged between 0.035 and 0.186 1 min-1 and the standardised uptake value (SUV) between 2.0 and 11.4. The uptake of 11C-methionine into the metastases decreased when clinical objective stability or regression of the metastases was later obtained and increased in cases where progressive disease was seen during treatment. We conclude that metabolic changes in the amino acid metabolism detected by PET precede the clinical response, and may be of clinical value in predicting the treatment response. Images Figure 1 PMID:8471437

  19. Independent and additive effects of glutamic acid and methionine on yeast longevity.

    PubMed

    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.

  20. PET evaluation of spinal cord tumor using sup 11 C-methionine

    SciTech Connect

    Higano, S.; Shishido, F.; Nagashima, M.; Tomura, N.; Murakami, M.; Inugami, A.; Fujita, H.; Tabata, K.; Yasui, N.; Uemura, K. )

    1990-03-01

    A cervical cord tumor was examined with positron emission tomography using L-methyl-({sup 11}C)methionine. The radioactive tracer accumulated in the solid parts (but not in the associated cysts) of the neoplasm, which at histology was found to be an ependymoma.

  1. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    PubMed Central

    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

  2. Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pi...

  3. Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2†

    PubMed Central

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min−1 mg−1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases. PMID:9726878

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

    PubMed Central

    Pérez-Sepúlveda, Alejandra; España-Perrot, Pedro P.; Fernández B, Ximena; Ahumada, Verónica; Bustos, Vicente; Arraztoa, José Antonio; Dobierzewska, Aneta; Figueroa-Diesel, Horacio; Rice, Gregory E.; Illanes, Sebastián E.

    2013-01-01

    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. PMID:24024209

  5. Coupling Oxidative Signals to Protein Phosphorylation via Methionine Oxidation in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mechanisms involved in sensing oxidative signaling molecules such as H2O2 in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of methionine (Met) to Met sulfoxide (MetSO) can couple oxidative signals to changes in protein phosphor...

  6. Overexpression of methionine-R-sulfoxide reductases has no influence on fruit fly aging

    PubMed Central

    Shchedrina, Valentina A.; Vorbrüggen, Gerd; Cheon Lee, Byung; Kim, Hwa-Young; Kabil, Hadise; Harshman, Lawrence G.; Gladyshev, Vadim N.

    2009-01-01

    Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine residues in proteins. This function implicated Msrs in antioxidant defense and the regulation of aging. There are two known Msr types in animals: MsrA specific for the reduction of methionine-S-sulfoxide, and MsrB that catalyzes the reduction of methionine-R-sulfoxide. In a previous study, overexpression of MsrA in the nervous system of Drosophila was found to extend lifespan by 70%. Overexpression of MsrA in yeast also extended lifespan, whereas MsrB overexpression did so only under calorie restriction conditions. The effect of MsrB overexpression on lifespan has not yet been characterized in any animal model systems. Here, the GAL4-UAS binary system was used to drive overexpression of cytosolic Drosophila MsrB and mitochondrial mouse MsrB2 in whole body, fatbody, and the nervous system of flies. In contrast to MsrA, MsrB overexpression had no consistent effect on the lifespan of fruit flies on both corn meal and sugar yeast diets. Physical activity, fecundity, and stress resistance were also similar in MsrB-overexpressing and control flies. Thus, MsrA and MsrB, the two proteins with identical function in antioxidant protein repair, have different effects on aging in fruit flies. PMID:19409408

  7. Evaluating a quantitative methionine requirement for juvenile Pacific white shrimp Litopenaeus vannamei

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 10-wk feeding trial was conducted as a third study (all conducted in our laboratory) to determine a quantitative requirement of juvenile Litopenaeus vannamei for sulfur amino acid methionine. Juvenile shrimp (mean weight 0.61 +/- 0.13 g) were reared in 110-L aquaria in a seawater recirculating sy...

  8. Purification and biochemical characterization of methionine aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155.

    PubMed

    Narayanan, Sai Shyam; Ramanujan, Ajeena; Krishna, Shyam; Nampoothiri, Kesavan Madhavan

    2008-12-01

    The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.

  9. Prediction of (L)-methionine VCD spectra in the gas phase and water solution.

    PubMed

    Rode, Joanna E; Dobrowolski, Jan Cz; Sadlej, Joanna

    2013-11-21

    In this paper we provide a computational study of the l-methionine conformational landscape and VCD spectra in the gas phase and a water environment simulated by implicit PCM and the hybrid model, i.e., a combination of explicit "microsolvation" and implicit models. In the gas phase, two groups of conformers differing in H-bonding, i.e., OH···NH2 and NH···O═C, could be distinguished based solely on the IR ν(OH) and ν(NH) stretching vibrations range. On the other hand, VCD better reflected chain differences. The most stable OH···NH2 conformer was predicted to be easily detected, and the presence of two out of four NH···O═C conformers could be confirmed. Three zwitterionic methionine conformers were shown to dominate in water. Their VCD spectra, simulated within the hybrid model at the B3LYP-IEF-PCM/aug-cc-pVDZ level of theory, indicated that they could be recognized in the mixture. Use of the hybrid model is crucial for good reproduction of the hydrogen bonding pattern in the VCD spectra of methionine in water solution. However, the 1300-800 cm(-1) region of the skeleton vibrations of methionine appeared to be relatively insensitive to the model of the solvent.

  10. Convergent signaling pathways – interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxidation of Methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible post-translational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been establi...

  11. Characteristics of viable Brevibacterium linens cells, methionine and cysteine in milkfat-coated microcapsules.

    PubMed

    Kim, S C; Olson, N F

    1985-01-01

    The potential for microencapsulation of viable Brevibacterium linens with methionine or cysteine in milkfat to produce sulphur compounds was examined in this study. More than 80 per cent of B. linens cells were encapsulated and then numbers inside capsules increased about three-fold during 48 h at 26 degrees C under anaerobic conditions before a slow decline. Most of micro-organisms (7 x 10(7)/ml) were still viable in the capsules after 15 days. More than 90 per cent of cysteine and methionine were encapsulated and 90, 80 and 60 per cent of the encapsulated amino acids were maintained in the capsules at 4, 12 and 26 degrees C, respectively, after 24 h. Most of the cysteine was oxidized to cystine during microencapsulation but still available to micro-organisms whereas methionine remained in the reduced form. Partition coefficients of methionine and cysteine to milkfat were 0.117 and 0.275, respectively, indicating that most of these substrates would be available to cells of B. linens in the capsules.

  12. Purification and characterization of L-methionine gamma-lyase from brevibacterium linens BL2

    PubMed

    Dias; Weimer

    1998-09-01

    L-Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the alpha,gamma elimination of methionine to produce methanethiol, alpha-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25 degreesC but was active between 5 and 50 degreesC. Activity was inhibited by carbonyl reagents, completely inactivated by DL-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of L-methionine gamma-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 &mgr;mol min-1 mg-1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

  13. Independent and Additive Effects of Glutamic Acid and Methionine on Yeast Longevity

    PubMed Central

    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. PMID:24244480

  14. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    EPA Science Inventory

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
    S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE

    Stephen 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...

  15. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    EPA Science Inventory

    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...

  16. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    PubMed

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  17. Methionine as a potential precursor for halogenated compounds by the reaction with iron minerals

    NASA Astrophysics Data System (ADS)

    Tubbesing, C.; Krause, T.; Mulder, I.; Kotte, K.; Schöler, H. F.

    2012-04-01

    Volatile halogenated compounds (VOX) play an important role in different photochemical reactions within the troposphere and the stratosphere. Soils and sediments seem to act as a major natural source for VOX, but investigations of the reaction mechanisms are rather scarce. To get further information on potential intermediates the reaction of the amino acid methionine with the ferrous and ferric iron minerals pyrite and ferrihydrite as well as solute ferrous sulfate was studied using a gas chromatography-flame ionization detector (GC-FID). Methionine is an important amino acid in the biosynthesis of plants used as a starting compound for the messenger ethene with aminocyclopropane carboxylic acid as an intermediate product. This pathway may also occur under abiotic conditions. Ethene is assumed as precursor for various halogenated C2-compounds like vinyl chloride and dichloroethene. Due to its ubiquity by an average concentration of 10 to 290 ng/g soil and its potential to regenerate in soils and organic litter by microorganisms, methionine may be an important educt for both abiotic and biotic terrestrial halogenation processes. In laboratory tests methionine was exposed to different iron species like pyrite, iron sulfate or ferrihydrite. The oxidant H2O2 was used to start the reaction. Production values of methyl chloride and other halogenated compounds are discussed in the context of methionine as their potential precursor and several Fe-minerals as soil-borne catalysers. Several possible intermediates for the production of VOX have been detected e.g. methane, ethene or propane. A formation of isobutylene is noteworthy for some cases. In addition to VOC the production of methyl chloride and dimethyl sulfide (DMS) was observed. Only the DMS bears upon a specific mineral. The samples containing pyrite reveal the highest concentrations. To get a better assessment of methionine, respectively VOC released from methionine as precursors for halogenated compounds

  18. Influence of methionine oxidation on the aggregation of recombinant human growth hormone.

    PubMed

    Mulinacci, Filippo; Poirier, Emilie; Capelle, Martinus A H; Gurny, Robert; Arvinte, Tudor

    2013-09-01

    Oxidation of methionine (Met) residues is one of the major chemical degradations of therapeutic proteins. This chemical degradation can occur at various stages during production and storage of a biotherapeutic drug. During the oxidation process, the side chain of methionine residue undergoes a chemical modification, with the thioether group substituted by a sulfoxide group. In previous papers, we showed that oxidation of the two most accessible methionine residues of recombinant human growth hormone (r-hGH), Met¹⁴ and Met¹²⁵, has no influence on the conformation of the protein [1]. However, the oxidized r-hGH is less thermally stable than the native protein [2]. In the current work, the consequences of the oxidation of these two methionine residues on the aggregation of r-hGH were investigated. The aggregation properties and kinetics of the native and oxidized r-hGH were measured in different buffers with both spectroscopic and chromatographic methods. Stabilities of oxidized and non-oxidized r-hGH were studied after storage at 37°C and freeze/thawing cycles. Methionine oxidation influenced the aggregation properties of r-hGH. In accelerated stability studies at 37°C, oxidized hormone aggregated more and faster than non-oxidized hormone. In freezing/thawing stability studies, it was found that oxidized r-hGH was less stable than its non-oxidized counterpart. In case of hGH, we have shown that chemical degradations such as oxidation can affect its physical stability and can induce aggregation.

  19. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    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 {sup 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.

  20. Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig.

    PubMed

    Halsted, Charles H; Villanueva, Jesus A; Devlin, Angela M; Niemelä, Onni; Parkkila, Seppo; Garrow, Timothy A; Wallock, Lynn M; Shigenaga, Mark K; Melnyk, Stepan; James, S Jill

    2002-07-23

    Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2'-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.

  1. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: potential roles in toxicity.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2009-05-01

    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 degrees 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.

  2. THE DISTRIBUTION OF METHIONINE-ENKEPHALIN AND LEUCINE-ENKEPHALIN IN THE BRAIN AND PERIPHERAL TISSUES

    PubMed Central

    Hughes, J; Kosterlitz, HW; Smith, TW

    1997-01-01

    A method is described for the rapid extraction of opioid peptides from the brain and other tissues. The method is based on acid extraction of tissues followed by adsorption of the extract onto Amberlite XAD-2 resin. Elution with methanol separates the enkephalins and α-endorphin from β-endorphin. Over 90% of the opioid peptide activity isolated from brain and gut of several species by our method was due to methionine- and leucine-enkephalin. In contrast, the major opioid peptide activity recovered from the pituitary was due to peptides of much greater mol. wt. than the enkephalins. An opioid peptide with properties unlike those of the known endorphins or enkephalins was present in brain extracts. This peptide, termed ∈-endorphin, has an apparent mol. wt. of 700 to 1200; it constituted between 5 to 10% of the total opioid activity in our extracts. A differential assay of methionine- and leucine-enkephalin was made either by destroying methionine-enkephalin activity with cyanogen bromide or by separating the peptides by thin layer chromatography. The ratio of methionine-enkephalin to leucine-enkephalin varied greatly in different brain regions. The highest proportions of leucine-enkephalin were found in the cerebral cortex and hippocampus. Formaldehyde perfusion and fixation of the brain in vivo had no significant effect on the brain content of enkephalin, indicating that proteolytic breakdown is not a major problem in the extraction of these peptides. It is suggested that the enkephalins may have a neurotransmitter role in both brain and peripheral tissues and that methionine- and leucine-enkephalin may subserve separate neuronal functions. PMID:9142421

  3. Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts.

    PubMed

    Tan, Amelia W J; Lee, Pin-Rou; Seow, Yi-Xin; Ong, Peter K C; Liu, Shao-Quan

    2012-08-01

    Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of L: -methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with L: -methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different L: -methionine concentrations (0.00-0.20%) and under various inorganic sulphate (0.00-0.20%) and nitrogen (ammonia) supplementation (0.00-0.20%), respectively. Optimal VSC production was obtained with 0.1% of L: -methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of L: -methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-01

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

  6. The importance of transmethylation reactions to methionine metabolism in sheep: effects of supplementation with creatine and choline.

    PubMed

    Lobley, G E; Connell, A; Revell, D

    1996-01-01

    The influence of administering the methylated products choline and creatine on methionine irreversible-loss rate (ILR) and recycling from homocysteine has been investigated in sheep fed close to energy and N equilibrium. Two methods to estimate methionine recycling were compared. The first involved [U-13C]methionine infused as part of a labelled amino acid mixture obtained from hydrolysed algal protein. In this approach the isotope dilution of methionine with all five C atoms labelled (m + 5) will represent the ILR which does not recycle through homocysteine, while that which includes molecules with C-1-C-4 labelled will allow for loss of the labelled methyl (5)-C atom and replacement by an unlabelled moiety in the remethylation of homocysteine. The second method involved a combined infusion of [1-13C]- and [S-methyl-2H3]methionine. These two approaches gave similar data for methionine ILR which does not include label recycled to the amino acid from homocysteine but differed for recycled methionine fluxes. Consequently the two procedures differed in the calculated extent of homocysteine methylation under control conditions (6 v. 28%). These extents of remethylation are within the range observed for the fed human subject, despite the fact that fewer dietary methyl groups are available for the ruminant. Using combined data from the infusions, significant depression of methionine recycling occurred in blood (P < 0.05), with a similar trend for plasma (P = 0.077), when choline plus creatine were infused. Wool growth, assessed by intradermal injection of [35S]cysteine, was not altered by supplementation with the methylated products. From changes in the label pattern of free methionine in aortal, hepatic portal and hepatic venous blood during U-13C-labelled algal hydrolysate infusion, the major sites of homocysteine remethylation appear to be the portal-drained viscera and the liver. This was confirmed by analysis of free methionine enrichments in various tissues

  7. Quantitative study in vivo of methionine cycle in humans using (methyl-/sup 2/H/sub 3/)- and (1-/sup 13/C)methionine

    SciTech Connect

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

    1988-09-01

    Kinetic aspects of body methionine (MET) metabolism were examined in healthy young men during the fed and postabsorptive (PA) states. Rates of MET incorporation (S) into and release (B) from body proteins; transmethylation (TM); and remethylation (RM) and transsulfuration (TS) of homocysteine (HCY) were estimated with the aid of a 5-h constant intravenous infusion of (methyl-2H3)- and (1-13C)methionine. The isotopic data (plasma methionine labeling and 13C enrichment of expired air) were submitted to a stochastic model of amino acid metabolism. During the fed state, the subjects (n = 4) received, at 20-min intervals, small isonitrogenous isocaloric meals containing a complete L-amino acid mixture supplying MET at a rate equivalent to 198 mumol.kg body wt-1.day-1. The PA subjects (n = 4) received the isotope after a 10-h overnight fast. For the PA group, the components of MET metabolism were as follows: S, 20 +/- 0.5; B, 24 +/- 0.5; TM, 5.8 +/- 0.6; RM, 1.8 +/- 0.4; and TS, 4.0 +/- 0.4 (+/-SE) mumol.kg-1.h-1. During the fed state the values were S, 26 +/- 2.5; B, 18 +/- 2; TM, 14 +/- 1.3; RM, 5.7 +/- 0.9; and TS 8.3 +/- 0.6 mumol.kg-1.h-1. The meal-induced changes in B, TM, RM, and TS were significant (P less than 0.05). Comparison of the partitioning of MET between S and TM (these two pathways of MET disposal constitute the ''methionine locus'') in the PA and in the fed states indicates that the MET locus is of regulatory importance in MET homeostasis. A twofold increase in the partitioning of MET to TM was observed in the fed state. The increase in HCY recycling, relative to TS (these two pathways of HCY disposal constitute the ''HCY locus''), in the fed state did not reach statistical significance when compared with the PA state. Total daily TM are estimated to be 238 +/- 22 mumol/kg. This is similar to the estimate generated by the methyl balance model of Mudd and Poole which approximated 241 mumol/kg.

  8. Effect of dietary chromium-L-methionine on glucose metabolism of beef steers.

    PubMed

    Kegley, E B; Galloway, D L; Fakler, T M

    2000-12-01

    Thirty-six crossbred steers (288 +/- 3.7 kg initial BW) were used to determine the effect of Cr, as chromium-L-methionine, on glucose tolerance and insulin sensitivity in beef calves. Calves were fed a control diet or the diet supplemented with 400 or 800 microg Cr/kg of diet as chromium-L-methionine. Calves were kept in drylots (six calves/pen; two pens/dietary treatment). Steers were caught twice a day in locking headgates and individually fed their respective diets for a period of 22, 23, or 24 d prior to the metabolic challenges. Calves received a totally mixed diet containing 54% corn, 38% cottonseed hulls, and 5% soybean meal. On d 21, 22, and 23, four calves/dietary treatment were fitted with an indwelling jugular catheter. Approximately 24 h after catheterization, an intravenous glucose tolerance test (500 mg glucose/kg of BW), followed 5 h later by an intravenous insulin challenge test (0.1 IU insulin/kg of BW), was conducted. There was no effect (P > 0.10) of dietary treatment on ADG or ADFI. During the intravenous glucose tolerance test, serum insulin concentrations were increased by supplemental chromium-L-methionine (linear effect of Cr, P < 0.05). There was a time x treatment interaction (P < 0.05) on plasma glucose concentrations after the glucose infusion. Plasma glucose concentrations of calves fed 400 microg Cr/kg of diet were lower than those of controls and calves supplemented with 800 microg Cr/kg of diet (quadratic effect of Cr, P < 0.05) 5 and 10 min after the glucose infusion. Supplemental chromium-L-methionine increased the glucose clearance rate from 5 to 10 min after the insulin challenge test (linear effect of Cr, P < 0.05). Glucose half-life from 5 to 10 min after the insulin infusion was also decreased by supplemental chromium-L-methionine (linear effect of Cr, P < 0.10). These data indicate that supplemental Cr, as chromium-L-methionine, increased glucose clearance rate after an insulin infusion and increased the insulin response to

  9. Effects of methionine on the cytoplasmic distribution of actin and tubulin during neural tube closure in rat embryos

    PubMed Central

    Moephuli, Shadrack R.; Klein, Norman W.; Baldwin, Michael T.; Krider, Hallie M.

    1997-01-01

    Research has previously shown that, without methionine supplements, neural tube proteins of rat embryos cultured on bovine sera were hypomethylated and neural tubes failed to close. In the present study, to identify the proteins that became methylated during neurulation, rat embryos were first cultured on methionine-deficient bovine serum for 40 hr, then incubated with puromycin for 1 hr, and, finally, incubated with [methyl-14C]methionine and puromycin for 5 hr. On the basis of molecular weights, isoelectric points, and Western immunoblots, the methyl-14C-labeled proteins were identified as actin, αβ-tubulin, and neurofilament L. Indirect immunofluorescence studies indicated that without the addition of methionine to the culture, localization of actin and αβ-tubulin in the basal cytoplasm did not occur and these neuroepithelial cells lost their columnar morphology. PMID:9012820

  10. Identification and characterization of a methionine γ-lyase in the calicheamicin biosynthetic cluster of Micromonospora echinospora.

    PubMed

    Song, Haigang; Xu, Ri; Guo, Zhihong

    2015-01-02

    CalE6 is a previously uncharacterized protein involved in the biosynthesis of calicheamicins in Micromonospora echinospora. It is a pyridoxal-5'-phosphate-dependent enzyme and exhibits high sequence homology to cystathionine γ-lyases and cystathionine γ-synthases. However, it was found to be active towards methionine and to convert this amino acid into α-ketobutyrate, ammonium, and methanethiol. The crystal structure of the cofactor-bound holoenzyme was resolved at 2.0 Å; it contains two active site residues, Gly105 and Val322, specific for methionine γ-lyases. Modeling of methionine into the active site allows identification of the active site residues responsible for substrate recognition and catalysis. These findings support that CalE6 is a putative methionine γ-lyase producing methanethiol as a building block in biosynthesis of calicheamicins.

  11. One-pot modification of 5'-capped RNA based on methionine analogs.

    PubMed

    Muttach, Fabian; Rentmeister, Andrea

    2016-09-01

    This paper outlines chemically and enzymatically synthesized S-adenosylmethionine (AdoMet) analogs and their use in the site-specific modification of RNA by methyltransferases, enabling the facile attachment of clickable moieties to the nucleic acid. We then focus on methodological aspects of setting up a methyltransferase-based enzymatic cascade reaction starting from methionine analogs. This strategy is applied to the one-pot modification of the mRNA cap which is subsequently derivatized in copper-free and copper-catalyzed click reactions. We show that high transfer efficiencies to the cap are obtained using Se-propargyl-, hexenynyl- and azido-bearing methionine analogs. By switching to other methyltransferases our one-pot modification approach should be directly applicable to the regiospecific modification of other target molecules including nucleic acids, proteins and small molecules.

  12. Isolation and primary structure of a methionine- and cystine-rich seed protein of Cannabis sativa.

    PubMed

    Odani, S; Odani, S

    1998-04-01

    A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant food-stuffs.

  13. Stabilization of methionine-rich protein in Saccharomyces cerevisiae: targeting of BZN protein into the peroxisome.

    PubMed

    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.

  14. Structural Transitions Induced by a Recombinant Methionine-Trigger in Silk Spidroin

    NASA Astrophysics Data System (ADS)

    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.

  15. Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions.

    PubMed

    Chugha, Preeti; Sage, Harvey J; Oas, Terrence G

    2006-03-01

    Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

  16. A Methionine Deficient Diet Enhances Adipose Tissue Lipid Metabolism and Alters Anti-Oxidant Pathways in Young Growing Pigs

    PubMed Central

    Castellano, Rosa; Perruchot, Marie-Hélène; Conde-Aguilera, José Alberto; van Milgen, Jaap; Collin, Anne; Tesseraud, Sophie; Mercier, Yves; Gondret, Florence

    2015-01-01

    Methionine is a rate-limiting amino-acid for protein synthesis but non-proteinogenic roles on lipid metabolism and oxidative stress have been demonstrated. Contrary to rodents where a dietary methionine deficiency led to a lower adiposity, an increased lipid accretion rate has been reported in growing pigs fed a methionine deficient diet. This study aimed to clarify the effects of a dietary methionine deficiency on different aspects of tissue lipid metabolism and anti-oxidant pathways in young pigs. Post-weaned pigs (9.8 kg initial body weight) were restrictively-fed diets providing either an adequate (CTRL) or a deficient methionine supply (MD) during 10 days (n=6 per group). At the end of the feeding trial, pigs fed the MD diet had higher lipid content in subcutaneous adipose tissue. Expression levels of genes involved in glucose uptake, lipogenesis but also lipolysis, and activities of NADPH enzyme suppliers were generally higher in subcutaneous and perirenal adipose tissues of MD pigs, suggesting an increased lipid turnover in those pigs. Activities of the anti-oxidant enzymes superoxide dismutase, catalase and glutathione reductase were increased in adipose tissues and muscle of MD pigs. Expression level and activity of the glutathione peroxidase were also higher in liver of MD pigs, but hepatic contents in the reduced and oxidized forms of glutathione and glutathione reductase activity were lower compared with control pigs. In plasma, superoxide dismutase activity was higher but total anti-oxidant power was lower in MD pigs. These results show that a dietary methionine deficiency resulted in increased levels of lipogenesis and lipolytic indicators in porcine adipose tissues. Decreased glutathione content in the liver and coordinated increase of enzymatic antioxidant activities in adipose tissues altered the cellular redox status of young pigs fed a methionine-deficient diet. These findings illustrate that a rapidly growing animal differently adapts tissue

  17. Effects of methionine supplementation on the expression of oxidative stress-related genes in acute heat stress-exposed broilers.

    PubMed

    Del Vesco, Ana Paula; Gasparino, Eliane; Grieser, Daiane de Oliveira; Zancanela, Vittor; Soares, Maria Amélia Menck; Neto, Adhemar Rodrigues de Oliveira

    2015-02-28

    The aim of the present study was to evaluate the effects of heat stress (HS) and methionine supplementation on the markers of stress and on the gene expression levels of uncoupling proteins (UCP), betaine-homocysteine methyltransferase (BHMT), cystathionine β-synthase (CBS), glutathione synthetase (GSS) and glutathione peroxidase 7 (GPx7). Broilers from 1 to 21 d and from 22 to 42 d of age were divided into three treatment groups related to methionine supplementation: without methionine supplementation (MD); recommended level of methionine supplementation (DL1); excess methionine supplementation (DL2). The broilers were either kept at a comfortable thermal temperature or exposed to HS (38°C for 24 h). During the starter period, we observed the effects of the interaction between diet and environment on the gene expression levels of UCP, BHMT and GSS. Higher gene expression levels of UCP and BHMT were observed in broilers that were maintained at thermal comfort conditions and received the MD diet. HS broilers fed the DL1 and DL2 diets had the highest expression level of GSS. The expression levels of the CBS and GPx7 genes were influenced by both the environment and methionine supplementation. During the grower period, the gene expression levels of BHMT, CBS, GSS and GPx7 were affected by the diet × environment interaction. A higher expression level of BHMT was observed in broilers maintained at thermal comfort conditions and on the MD diet. HS induced higher expression levels of CBS, GSS and GPx7 in broilers that received the DL1 and DL2 diets. The present results suggest that under HS conditions, methionine supplementation could mitigate the effects of stress, since methionine contributed to the increased expression levels of genes related to antioxidant activity.

  18. Clinical Trials: D-Methionine to Reduce Noise-Induced Hearing Loss. Phase 3

    DTIC Science & Technology

    2014-03-01

    placebo-controlled Phase 3 clinical trial of oral D-met to reduce noise-induced hearing loss (NIHL) and tinnitus . The goal of the study is to...primary objective of this study is to determine the efficacy of D-Met in preventing NIHL or reducing tinnitus secondary to a minimum of 500 rounds...an oral, orange flavored suspension of D-methionine can prevent noise-induced hearing loss (NIHL) and tinnitus in our troops. Hypotheses

  19. Phase 3 Clinical Trials: D-Methionine to Reduce Noise-Induced Hearing Loss

    DTIC Science & Technology

    2013-03-01

    Phase 3 clinical trial of oral D-met to reduce noise-induced hearing loss (NIHL) and tinnitus . The goal of the study is to develop a safe, oral...this study is to determine the efficacy of D- Met in preventing NIHL or reducing tinnitus secondary to a minimum of 500 rounds of M-16 weapons training...and tinnitus in our troops. Hypotheses: Primary Hypothesis: Administration of oral D-methionine prior to and during weapons training will

  20. Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

    PubMed

    Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji

    2016-12-21

    l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

  1. Genetic Studies of Sulfadiazine-resistant and Methionine-requiring Neisseria Isolated From Clinical Material

    PubMed Central

    Catlin, B. Wesley

    1967-01-01

    Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met+) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met+). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met−). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B12, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met+ and Sul-r/Met− loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met− properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met− clones tested against recipients having nonidentical Sul-r/Met− mutant sites yielded Sul-s Met+ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species. Images PMID:4962305

  2. 11C-Methionine PET of Myocardial Inflammation in a Rat Model of Experimental Autoimmune Myocarditis.

    PubMed

    Maya, Yoshifumi; Werner, Rudolf A; Schütz, Claudia; Wakabayashi, Hiroshi; Samnick, Samuel; Lapa, Constantin; Zechmeister, Christina; Jahns, Roland; Jahns, Valérie; Higuchi, Takahiro

    2016-12-01

    Myocarditis represents a major cause of dilated cardiomyopathy and sudden cardiac death in younger adults. Currently, definitive diagnosis of myocarditis requires endomyocardial biopsy, which is highly invasive and has the drawback of variable sensitivity due to inherent sampling error. Therefore, reliable noninvasive methods to detect and monitor cardiac inflammation are clinically relevant. In this study, we explored the potential of radiolabeled methionine to assess myocardial inflammatory activity in a rat model of experimental autoimmune myocarditis (EAM).

  3. Biochemical and histologic presentations of female Wistar rats administered with different doses of paracetamol/methionine.

    PubMed

    Iyanda, A A; Adeniyi, F A A

    2011-12-20

    This study was carried out to compare the hepatoprotective effect of methionine on paracetamol treated rats at both the peaks of toxicity and absorption. Female Wistar rats were divided into 17 groups consisting of eight rats per group and treated with different doses of paracetamol/methionine (5:1). Each control rat received 5 ml of physiologic saline. The study was terminated at two different end points -the 4th and 16th hours. Results show that rats administered with toxic doses (1000 mg/kg, 3000 mg/kg, 5000 mg/kg BW) of paracetamol exhibited significant increases in the levels of ALT, AST, γ- GT compared with controls. These increases were much higher at the 16th than 4th hour but serum total protein, albumin and globulin were significantly decreased by the end of the 16th hour. Histology results of rats in the 3000 and 5000 mg/kg (by the end of the 16th hour) confirmed hepatic damage, light microscopic evaluation of liver showed remarkable centrilobular necrosis. Moreover, the presence of mononuclear cells in liver section of rats intoxicated with APAP (5000 mg/kg) suggests a possible involvement of inflammatory process which resulted in regurgitation of bilirubin leading to its elevated level as well as increase activity of ALP. The hepatoprotective effect of methionine, on the other hand, was demonstrated in these rats at the 4th and 16th hours, and both results were comparable and therefore not significantly different but elevation in GGT level still persisted. In conclusion, data obtained from this study suggest that these agents may be capable of inducing GGT, although further study is required to establish a possible relationship between methionine and this enzyme in some other animal species.

  4. Dietary B vitamin and methionine intakes and lung cancer risk among female never smokers in China

    PubMed Central

    Takata, Yumie; Cai, Qiuyin; Beeghly-Fadiel, Alicia; Li, Honglan; Shrubsole, Martha J.; Ji, Bu-Tian; Yang, Gong; Chow, Wong-Ho; Gao, Yu-Tang; Zheng, Wei; Shu, Xiao-Ou

    2012-01-01

    Purpose B vitamins and methionine have been postulated to have potential effects on carcinogenesis; however, findings from previous epidemiologic studies on B vitamins, methionine, and lung cancer risk are inconsistent. We investigated associations of dietary intakes of B vitamins (i.e., riboflavin, niacin, vitamin B6, folate, and vitamin B12) and methionine with lung cancer risk among female never smokers. Methods The Shanghai Women’s Health Study, a population-based, prospective cohort study, included 74,941 women. During a median follow-up of 11.2 years, 428 incident lung cancer cases accrued among 71,267 women with no history of smoking or cancer at baseline. Baseline dietary intakes were derived from a validated, interviewer-administered food frequency questionnaire. Cancer incidence and vital status were ascertained through annual linkage to the Shanghai Cancer Registry and Shanghai Vital Statistics Registry databases and through biennial in-person follow-ups with participants. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were calculated using Cox regression. Results Dietary riboflavin intake was inversely associated with lung cancer risk (HR = 0.62; 95% CI = 0.43–0.89; P-trend = 0.03 for the highest quartile compared with the lowest). A higher than median intake of methionine was associated with lower risk of lung cancer (HR = 0.78; 95% CI = 0.60–0.99), however, there was no dose-response relation. Intakes of other B vitamins were not associated with lung cancer risk. Conclusions Our study suggests that dietary riboflavin intake may be inversely associated with lung cancer risk among female never smokers, which warrants further investigation. PMID:23065072

  5. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    PubMed Central

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W.

    2010-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell.1,2 The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions.3,4 No prior 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 important new structural information about the HME sub-family 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. Intriguingly, 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, utilizing these methionine pairs/clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites. PMID:20865003

  6. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    SciTech Connect

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W.

    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.

  7. Evidence for distinct L-methionine catabolic pathways in the yeast Geotrichum candidum and the bacterium Brevibacterium linens.

    PubMed

    Arfi, Kenza; Landaud, Sophie; Bonnarme, Pascal

    2006-03-01

    Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and alpha-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.

  8. Predicting side-chain conformations of methionine using a hard-sphere model with stereochemical constraints

    NASA Astrophysics Data System (ADS)

    Virrueta, A.; Gaines, J.; O'Hern, C. S.; Regan, L.

    2015-03-01

    Current research in the O'Hern and Regan laboratories focuses on the development of hard-sphere models with stereochemical constraints for protein structure prediction as an alternative to molecular dynamics methods that utilize knowledge-based corrections in their force-fields. Beginning with simple hydrophobic dipeptides like valine, leucine, and isoleucine, we have shown that our model is able to reproduce the side-chain dihedral angle distributions derived from sets of high-resolution protein crystal structures. However, methionine remains an exception - our model yields a chi-3 side-chain dihedral angle distribution that is relatively uniform from 60 to 300 degrees, while the observed distribution displays peaks at 60, 180, and 300 degrees. Our goal is to resolve this discrepancy by considering clashes with neighboring residues, and averaging the reduced distribution of allowable methionine structures taken from a set of crystallized proteins. We will also re-evaluate the electron density maps from which these protein structures are derived to ensure that the methionines and their local environments are correctly modeled. This work will ultimately serve as a tool for computing side-chain entropy and protein stability. A. V. is supported by an NSF Graduate Research Fellowship and a Ford Foundation Fellowship. J. G. is supported by NIH training Grant NIH-5T15LM007056-28.

  9. Metformin Retards Aging in C. elegans by Altering Microbial Folate and Methionine Metabolism

    PubMed Central

    Cabreiro, Filipe; Au, Catherine; Leung, Kit-Yi; Vergara-Irigaray, Nuria; Cochemé, Helena M.; Noori, Tahereh; Weinkove, David; Schuster, Eugene; Greene, Nicholas D.E.; Gems, David

    2013-01-01

    Summary The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy—and also to its side effects, which include folate deficiency and gastrointestinal upset. PaperClip PMID:23540700

  10. Analysis of methionine synthase reductase polymorphism (A66G) in Indian Muslim population

    PubMed Central

    Rai, Vandana; Yadav, Upendra; Kumar, Pradeep; Yadav, Sushil Kumar

    2013-01-01

    BACKGROUND AND OBJECTIVES: Methionine synthase reductase (MTRR) is a vital enzyme of homocysteine/methionine metabolic pathway and is required for the conversion of inactive form of methionine synthase (MTR) to its active form. A clinically important allelic variant of MTRR A66G, with less enzymatic activity is reported with worldwide prevalence rate of ~ 30%. The present study was designed to determine the frequency of MTRR A66G polymorphism in rural Sunni Muslim population of Eastern Uttar Pradesh. MATERIALS AND METHODS: Total 56 subjects were analyzed for MTRR A66G polymorphism. A66G mutation analysis was carried out according to the polymerase chain reaction-restriction fragment length polymorphism method of Wilson et al. [1] amplification with MTRR specific primers followed by amplicon digestion with NdeI enzyme was used for the identification of different MTRR genotypes in subjects. RESULTS AND DISCUSSION: The AA genotype was found in 5 subjects, AG in 23 subjects, and GG genotype in 28 subjects. Genotype frequencies of AA, AG, and GG were 0.089, 0.41, and 0.5 respectively. The allele frequency of A allele was found to be 0.298 and G allele was 0.705. CONCLUSION: It is evident from the present study that the percentage of homozygous genotype GG and frequency of G allele is high in the target Muslim population. PMID:24019620

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

    PubMed Central

    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. 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. PMID:23401556

  12. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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.

  14. Stability of DNA methylation patterns in mouse spermatogonia under conditions of MTHFR deficiency and methionine supplementation.

    PubMed

    Garner, Justine L; Niles, Kirsten M; McGraw, Serge; Yeh, Jonathan R; Cushnie, Duncan W; Hermo, Louis; Nagano, Makoto C; Trasler, Jacquetta M

    2013-11-01

    Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome

  15. Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

    NASA Astrophysics Data System (ADS)

    Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

    2016-01-01

    Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

  16. Effects of Methionine Supplementation on the Expression of Protein Deposition-Related Genes in Acute Heat Stress-Exposed Broilers

    PubMed Central

    Grieser, Daiane Oliveira; Zancanela, Vittor; Voltolini, Débora Marques; Khatlab, Angélica Souza; Guimarães, Simone Eliza Facioni; Soares, Maria Amélia Menck; Neto, Adhemar Rodrigues Oliveira

    2015-01-01

    The objective of this study was to evaluate the effect of heat stress and methionine supplementation on the gene expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), phosphatidylinositol 3-kinase, and regulatory 1 (PI3KR1) in the liver, as well as the expression of the atrogin 1 and cathepsin L2 (CTSL2) genes in the breast muscle of broilers. Broilers from 1–21 and 22–42 days of age were divided into three treatments related to methionine supplementation as follows: without methionine supplementation (MD), recommended level of methionine (DL1), and excess supplementation of methionine (DL2). The animals were either maintained at a thermal comfort temperature or exposed to heat stress (HS) (38°C for 24 hours, starting on day 20 or day 41 for experiments 1 and 2, respectively). The heat stress increased the body temperature at both ages. Starter period: The HS animals presented increased plasma creatinine content (P<0.0001) and the highest CTSL2 gene expression (P<0.0001). The methionine supplementation increased the IGF-I (P = 0.0144) and GHR (P = 0.0011) gene expression and decreased the CTSL2 (P = 0.0004) and atrogin 1 (P = 0.0012) gene expression. Grower period: Significant effects for the interaction between supplementation and environment were observed for GHR (P = 0.0252) and CTSL2 (P = 0.0011) gene expression. The highest GHR expression was observed in animals that remained in thermal comfort on the DL2 diet, and the lowest expression occurred in the HS animals fed the MD diet. For CTSL2, the HS animals fed the MD diet presented the highest CTSL2 gene expression, and the lowest expression was observed in the animals maintained at thermal comfort on DL1 and DL2 diets. Only methionine supplementation had effect on atrogin-1 gene expression (P<0.0001), with higher methionine content in the diet lower atrogin-1 gene expression was observed. Our results suggest that heat stress induces greater protein degradation and that

  17. 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

    PubMed Central

    Chiba, Tsuyoshi; Suzuki, Sachina; Sato, Yoko; Itoh, Tatsuki; Umegaki, Keizo

    2016-01-01

    Aim 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. Methods 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. Results 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. Conclusions The 0.2% methionine in HFCD diet for 12 weeks was able to induce NASH without weight loss. PMID:27723801

  18. Mechanistic and Kinetic Study of Singlet O2 Oxidation of Methionine by On-Line Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Fangwei; Lu, Wenchao; Yin, Xunlong; Liu, Jianbo

    2016-01-01

    We report a reaction apparatus developed to monitor singlet oxygen (1O2) reactions in solution using on-line ESI mass spectrometry and spectroscopy measurements. 1O2 was generated in the gas phase by the reaction of H2O2 with Cl2, detected by its emission at 1270 nm, and bubbled into aqueous solution continuously. 1O2 concentrations in solution were linearly related to the emission intensities of airborne 1O2, and their absolute scales were established based on a calibration using 9,10-anthracene dipropionate dianion as an 1O2 trapping agent. Products from 1O2 oxidation were monitored by UV-Vis absorption and positive/negative ESI mass spectra, and product structures were elucidated using collision-induced dissociation-tandem mass spectrometry. To suppress electrical discharge in negative ESI of aqueous solution, methanol was added to electrospray via in-spray solution mixing using theta-glass ESI emitters. Capitalizing on this apparatus, the reaction of 1O2 with methionine was investigated. We have identified methionine oxidation intermediates and products at different pH, and measured reaction rate constants. 1O2 oxidation of methionine is mediated by persulfoxide in both acidic and basic solutions. Persulfoxide continues to react with another methionine, yielding methionine sulfoxide as end-product albeit with a much lower reaction rate in basic solution. Density functional theory was used to explore reaction potential energy surfaces and establish kinetic models, with solvation effects simulated using the polarized continuum model. Combined with our previous study of gas-phase methionine ions with 1O2, evolution of methionine oxidation pathways at different ionization states and in different media is described.

  19. Methionine choline reverses lead-induced cognitive and N-methyl-d-aspartate receptor subunit 1 deficits.

    PubMed

    Fan, Guangqin; Feng, Chang; Wu, Fengyun; Ye, Weiwei; Lin, Fen; Wang, Chunhong; Yan, Ji; Zhu, Gaochun; Xiao, Yuanmei; Bi, Yongyi

    2010-06-04

    The principal effects of Pb(2+) exposure in children are attention, memory and learning deficits that persist into adulthood. The application of the conventional chelators in children is somewhat prohibited by adverse health effects and is not effective in reversing learning deficits once they have occurred. In this study, we applied the nutrients, methionine and choline, to prevent Pb(2+)-induced cognitive impairment. Male weanling Sprague-Dawley rats were divided into five groups. Three groups of rats were exposed to Pb(2+) in drinking water containing 400mg/L Pb(2+) acetate, of which two groups were concurrently administered by oral gavage once a day, 6 days per week, with low or high doses of methionine and choline for 60 days. The normal control group received distilled water alone, and the reagent control received methionine choline chloride alone. Methionine choline treatment reversed long-term deficits in spatial learning and memory caused by Pb(2+) exposure in rats. Enhanced learning performance of Pb(2+)-exposed rats was associated with recovery of deficits in N-methyl-d-aspartate receptor (NMDAR) subunit 1 (NR1) mRNA and protein expression in the hippocampus. The effect of methionine choline on NR1 gene and protein expression was somewhat specific to Pb(2+)-exposed rats and did not affect the NR2A and NR2B subunits of the NMDAR measured in the same animals. Moreover, methionine choline treatment did not lower brain Pb(2+) content in Pb(2+)-exposed rats, although it reduced blood and bone Pb(2+) content. Methionine and choline reversed cognitive and NR1 deficits induced by Pb(2+) exposure, a beneficial effect that has significant clinical implications for the treatment of childhood Pb(2+) intoxication.

  20. Regulation of Protein Function by Reversible Methionine Oxidation and the Role of Selenoprotein MsrB1

    PubMed Central

    Kaya, Alaattin

    2015-01-01

    Abstract Significance: Protein structure and function can be regulated via post-translational modifications by numerous enzymatic and nonenzymatic mechanisms. Regulation involving oxidation of sulfur-containing residues emerged as a key mechanism of redox control. Unraveling the participants and principles of such regulation is necessary for understanding the biological significance of redox control of cellular processes. Recent Advances: Reversible oxidation of methionine residues by monooxygenases of the Mical family and subsequent reduction of methionine sulfoxides by a selenocysteine-containing methionine sulfoxide reductase B1 (MsrB1) was found to control the assembly and disassembly of actin in mammals, and the Mical/MsrB pair similarly regulates actin in fruit flies. This finding has opened up new avenues for understanding the use of stereospecific methionine oxidation in regulating cellular processes and the roles of MsrB1 and Micals in regulation of actin dynamics. Critical Issues: So far, Micals have been the only known partners of MsrB1, and actin is the only target. It is important to identify additional substrates of Micals and characterize other Mical-like enzymes. Future Directions: Oxidation of methionine, reviewed here, is an emerging but not well-established mechanism. Studies suggest that methionine oxidation is a form of oxidative damage of proteins, a modification that alters protein structure or function, a tool in redox signaling, and a mechanism that controls protein function. Understanding the functional impact of reversible oxidation of methionine will require identification of targets, substrates, and regulators of Micals and Msrs. Linking the biological processes, in which these proteins participate, might also lead to insights into disease conditions, which involve regulation of actin by Micals and Msrs. Antioxid. Redox Signal. 23, 814–822. PMID:26181576

  1. Mechanistic and Kinetic Study of Singlet O2 Oxidation of Methionine by On-Line Electrospray Ionization Mass Spectrometry.

    PubMed

    Liu, Fangwei; Lu, Wenchao; Yin, Xunlong; Liu, Jianbo

    2016-01-01

    We report a reaction apparatus developed to monitor singlet oxygen ((1)O2) reactions in solution using on-line ESI mass spectrometry and spectroscopy measurements. (1)O2 was generated in the gas phase by the reaction of H2O2 with Cl2, detected by its emission at 1270 nm, and bubbled into aqueous solution continuously. (1)O2 concentrations in solution were linearly related to the emission intensities of airborne (1)O2, and their absolute scales were established based on a calibration using 9,10-anthracene dipropionate dianion as an (1)O2 trapping agent. Products from (1)O2 oxidation were monitored by UV-Vis absorption and positive/negative ESI mass spectra, and product structures were elucidated using collision-induced dissociation-tandem mass spectrometry. To suppress electrical discharge in negative ESI of aqueous solution, methanol was added to electrospray via in-spray solution mixing using theta-glass ESI emitters. Capitalizing on this apparatus, the reaction of (1)O2 with methionine was investigated. We have identified methionine oxidation intermediates and products at different pH, and measured reaction rate constants. (1)O2 oxidation of methionine is mediated by persulfoxide in both acidic and basic solutions. Persulfoxide continues to react with another methionine, yielding methionine sulfoxide as end-product albeit with a much lower reaction rate in basic solution. Density functional theory was used to explore reaction potential energy surfaces and establish kinetic models, with solvation effects simulated using the polarized continuum model. Combined with our previous study of gas-phase methionine ions with (1)O2, evolution of methionine oxidation pathways at different ionization states and in different media is described.

  2. Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine.

    PubMed

    Altenbach, S B; Pearson, K W; Leung, F W; Sun, S S

    1987-05-01

    The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.

  3. Influence of dietary excess methionine on the relationship between dietary copper and the concentration of copper and iron in organs of broiler chicks.

    PubMed

    Ekperigin, H E; Vohra, P

    1981-09-01

    Four experiments were conducted to study the nature of the relationship between methionine, copper and iron. The day-old broiler chicks used were adapted to an isolated soyprotein-cornstarch diet containing 20% protein, 3,300 kcal metabolizable energy (ME)/kg, normal levels of minerals and 0.59% methionine. They were then fed the basal diet, alone or supplemented with 1.5% DL-methionine, 2,000 ppm molybdenum (Mo), 4,000 ppm Mo, or 100, 250, 500 or 1,000 ppm copper for 1 or 3 weeks. In contrast to 1.5% excess methionine, excess Mo increased plasma copper concentrations significantly, had no effect on spleen or liver iron and did not induce neurological changes. Dietary copper or methionine significantly influenced body weight gain, and the concentrations of copper in plasmas or livers. Supplements of 1.5% DL-methionine, 500 ppm or 1,000 ppm copper retarded body weight. A direct relationship between dietary copper, and the concentration of copper in livers or plasmas was demonstrated in chicks fed either the basal diet or the basal diet supplemented with 1.5% DL-methionine. However, chicks fed excess methionine had lower levels of copper in their plasmas and livers at every level of excess dietary copper. Liver and spleen iron concentrations were increased by 1.5% excess DL-methionine, but were not significantly altered by supplementary copper.

  4. Chronic administration of methionine and/or methionine sulfoxide alters oxidative stress parameters and ALA-D activity in liver and kidney of young rats.

    PubMed

    Soares, Mayara Sandrielly Pereira; Oliveira, Pathise Souto; Debom, Gabriela Nogueira; da Silveira Mattos, Bruna; Polachini, Carla Roberta; Baldissarelli, Jucimara; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina; Tavares, Rejane Giacomelli; Stefanello, Francieli Moro; Spanevello, Roselia Maria

    2017-01-01

    High levels of methionine (Met) and methionine sulfoxide (MetO) are found in several genetic abnormalities. Oxidative stress is involved in the pathophysiology of many inborn errors of metabolism. However, little is known about the role of oxidative damage in hepatic and renal changes in hypermethioninemia. We investigated the effect of chronic treatment with Met and/or MetO on oxidative stress parameters in liver and kidney, as lipid peroxidation (TBARS), total sulfhydryl content (SH), reactive oxygen species (ROS) and enzymes activities superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and delta aminolevulinic dehydratase (ALA-D). Serum biochemical parameters were evaluated. Wistar rats were treated daily with two subcutaneous injections of saline (control), Met (0.2-0.4 g/kg), MetO (0.05-0.1 g/kg) and the association between these (Met plus MetO) from the 6th to the 28th day of life. Our data demonstrated an increase of glucose and urea levels in all experimental groups. Cholesterol (MetO and Met plus MetO) were decreased and triglycerides (MetO) were increased. SOD (MetO and Met plus MetO) and CAT (Met, MetO and Met plus MetO) activities were decreased, while GPx was enhanced by MetO and Met plus MetO treatment in liver. In kidney, we observed a reduction of SH levels, SOD and CAT activities and an increase of TBARS levels in all experimental groups. ROS levels in kidney were increased in MetO and Met plus MetO groups. ALA-D activity was enhanced in liver (MetO and Met plus MetO) and kidney (Met plus MetO). These findings help to understand the pathophysiology of hepatic and renal alterations present in hypermethioninemia.

  5. S-adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3.

    PubMed

    Jayaram, Hariharan; Hoelper, Dominik; Jain, Siddhant U; Cantone, Nico; Lundgren, Stefan M; Poy, Florence; Allis, C David; Cummings, Richard; Bellon, Steven; Lewis, Peter W

    2016-05-31

    Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

  6. Discovery of a New Genetic Variant of Methionine Aminopeptidase from Streptococci with Possible Post-Translational Modifications: Biochemical and Structural Characterization

    PubMed Central

    Arya, Tarun; Kishor, Chandan; Saddanapu, Venkateshwarlu; Reddi, Ravikumar; Addlagatta, Anthony

    2013-01-01

    Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the ‘insert one’. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M. tuberculosis and human Type I MetAPs as well as the 60 amino acid insert in the human Type II enzyme suggesting possible common function. In addition, one of the β-hairpins within in the catalytic domain undergoes a flip placing a residue which is essential for enzyme activity away from the active site and the β-hairpin loop of this secondary structure in the active site obstructing substrate binding. This is the first example of a MetAP crystallizing in the inactive form. PMID:24124477

  7. Functioning methionine sulfoxide reductases A and B are present in human epidermal melanocytes in the cytosol and in the nucleus

    SciTech Connect

    Schallreuter, Karin U.; Chavan, Bhaven; Gillbro, Johanna M.

    2006-03-31

    Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for First time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.

  8. Endosperm protein synthesis and L-(/sup 35/S)methionine incorporation in maize kernels cultured in vitro

    SciTech Connect

    Cully, D.E.; Gengenbach, B.G.; Smith, J.A.; Rubenstein, I.; Connely, J.A.; Park, W.D.

    1984-02-01

    This study was conducted to examine protein synthesis and L-(/sup 35/S)methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries L-(/sup 35/S)methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endosperm for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages. Sodium, dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing patterns for endosperm proteins were similar for field and cultured kernels throughout development. By 15 days, over 70% of the L-(/sup 35/S)methionine taken up was present in endosperm proteins. Label incorporation visualized by fluorography generally followed the protein intensity of the stained gels. The high methionine content, low molecular weight zeins (i.e. 15 and 9 kilodaltons) were highly labeled. All of the radioactivity in hydrolyzed zein samples was recovered in the methionine peak indicating minimal conversion to L-(/sup 35/S)cysteine. The procedure described here is suitable for long term culture and labeling experiments in which continued kernel development is required.

  9. Effects of calcium soaps of rapeseed fatty acids and protected methionine on milk yield and composition in dairy cows.

    PubMed

    Kowalski, Z M; Pisulewski, P M; Spanghero, M

    1999-11-01

    The objective of this study was to determine the effects of supplementing the diets of dairy cows with Ca soaps of rapeseed fatty acids (CSRFA) and rumen-protected (RP) methionine on their milk yield and composition, including milk protein fractions and fatty acids. Twelve Polish Red Lowland cows were used in a complete balanced two period changeover experiment. The four treatment diets were a control consisting of a total mixed ration of grass silage and concentrates, and the total mixed ration supplemented with RP methionine, CSRFA or RP methionine plus CSRFA. Dry matter intake was not affected by diet. Milk yield increased when cows were given the diet with CSRFA, but supplementation of diets with RP methionine did not affect milk yield. Milk protein content, but not milk protein yield, decreased when CSRFA was given. The addition of RP methionine to the control diet and the CSRFA diet produced similar increases in the milk protein content. Supplementation of the diet with CSRFA significantly changed the milk fatty acid profile: the proportions of 10:0, 12:0, 14:0, 15:0 and 16:0 in milk fat decreased, but those of 18:0 and cis-18:1 increased. We conclude that CSRFA can be used in practical dairy diets to increase milk yield and manipulate its fatty acid composition.

  10. Immune function and hematology of male cotton rats (Sigmodon hispidus) in response to food supplementation and methionine

    USGS Publications Warehouse

    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. ?? 2003 Elsevier Inc. All rights reserved.

  11. Insights into function, catalytic mechanism, and fold evolution of selenoprotein methionine sulfoxide reductase B1 through structural analysis.

    PubMed

    Aachmann, Finn L; Sal, Lena S; Kim, Hwa-Young; Marino, Stefano M; Gladyshev, Vadim N; Dikiy, Alexander

    2010-10-22

    Methionine sulfoxide reductases protect cells by repairing oxidatively damaged methionine residues in proteins. Here, we report the first three-dimensional structure of the mammalian selenoprotein methionine sulfoxide reductase B1 (MsrB1), determined by high resolution NMR spectroscopy. Heteronuclear multidimensional spectra yielded NMR spectral assignments for the reduced form of MsrB1 in which catalytic selenocysteine (Sec) was replaced with cysteine (Cys). MsrB1 consists of a central structured core of two β-sheets and a highly flexible, disordered N-terminal region. Analysis of pH dependence of NMR signals of catalytically relevant residues, comparison with the data for bacterial MsrBs, and NMR-based structural analysis of methionine sulfoxide (substrate) and methionine sulfone (inhibitor) binding to MsrB1 at the atomic level reveal a mechanism involving catalytic Sec(95) and resolving Cys(4) residues in catalysis. The MsrB1 structure differs from the structures of Cys-containing MsrBs in the use of distal selenenylsulfide, residues needed for catalysis, and the mode in which the active form of the enzyme is regenerated. In addition, this is the first structure of a eukaryotic zinc-containing MsrB, which highlights the structural role of this metal ion bound to four conserved Cys. We integrated this information into a structural model of evolution of MsrB superfamily.

  12. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter - substrate binding protein complex

    PubMed Central

    Nguyen, Phong T.; Li, Qi Wen; Kadaba, Neena S.; Lai, Jeffrey Y.; Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ~40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system. PMID:25803078

  13. Methionine requirements of male white Peking ducks from twenty-one to forty-nine days of age.

    PubMed

    Xie, M; Hou, S S; Huang, W

    2006-04-01

    A dose-response experiment with 6 dietary methionine levels (0.20, 0.275, 0.35, 0.425, 0.50, and 0.575%) was conducted with male White Peking ducklings to estimate the methionine requirement of growing ducks from 21 to 49 d of age. One-day-old male White Peking ducklings were fed common starter diets from hatching to 21 d of age and then fed the experimental diets from 21 to 49 d of age. Three hundred thirty-six 21-d-old birds were allotted to 24 pens with 14 birds per pen according to similar pen weight. There were 6 dietary treatments, each containing 4 replicate pens. At 49 d of age, weight gain, feed intake, and feed/gain of ducks from each pen were measured, and 2 ducks selected randomly from each pen were slaughtered to evaluate the yields of abdominal fat, breast meat (including pectoralis major and pectoralis minor), and leg meat (including thigh and drum stick). Significant effects of dietary methionine on weight gain, breast meat, and abdominal fat were observed. Both weight gain and breast meat yield showed significant quadratic response to increasing dietary methionine, and abdominal fat decreased linearly (P < 0.05). According to the quadratic model, the optimal methionine requirement of male White Peking ducks from 21 to 49 d of age for maximum weight gain and breast meat yield were 0.377 and 0.379%, respectively.

  14. Effects of diet supplementation with white tea and methionine on lipid metabolism of gilthead sea bream juveniles (Sparus aurata).

    PubMed

    Pérez-Jiménez, Amalia; Peres, Helena; Rubio, Vera Cruz; Oliva-Teles, Aires

    2013-06-01

    A growth trial was performed with gilthead sea bream juveniles (Sparus aurata) to evaluate the effect of diet supplementation with white tea and methionine on fish performance and lipid metabolism. For that purpose, four diets were formulated: a fish meal-based diet (Control) and diets identical to the control diet but supplemented with 2.9 % white tea (Tea), 0.3 % methionine (Met) or 2.9 % white tea plus 0.3 % methionine (Tea + Met). Growth performance and feed efficiency parameters, whole-body and liver composition, plasma metabolites concentration and liver glucose 6-phosphate dehydrogenase (G6PDH), malic enzyme (ME) and fatty acid synthetase (FAS) activities were determined. Feed intake was higher in fish fed methionine-supplemented diets, whereas this parameter and growth was decreased in fish fed white tea supplementation. Feed efficiency and protein efficiency ratio were not affected by diet composition. Plasma HDL cholesterol and total lipids concentration were higher in fish fed white tea-supplemented diets. Whole-body lipid, plasma glucose, liver glycogen concentration and liver G6PDH, ME and FAS activities were lower in fish fed white tea-supplemented diets. Results of the present study indicate that methionine seems to act as a feed attractant in diets for sea bream juveniles. Additionally, white tea is an important modulator of lipid metabolism in sea bream juveniles.

  15. Methionine oxidation within the cerebroside-sulfate activator protein (CSAct or Saposin B).

    PubMed Central

    Whitelegge, J. P.; Penn, B.; To, T.; Johnson, J.; Waring, A.; Sherman, M.; Stevens, R. L.; Fluharty, C. B.; Faull, K. F.; Fluharty, A. L.

    2000-01-01

    The cerebroside-sulfate activator protein (CSAct or Saposin B) is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed-phase high-performance liquid chromatography (RP-HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. High-resolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal. PMID:11045609

  16. Methionine sulfoxides on PrPSc: a prion-specific covalent signature.

    PubMed

    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.

  17. Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

    PubMed Central

    Elmallah, Mohammed IY; Borgmeyer, Uwe; Betzel, Christian; Redecke, Lars

    2013-01-01

    Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrPC into the infectious misfolded isoform PrPSc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrPSc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders. PMID:24121542

  18. Computational Analysis of Cysteine and Methionine Metabolism and Its Regulation in Dairy Starter and Related Bacteria

    PubMed Central

    Liu, Mengjin; Prakash, Celine; Nauta, Arjen; Siezen, Roland J.

    2012-01-01

    Sulfuric volatile compounds derived from cysteine and methionine provide many dairy products with a characteristic odor and taste. To better understand and control the environmental dependencies of sulfuric volatile compound formation by the dairy starter bacteria, we have used the available genome sequence and experimental information to systematically evaluate the presence of the key enzymes and to reconstruct the general modes of transcription regulation for the corresponding genes. The genomic organization of the key genes is suggestive of a subdivision of the reaction network into five modules, where we observed distinct differences in the modular composition between the families Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, on the one hand, and the family Streptococcaceae, on the other. These differences are mirrored by the way in which transcription regulation of the genes is structured in these families. In the Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, the main shared mode of transcription regulation is methionine (Met) T-box-mediated regulation. In addition, the gene metK, encoding S-adenosylmethionine (SAM) synthetase, is controlled via the SMK box (SAM). The SMK box is also found upstream of metK in species of the family Streptococcaceae. However, the transcription control of the other modules is mediated via three different LysR-family regulators, MetR/MtaR (methionine), CmbR (O-acetyl[homo]serine), and HomR (O-acetylhomoserine). Redefinition of the associated DNA-binding motifs helped to identify/disentangle the related regulons, which appeared to perfectly match the proposed subdivision of the reaction network. PMID:22522891

  19. Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion.

    PubMed

    Elmallah, Mohammed I Y; Borgmeyer, Uwe; Betzel, Christian; Redecke, Lars

    2013-01-01

    Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP(C) into the infectious misfolded isoform PrP(Sc). Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP(Sc) destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121-231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.

  20. Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate.

    PubMed

    Peskin, A V; Winterbourn, C C

    2001-03-01

    Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.

  1. Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

    PubMed

    Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

    2008-12-01

    The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

  2. Structural Basis for the Differential Regulation of DNA by the Methionine Repressor MetJ

    SciTech Connect

    Augustus, Anne; Reardon, Patrick; Heller, William T; Spicer, Leonard D.

    2006-01-01

    The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.

  3. Nutritional and technological evaluation of an enzymatically methionine-enriched soy protein for infant enteral formulas.

    PubMed

    de Regil, Luz María; de la Barca, Ana María Calderón

    2004-03-01

    Enzymatically modified soy proteins have the amino acid profile and functional properties required for dietary support. The objective of this study was to evaluate the nutritional and technological properties of an enzymatically modified soy protein ultrafiltered fraction with bound methionine (F(1-10)E) to be used as a protein ingredient for infant enteral formulas. F(1-10)E was chemically characterized and biologically evaluated. Thirty-six weaning Wistar rats were fed during 3 weeks with a 4% casein-containing diet. Rats were divided into three groups and recovered for 3 weeks with 18% protein-containing diets based on: (1) F(1-10)E, (2) casein or (3) soy isolate+methionine. Nutritional indicators were weight gain, protein efficiency ratio, plasma proteins, apparent digestibility and protein in the carcass. Additionally, F(1-10)E was added as a protein ingredient of an enteral formula, and its sensory and rheological properties were compared with a hydrolyzed-whey protein commercial formula. F(1-10)E contained 68% protein and 5% sulphur amino acids, with 60% of peptides 0.05) in weight gain (108 g and 118 g, respectively), protein efficiency ratio (2.7), apparent digestibility (93% and 95%), plasma proteins (5.7 mg/100 ml) and carcass protein (61%), and better than soy isolate-based+methionine diet (P<0.05). Viscosity of the commercial formula and our formula was similar during a 24-h period. Sensory acceptability was 8 for our formula and 3.5 for the commercial one, on a scale of 1-10 (P<0.05). Due to its nutritional, sensorial and rheological properties, F(1-10)E could be used as a protein source in infant enteral formulas.

  4. Methionine one-electron oxidation: Coherent contributions from radiolysis, IRMPD spectroscopy, DFT calculations and electrochemistry

    NASA Astrophysics Data System (ADS)

    Scuderi, Debora; Bergès, Jacqueline; de Oliveira, Pedro; Houée-Levin, Chantal

    2016-11-01

    Methionine is an essential amino acid, unfortunately prone to oxidation. The mechanism of its oxidation by •OH radicals has been studied for more than 40 years and still remains misunderstood. We have reinvestigated the oxidation of this residue in model peptides, aiming at i) improving the identification of free radicals by the use of more modern quantum chemistry methods; ii) reinvestigating the one-electron reduction potentials as a function of the position in the sequence; iii) identifying the final compounds, which were still unknown; iv) reinvestigating the intramolecular electron transfer (IET) involving this residue.

  5. Protective role of S-adenosyl-L-methionine against hydrochloric acid stress in Saccharomyces cerevisiae.

    PubMed

    Malakar, Dipankar; Dey, Anindya; Ghosh, Anil K

    2006-09-01

    S-adenosyl-L-methionine (AdoMet, 1mM) protects the stationary phase cells of Saccharomyces cerevisiae against the killing effect of acid (10mM HCl, pH approximately 2). Both the acid and the acid plus AdoMet treatment for 2h increased the plasma membrane H(+)-ATPase activity; thereafter it decreased to the basal level. AdoMet partially recovered the intracellular pH (pH(in)) that dropped in presence of acid. AdoMet treatment facilitated acid induced phospholipid biosynthesis as well as membrane proliferation, which was reflected in the cellular lipid composition.

  6. Catalytic Promiscuity of the Radical S-adenosyl-L-methionine Enzyme NosL

    PubMed Central

    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. PMID:27446906

  7. New metabolic labelling medium for Trichomonas vaginalis and Tritrichomonas foetus using 35S methionine

    SciTech Connect

    Torian, B.E.; Kenny, G.E.

    1986-04-01

    A metabolic labelling medium was devised for Trichomonas vaginalis and Tritrichomonas foetus utilizing 35S methionine. T. vaginalis cultured for 24h in the medium took up approximately 27% of the available label and increased greater than two fold in number. Counts per microgram of protein were 32,555 +/- 10% between different strains or identical strains in different labelling runs. T. foetus took up approximately 5% of the available label and increased greater than two fold in 24h. This resulted in specific labelling of 12,704 cpm/ug protein +/- 10% between different runs with the same strain.

  8. Identification of the molecular basis of inhibitor selectivity between the human and streptococcal type I methionine aminopeptidases.

    PubMed

    Arya, Tarun; Reddi, Ravikumar; Kishor, Chandan; Ganji, Roopa Jones; Bhukya, Supriya; Gumpena, Rajesh; McGowan, Sheena; Drag, Marcin; Addlagatta, Anthony

    2015-03-12

    The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound 15) that selectively inhibits the S. pneumonia MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the S. pneumonia MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity.

  9. Cobalamin inactivation by nitrous oxide produces severe neurological impairment in fruit bats: protection by methionine and aggravation by folates

    SciTech Connect

    van der Westhuyzen, J.; Fernandes-Costa, F.; Metz, J.

    1982-11-01

    Nitrous oxide, which inactivates cobalamin when administered to fruit bats, results in severe neurological impairment leading to ataxia, paralysis and death. This occurs after about 6 weeks in animals depleted of cobalamin by dietary restriction, and after about 10 weeks in cobalamin replete bats. Supplementation of the diet with pteroylglutamic acid caused acceleration of the neurological impairment--the first unequivocal demonstration of aggravation of the neurological lesion in cobalamin deficiency by pteroylglutamic acid. The administration of formyltetrahydropteroylglutamic acid produced similar aggravation of the neurological lesion. Supplementation of the diet with methionine protected the bats from neurological impairment, but failed to prevent death. Methionine supplementation protected against the exacerbating effect of folate, preventing the development of neurological changes. These findings lend support to the hypothesis that the neurological lesion in cobalamin deficiency may be related to a deficiency in the methyl donor S-adenosylmethionine which follows diminished synthesis of methionine.

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

    SciTech Connect

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

    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°.

  11. Mulberry (Morus L.) methionine sulfoxide rreductase gene cloning, sequence analysis, and expression in plant development and stress response.

    PubMed

    Tong, Wei; Zhang, Yinghua; Wang, Heng; Li, Feng; Liu, Zhaoyue; Wang, Yuhua; Fang, Rongjun; Zhao, Weiguo; Li, Long

    2013-01-01

    Methionine sulfoxide reductase plays a regulatory role in plant growth and development, especially in scavenging reactive oxygen species by restoration of the oxidation of methionine in protein. A full-length cDNA sequence encoding methionine sulfoxide reductase (MSR) from mulberry, which we designated MMSR, was cloned based on mulberry expressed sequence tags (ESTs). Sequence analysis showed that the MMSR is 810 bp long, encoding 194 amino acids with a predicted molecular weight of 21.6 kDa and an isoelectric point of 6.78. The expression level of the MMSR gene under conditions of drought and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.

  12. The in vivo sparing of methionine by cysteine in sulfur amino acid requirements in animal models and adult humans.

    PubMed

    Ball, Ronald O; Courtney-Martin, Glenda; Pencharz, Paul B

    2006-06-01

    Sulfur amino acid metabolism has been receiving increased attention because of the link to chronic diseases such as cardiovascular disease, Alzheimer's disease, and diabetes. In addition, the role of cysteine and optimal intakes for physiological substrates such as glutathione are currently of considerable interest in human health. Although the dietary indispensability of methionine is not in question, the ability of cysteine to substitute for a portion of its requirement has been the topic of much debate. Methionine is often the most limiting amino acid in the diets of the developing world's population because of its low concentration in cereal grains. Therefore, the ability of cysteine to substitute for methionine requirement is not just biologically interesting; it is also of considerable economic and social importance. The primary goal of this review is to discuss the available evidence on the effect of cysteine substitution for methionine to meet the total sulfur amino acid requirement in adult humans, including an assessment of the methodological features of experiments with conflicting results. Assessment of the requirement experiments for amino acids with complex metabolism such as methionine and cysteine must begin with a careful definition of requirements and what substitution means. As a result of these definitions, a set of criteria for the intakes of methionine that will allow demonstration of the substitution effect have been developed. Some recent publications are assessed using these definitions and criteria, and a possible reason for the conflicting results in the literature is proposed. An approach to estimating tolerable upper intakes is also proposed. Research on in vivo sulfur amino acid metabolism in humans is tremendously difficult, and therefore, we do not wish to be overly critical of the high-quality work of the ambitious and highly intelligent men and women who have conducted various studies. Our goal is to objectively review the data for

  13. YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli.

    PubMed

    Liu, Qian; Liang, Yong; Zhang, Yun; Shang, Xiuling; Liu, Shuwen; Wen, Jifu; Wen, Tingyi

    2015-11-01

    Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical "5 + 5" transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.

  14. YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

    PubMed Central

    Liu, Qian; Liang, Yong; Zhang, Yun; Shang, Xiuling; Liu, Shuwen; Wen, Jifu

    2015-01-01

    Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering. PMID:26319875

  15. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Yamagata, Wataru; Kamei, Kaeko; Nozaki, Tomoyoshi; Harada, Shigeharu

    2006-10-01

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å{sup 3} Da{sup −1}. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

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

    SciTech Connect

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

    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.

  17. Formation of volatile sulfur compounds and metabolism of methionine and other sulfur compounds in fermented food.

    PubMed

    Landaud, Sophie; Helinck, Sandra; Bonnarme, Pascal

    2008-01-01

    The formation of volatile sulfur compounds (VSC) in fermented food is a subject of interest. Such compounds are essential for the aroma of many food products like cheeses or fermented beverages, in which they can play an attractive or a repulsive role, depending on their identity and their concentration. VSC essentially arise from common sulfur-bearing precursors, methionine being the most commonly found. In the first section of this paper, the main VSC found in cheese, wine, and beer are reviewed. It is shown that a wide variety of VSC has been evidenced in these food products. Because of their low odor threshold and flavor notes, these compounds impart essential sensorial properties to the final product. In the second section of this review, the main (bio)chemical pathways leading to VSC synthesis are presented. Attention is focused on the microbial/enzymatic phenomena-which initiate sulfur bearing precursors degradation-leading to VSC production. Although chemical reactions could also play an important role in this process, this aspect is not fully developed in our review. The main catabolic pathways leading to VSC from the precursor methionine are presented.

  18. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  19. Engineered Citrobacter freundii methionine γ-lyase effectively produces antimicrobial thiosulfinates.

    PubMed

    Morozova, Elena A; Kulikova, Vitalia V; Rodionov, Alexei N; Revtovich, Svetlana V; Anufrieva, Natalya V; Demidkina, Tatyana V

    2016-01-01

    Antimicrobial activity of thiosulfinates in situ produced by mixtures of Citrobacter freundii methionine γ-lyase (MGL) with new substrates, l-methionine and S-(alkyl/allyl)-l-cysteine sulfoxides has been recently demonstrated (Anufrieva et al., 2015). This opens a way to the rational design of a new biotechnologically relevant antimicrobial drug producer. To increase the efficiency of the enzyme toward sulfoxides, the mutant forms of MGL, with the replacements of active site cysteine 115 with alanine (C115A MGL) and histidine (C115H MGL) were obtained. The replacement of cysteine 115 by histidine results in the loss of activity of the mutant enzyme in the γ-elimination reaction of physiological substrate, whereas the activity in the β-elimination reaction of characteristic substrates persists. However, the catalytic efficiency of C115H MGL in the β-elimination reaction of S-substituted l-cysteine sulfoxides is increased by about an order of magnitude compared to the wild type MGL. The antibacterial activity of C115H MGL mixtures with a number of sulfoxides was assessed against Gram-positive and Gram-negative bacteria. The bacteriostatic effect was more pronounced against Gram-positive than against Gram-negative bacteria, while antibacterial potential proved to be quite similar. Thus, the mutant enzyme C115H MGL is an effective catalyst, in particular, for decomposition of sulfoxides and the pharmacological couples of the mutant form with sulfoxides might be new antimicrobial agents.

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

    SciTech Connect

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

    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.

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

    PubMed

    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.

  2. Calorie restriction and methionine restriction in control of endogenous hydrogen sulfide production by the transsulfuration pathway

    PubMed Central

    Hine, Christopher; Mitchell, James R.

    2015-01-01

    H2S is a gas easily identified by its distinctive odor. Although environmental exposure to H2S has been viewed alternately as therapeutic or toxic through the centuries, H2S has recently regained recognition for its numerous beneficial biological effects. Most experiments documenting such benefits, including improved glucose tolerance, increased stress resistance, and even lifespan extension, are based on exposure of experimental organisms to exogenous sources of H2S. However, appreciation is growing for the importance of H2S produced endogenously by the evolutionary conserved transsulfuration pathway (TSP) in health and longevity. Recent data implicate H2S produced by the TSP in pleiotropic benefits of dietary restriction (DR), or reduced nutrient/energy intake without malnutrition. DR, best known as the most reliable way to extend lifespan in a wide range of experimental organisms, includes various regimens aimed at either reducing overall calorie intake (calorie restriction, intermittent/ every-other-day fasting) or reducing particular nutrients such as protein or the essential amino acid, methionine (methionine restriction), with overlapping functional benefits on stress resistance, metabolic fitness and lifespan. Here we will review the small but growing body of literature linking the TSP to the functional benefits of DR in part through the production of endogenous H2S, with an emphasis on regulation of the TSP and H2S production by diet and mechanisms of beneficial H2S action. PMID:25523462

  3. Betaine homocysteine S-methyltransferase emerges as a new player of the nuclear methionine cycle.

    PubMed

    Pérez-Miguelsanz, Juliana; Vallecillo, Néstor; Garrido, Francisco; Reytor, Edel; Pérez-Sala, Dolores; Pajares, María A

    2017-03-10

    The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.

  4. Whey protein supplementation increases methionine intake but not homocysteine plasma concentration in rats.

    PubMed

    Deminice, Rafael; Comparotto, Hugo; Jordao, Alceu Afonso

    2015-01-01

    The purpose of this study was to examine the effects of whey protein supplementation on homocysteine (Hcy) metabolism and liver oxidative stress in rats. Twenty-four rats were divided into 3 groups (n = 8) to receive one of the following diets for 4 weeks: control diet (C), whey protein-composed diet (WP), and whey protein-supplemented diet (WPS). The C and WP diets consisted of AIN-93 with 20% casein and 20% whey protein as protein source, respectively. WPS was AIN-93 (20% casein) supplemented by the addition of 20% (w/w) whey protein. Four weeks of ingesting a WPS diet resulted in a significantly higher (P < 0.05) total protein and methionine intakes. Although a significant increase (P < 0.05) in the hepatic S-adenosylmethionine and S-adenosylhomocysteine levels occurred in WPS group compared with C and WP, no significant change was observed in plasma Hcy concentration between groups. Furthermore, the levels of lipid hydroperoxides and advanced oxidation protein products, known liver oxidative stress markers, were increased in the WPS group compared with the C group. In addition, no change in glutathione liver concentration was observed in any of the groups studied. In conclusion, whey protein supplementation increases methionine intake substantially; however, it does not change plasma Hcy concentrations. On the other hand, increased hepatic oxidative stress markers were observed in whey protein supplemented rats were probably due to high protein intake.

  5. Evidence for the dimerization-mediated catalysis of methionine sulfoxide reductase A from Clostridium oremlandii.

    PubMed

    Lee, Eun Hye; Lee, Kitaik; Kwak, Geun-Hee; Park, Yeon Seung; Lee, Kong-Joo; Hwang, Kwang Yeon; Kim, Hwa-Young

    2015-01-01

    Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.

  6. Expression and biochemical characterization of a type I methionine aminopeptidase of Plasmodium vivax.

    PubMed

    Kang, Jung-Mi; Ju, Jung-Won; Kim, Jung-Yeon; Ju, Hye-Lim; Lee, Jinyoung; Lee, Kon Ho; Lee, Won-Ja; Sohn, Woon-Mok; Kim, Tong-Soo; Na, Byoung-Kuk

    2015-04-01

    Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Plasmodium vivax (PvMetAP1). PvMetAP1 had the typical amino acid residues essential for metal binding and substrate binding sites, which are well conserved in the type I MetAP family enzymes. Recombinant PvMetAP1 showed activity in a broad range of neutral pHs, with optimum activity at pH 7.5. PvMetAP1 was stable under neutral and alkaline pHs, but was relatively unstable under acidic conditions. PvMetAP1 activity was highly increased in the presence of Mn(2+), and was effectively inhibited by a metal chelator, EDTA. Fumagillin and aminopeptidase inhibitors, amastatin and bestatin, also showed an inhibitory effect on PvMetAP1. The enzyme had a highly specific hydrolytic activity for N-terminal methionine. These results collectively suggest that PvMetAP1 belongs to the family of type I MetAPs and may play a pivotal role for the maintenance of P. vivax physiology by mediating protein maturation and processing of the parasite.

  7. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  8. Association of methionine synthase rs1801394 and methionine synthase reductase rs1805087 polymorphisms with meningioma in adults: A meta-analysis

    PubMed Central

    ZENG, XIAN-TAO; LU, JUN-TI; TANG, XIANG-JUN; WENG, HONG; LUO, JIE

    2014-01-01

    Several epidemiological studies suggested that methionine synthase (MTRR) rs1801394 and methionine synthase reductase (MTR) rs1805087 polymorphisms may be involved in the risk of meningioma in adults; however, the results from different case-control studies have been inconsistent. Therefore, we performed a meta-analysis to investigate the association of MTRR and MTR polymorphisms with meningioma. PubMed, Web of Knowledge, China National Knowledge Infrastructure and Wanfang databases were searched up to October 30, 2013 and 3 publications, involving 7 case-control studies, were finally included. Following data extraction, a meta-analysis was conducted using Stata 12.0 software. The pooled results based on the fixed effects model demonstrated that the MTRR rs1801394 polymorphism was associated with an increased risk of meningioma [odds ratio (OR)=1.18, 95% confidence interval (CI): 1.05–1.32 for G vs. A; OR=1.41, 95% CI: 1.12–1.77 for GG vs. AA; OR=1.08, 95% CI: 0.94–1.33 for AG vs. AA; OR=1.19, 95% CI: 1.01–1.40 for (AG+GG) vs. AA; and OR=1.32, 95% CI: 1.07–1.63 for GG vs. (AG+AA)]; however, an association between the MTR rs1805087 polymorphism and the risk of meningioma was not identified [OR=0.99, 95% CI: 0.88–1.12 for G vs. A; OR=1.09, 95% CI: 0.80–1.48 for GG vs. AA; OR=0.95, 95% CI: 0.82–1.11 for AG vs. AA; OR=0.97, 95% CI: 0.84–1.13 for (AG+GG) vs. AA; and OR=1.09, 95% CI: 0.80–1.48 for GG vs. (AG+AA)]. Therefore, the currently available evidence suggests that the MTRR rs1801394 polymorphism may increase the risk of meningioma, whereas the MTRR rs1801394 polymorphism is not associated with meningioma. PMID:24748989

  9. Methionine Sulfoxide Reductase A Negatively Controls Microglia-Mediated Neuroinflammation via Inhibiting ROS/MAPKs/NF-κB Signaling Pathways Through a Catalytic Antioxidant Function

    PubMed Central

    Fan, Hua; Wu, Peng-Fei; Zhang, Ling; Hu, Zhuang-Li; Wang, Wen; Guan, Xin-Lei; Luo, Han; Ni, Ming; Yang, Jing-Wen; Li, Ming-Xing

    2015-01-01

    Abstract Aims: Oxidative burst is one of the earliest biochemical events in the inflammatory activation of microglia. Here, we investigated the potential role of methionine sulfoxide reductase A (MsrA), a key antioxidant enzyme, in the control of microglia-mediated neuroinflammation. Results: MsrA was detected in rat microglia and its expression was upregulated on microglial activation. Silencing of MsrA exacerbated lipopolysaccharide (LPS)-induced activation of microglia and the production of inflammatory markers, indicating that MsrA may function as an endogenous protective mechanism for limiting uncontrolled neuroinflammation. Application of exogenous MsrA by transducing Tat-rMsrA fusion protein into microglia attenuated LPS-induced neuroinflammatory events, which was indicated by an increased Iba1 (a specific microglial marker) expression and the secretion of pro-inflammatory cytokines, and this attenuation was accompanied by inhibiting multiple signaling pathways such as p38 and ERK mitogen-activated protein kinases (MAPKs) and nuclear factor kappaB (NF-κB). These effects were due to MsrA-mediated reactive oxygen species (ROS) elimination, which may be derived from a catalytic effect of MsrA on the reaction of methionine with ROS. Furthermore, the transduction of Tat-rMsrA fusion protein suppressed the activation of microglia and the expression of pro-inflammatory factors in a rat model of neuroinflammation in vivo. Innovation: This study provides the first direct evidence for the biological significance of MsrA in microglia-mediated neuroinflammation. Conclusion: Our data provide a profound insight into the role of endogenous antioxidative defense systems such as MsrA in the control of microglial function. Antioxid. Redox Signal. 22, 832–847. PMID:25602783

  10. Analyses of Fruit Flies That Do Not Express Selenoproteins or Express the Mouse Selenoprotein, Methionine Sulfoxide Reductase B1, Reveal a Role of Selenoproteins in Stress Resistance*

    PubMed Central

    Shchedrina, Valentina A.; Kabil, Hadise; Vorbruggen, Gerd; Lee, Byung Cheon; Turanov, Anton A.; Hirosawa-Takamori, Mitsuko; Kim, Hwa-Young; Harshman, Lawrence G.; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2011-01-01

    Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with 75Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms. PMID:21622567

  11. Gene polymorphisms involved in folate and methionine metabolism and increased risk of sporadic colorectal adenocarcinoma.

    PubMed

    Guimarães, José Luiz Miranda; Ayrizono, Maria de Lurdes; Coy, Cláudio Saddy Rodrigues; Lima, Carmen Silvia Passos

    2011-10-01

    This pilot study has compared the polymorphic genotype frequencies of methylenetetrahydrofolate reductase (MTHFR A1298C and C677T), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G), and thymidylate synthase (TS 2R/3R) in 113 patients with sporadic colorectal adenocarcinoma (SCA) and 188 healthy blood donors, used as matched controls. The aim was to assess the role of these genotypes in the increased risk of SCA among the southeastern Brazilian population. Carriers of genotype MTRR 66GG, or the combined variants MTHFR 1298AC + CC plus 677CT + TT, or MTHFR 677CT + TT plus MTR 2756AG + GG, or MTHFR 1298AC + CC plus 677CT + TT plus MTR 2756AG + GG, or yet, MTHFR 1298AC + CC plus 677CT + TT plus MTRR 66AG + GG, respectively, showed an increased risk of the order of 1.99-, 3.26-, 2.22-, 10.92-, and 14.88-fold of developing SCA when compared with carriers of the other studied polymorphic genotypes, whether in isolation or in combination. In addition, individuals with the MTHFR 677CT + TT or the MTR 2756AG + GG genotypes had a 2.12- and a 1.42-fold increased risks of SCA onset before 50 years of age. African-Brazilians with the MTRR 66GG genotype had a 1.98-fold increased risk of SCA while individuals with the MTR 2756AG + GG and the MTHFR 677CT + TT genotypes showed a 2.11- and a 1.62-fold increased risk of undifferentiated and advanced tumors at diagnosis, respectively. Carriers of genotype MTHFR 1298AC + CC or MTHFR 1298AC + CC plus MTRR 66AG + GG had a 1.42- and a 3.07-fold increased risk of rectal tumor, respectively. Additionally, carriers of MTHFR 677CT + TT or MTHFR 677CT + TT plus TS 2R/3R + 3R/3R had a 1.55- and a 5.39-fold increased risk for colon tumor, respectively, in comparison with carriers of the wild genotypes. These data suggest that all polymorphisms coding for folate and methionine-dependent enzymes, particularly when present in combination with

  12. Dimethylsulfoniopropionate and methanethiol are important precursors of methionine and protein-sulfur in marine bacterioplankton.

    PubMed

    Kiene, R P; Linn, L J; González, J; Moran, M A; Bruton, J A

    1999-10-01

    Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [(35)S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [(35)S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the alpha-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine gamma-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-(3)H]MeSH and [(35)S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a

  13. Methionine sulfoxide reductase A affects β-amyloid solubility and mitochondrial function in a mouse model of Alzheimer's disease

    PubMed Central

    Du, Fang; Bowman, Connor F.; Yan, Shirley S.

    2016-01-01

    Accumulation of oxidized proteins, and especially β-amyloid (Aβ), is thought to be one of the common causes of Alzheimer's disease (AD). The current studies determine the effect of an in vivo methionine sulfoxidation of Aβ through ablation of the methionine sulfoxide reductase A (MsrA) in a mouse model of AD, a mouse that overexpresses amyloid precursor protein (APP) and Aβ in neurons. Lack of MsrA fosters the formation of methionine sulfoxide in proteins, and thus its ablation in the AD-mouse model will increase the formation of methionine sulfoxide in Aβ. Indeed, the novel MsrA-deficient APP mice (APP+/MsrAKO) exhibited higher levels of soluble Aβ in brain compared with APP+ mice. Furthermore, mitochondrial respiration and the activity of cytochrome c oxidase were compromised in the APP+/MsrAKO compared with control mice. These results suggest that lower MsrA activity modifies Aβ solubility properties and causes mitochondrial dysfunction, and augmenting its activity may be beneficial in delaying AD progression. PMID:26786779

  14. Oxidation of methionine in PrP is dependent upon the oxidant and the amino acid two positions removed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. Methionine oxidation has been shown both to be associated with prion formation and implicated in the inhibition of amyloid formation in model systems. This work is based on model systems where hydrogen peroxide was used as an oxidant. Materials and Methods. We developed...

  15. Mice fed a lipogenic methionine-choline-deficient diet develop hypermetabolism coincident with hepatic suppression of SCD-1.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipogenic diets that are completely devoid of methionine and choline (MCD) induce hepatic steatosis. MCD feeding also provokes systemic weight loss, for unclear reasons. In this study, we found that MCD feeding causes profound hepatic suppression of the gene encoding stearoyl-coenzyme A desaturase-1...

  16. Feeding rumen-protected methionine pre- and post-partum increases milk protein content and yield in early lactation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to evaluate the effects of feeding rumen-protected methionine (MET) from 23 d (±12) before calving until 98 days in milk (DIM) on lactation performance, dry matter intake (DMI), body condition score (BCS) and body weight (BW) change of dairy cows. Multiparous Holstein cows (n = 223) ...

  17. Effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich d-zein in transgenic soybeans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of heterologous methionine-rich proteins to increase the overall sulfur amino acid content of soybean seeds has been only marginally successful, presumably due to low accumulation of transgenes in soybeans. Proteome rebalancing of seed proteins has been shown to promote the accumulation o...

  18. Zinc Methionine Supplementation Impacts Gene and Protein Expression in Calf-fed Holstein Steers with Miniaml Impact on Feedlot Performance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calf-fed Holstein steers were supplemented with a zinc (Zn) methionine supplement (ZnMet; ZINPRO®; Zinpro Corporation, Eden Prairie, MN) for 115±5 days prior to harvest along with zilpaterol hydrochloride (ZH; Zilmax®; Merck Animal Health, Summit, NJ) for the last 20 days with a 3 day withdrawal to ...

  19. Analysis of Methionine Oxidation in Myosin Isoforms in Porcine Skeletal Muscle by LC-MS/MS Analysis

    PubMed Central

    Jeong, Jin-Yeon; Jung, Eun-Young; Jeong, Tae-Chul; Yang, Han-Sul; Kim, Gap-Don

    2016-01-01

    The purpose of this study was to analyze oxidized methionines in the myosin isoforms of porcine longissimus thoracis, psoas major, and semimembranosus muscles by liquid chromatography (LC) and mass spectrometry (MS). A total of 836 queries matched to four myosin isoforms (myosin-1, -2, -4, and -7) were analyzed and each myosin isoform was identified by its unique peptides (7.3-13.3). Forty-four peptides were observed from all three muscles. Seventeen peptides were unique to the myosin isoform and the others were common peptides expressed in two or more myosin isoforms. Five were identified as oxidized peptides with one or two methionine sulfoxides with 16 amu of mass modification. Methionines on residues 215 (215), 438 (438), 853 (851), 856 (854), 1071 (1069), and 1106 (1104) of myosin-1 (myosin-4) were oxidized by the addition of oxygen. Myosin-2 had two oxidized methionines on residues 215 and 438. No queries matched to myosin-7 were observed as oxidized peptides. LC-MS/MS allows analysis of the oxidation of specific amino acids on specific residue sites, as well as in specific proteins in the food system. PMID:27194935

  20. Comparative toxicity of selenate, selenite, seleno-DL-methionine and seleno-DL-cystine to Daphnia magna

    SciTech Connect

    Maier, K.J.; Foe, C.G.; Knight, A.W. )

    1993-04-01

    Elevated concentrations of the trace element selenium (Se) have resulted in the degradation of several aquatic ecosystems. This study evaluated the comparative toxicity of several aqueous chemical species of selenium to an aquatic cladoceran, Daphnia magna. Responses to mixtures of these selenium forms, varying the sulfate concentration, were also examined. Initial experiments compared the toxicity of aqueous forms of selenate, selenite, seleno-DL-methionine, and seleno-DL-cystine to neonate Daphnia magna, resulting in 4-h LC50 values of 2.84, 0.55, 0.31, and 2.01 mg Se per liter, respectively. Immobilization was an acute sublethal response observed during exposure to the organic selenium forms only. The 48-h IC50 values were 0.045 and 0.52 mg Se per liter for seleno-DL-methionine and seleno-DL-cystine, respectively. Evaluation of the invertebrate response to various combinations of selenate, selenite, and seleno-DL-methionine demonstrated that the toxicities of these forms of selenium are additive. Increasing the concentration of sulfate decreased, varied, and left unaffected the toxicities of selenate, selenite, and seleno-DL-methionine, respectively. These results indicate that both the chemical form of selenium and the sulfate concentration can influence the toxicity of selenium.

  1. Protection against UVB-induced oxidative stress in human skin cells and skin models by methionine sulfoxide reductase A.

    PubMed

    Pelle, Edward; Maes, Daniel; Huang, Xi; Frenkel, Krystyna; Pernodet, Nadine; Yarosh, Daniel B; Zhang, Qi

    2012-01-01

    Environmental trauma to human skin can lead to oxidative damage of proteins and affect their activity and structure. When methionine becomes oxidized to its sulfoxide form, methionine sulfoxide reductase A (MSRA) reduces it back to methionine. We report here the increase in MSRA in normal human epidermal keratinocytes (NHEK) after ultraviolet B (UVB) radiation, as well as the reduction in hydrogen peroxide levels in NHEK pre-treated with MSRA after exposure. Further, when NHEK were pre-treated with a non-cytotoxic pentapeptide containing methionine sulfoxide (metSO), MSRA expression increased by 18.2%. Additionally, when the media of skin models were supplemented with the metSO pentapeptide and then exposed to UVB, a 31.1% reduction in sunburn cells was evident. We conclude that the presence of MSRA or an externally applied peptide reduces oxidative damage in NHEK and skin models and that MSRA contributes to the protection of proteins against UVB-induced damage in skin.

  2. Influence of oxygen and pH on methanethiol production from L-methionine by Brevibacterium lines CNRZ 918

    SciTech Connect

    Ferchichi, M.; Hemme, D.; Bouillanne, C.

    1986-04-01

    The effects of dissolved oxygen concentration and pH on the growth of Brevibacterium linens CNRZ 918 and its production of methanethiol from L-methionine were investigated. Optimal specific methanethiol production was obtained at 25% saturation of dissolved oxygen and at a pH between 8 and 9, whereas optimal cell growth occurred at 50% oxygen saturation and when the pH was maintained constantly at 7. Methanethiol production by nonproliferating bacteria required the presence of L-methionine (7 mM) in the culture medium. This was probably due to the induction of enzyme systems involved in the process. The intracellular concentration of L-methionine seemed to play a key role in this process. B. linens CNRZ 918 tolerated alkaline pHs with a maximal growth pH of approximately 9. Its orange pigmentation seemed to depend on the presence of L-methionine in the culture medium and on the concentration of dissolved oxygen.

  3. Influence of Oxygen and pH on Methanethiol Production from l-Methionine by Brevibacterium linens CNRZ 918.

    PubMed

    Ferchichi, M; Hemme, D; Bouillanne, C

    1986-04-01

    The effects of dissolved oxygen concentration and pH on the growth of Brevibacterium linens CNRZ 918 and its production of methanethiol from l-methionine were investigated. Optimal specific methanethiol production was obtained at 25% saturation of dissolved oxygen and at a pH between 8 and 9, whereas optimal cell growth occurred at 50% oxygen saturation and when the pH was maintained constantly at 7. Methanethiol production by nonproliferating bacteria required the presence of l-methionine (7 mM) in the culture medium. This was probably due to the induction of enzyme systems involved in the process. The intracellular concentration of l-methionine seemed to play a key role in this process. B. linens CNRZ 918 tolerated alkaline pHs with a maximal growth pH of approximately 9. Its orange pigmentation seemed to depend on the presence of l-methionine in the culture medium and on the concentration of dissolved oxygen.

  4. Comparison of methionine chelated versus sulfate trace minerals on rate and efficiency of gain and pregnancy rates in beef heifers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives of this experiment were to compare rate and efficiency of gain, and conception rates of yearling heifers supplemented with Cu, Zn, and Mn as either metal methionine hydroxy analogue chelated trace minerals (CTM; provided as MINTREX) or the same trace minerals in SO4 form. The experimental...

  5. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  6. Selective Gold Recovery and Catalysis in a Highly Flexible Methionine-Decorated Metal-Organic Framework.

    PubMed

    Mon, Marta; Ferrando-Soria, Jesús; Grancha, Thais; Fortea-Pérez, Francisco R; Gascon, Jorge; Leyva-Pérez, Antonio; Armentano, Donatella; Pardo, Emilio

    2016-06-29

    A novel chiral 3D bioMOF exhibiting functional channels with thio-alkyl chains derived from the natural amino acid l-methionine (1) has been rationally prepared. The well-known strong affinity of gold for sulfur derivatives, together with the extremely high flexibility of the thioether "arms" decorating the channels, account for a selective capture of gold(III) and gold(I) salts in the presence of other metal cations typically found in electronic wastes. The X-ray single-crystal structures of the different gold adsorbates Au(III)@1 and Au(I)@1 suggest that the selective metal capture occurs in a metal ion recognition process somehow mimicking what happens in biological systems and protein receptors. Both Au(III)@1 and Au(I)@1 display high activity as heterogeneous catalyst for the hydroalkoxylation of alkynes, further expanding the application of these novel hybrid materials.

  7. Effect of methionine sulfoximine on nitrogen metabolism and externally supplied ammonium assimilation in kidney bean.

    PubMed

    Sadunishvili, T; Gvarliani, N; Nutsubidze, N; Kvesitadze, G

    1996-06-01

    L-Methionine sulfoximine (MSO) at concentration 1.25 mM in vivo causes the inhibition of glutamine synthetase (GS) in both roots and leaves of young seedlings of kidney bean following the accumulation of high levels of ammonia and decrease in amounts of free amino acids that is more pronounced in leaves. The inhibition of GS by MSO in leaves in the case of externally supplied 5 mM (15NH4)2SO4 assimilation leads to ammonia accumulation and the decrease in the amounts of glutamine and glutamic acid and the intensity of the incorporation of 15N into them. In roots the inhibition of GS is not followed by the decrease of 15N content into glutamate. It is concluded that the pathway of ammonia primary assimilation in leaves is via GS and glutamate synthase (GOGAT), while in roots glutamate dehydrogenase also plays an important role in this process.

  8. Structure-Functional Study of Tyrosine and Methionine Dipeptides: An Approach to Antioxidant Activity Prediction

    PubMed Central

    Torkova, Anna; Koroleva, Olga; Khrameeva, Ekaterina; Fedorova, Tatyana; Tsentalovich, Mikhail

    2015-01-01

    Quantum chemical methods allow screening and prediction of peptide antioxidant activity on the basis of known experimental data. It can be used to design the selective proteolysis of protein sources in order to obtain products with antioxidant activity. Molecular geometry and electronic descriptors of redox-active amino acids, as well as tyrosine and methionine-containing dipeptides, were studied by Density Functional Theory method. The calculated data was used to reveal several descriptors responsible for the antioxidant capacities of the model compounds based on their experimentally obtained antioxidant capacities against ABTS (2,2′-Azino-bis-(3-ethyl-benzothiazoline-6-sulfonate)) and peroxyl radical. A formula to predict antioxidant activity of peptides was proposed. PMID:26512651

  9. Chemoenzymatic synthesis and in situ application of S-adenosyl-L-methionine analogs

    PubMed Central

    Thomsen, Marie; Vogensen, Stine B.; Buchardt, Jens; Burkart, Michael D.

    2013-01-01

    Analogs of S-adenosyl-L-methionine (SAM) are increasingly applied to the methyltransferase (MT) catalysed modification of biomolecules including proteins, nucleic acids, and small molecules. However, SAM and analogs suffer from an inherent instability, and their chemical synthesis is challenged by low yields and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymatic synthesis of a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases, SalL and FDAS. Molecular modelling studies are used to guide the rational design of mutants, and the enzymatic conversion of L-Met and other analogs into SAM analogs is demonstrated. We also apply this in situ enzymatic synthesis to the modification of a small peptide substrate by protein arginine methyltransferase 1 (PRMT1). This technique offers an attractive alternative to chemical synthesis and can be applied in situ to overcome stability and activity issues. PMID:24100405

  10. EPR study of gamma irradiated DL-methionine sulfone single crystals

    NASA Astrophysics Data System (ADS)

    Karabulut, Bünyamin; Yıldırım, İlkay

    2015-12-01

    Electron paramagnetic resonance (EPR) study of gamma irradiated dl-2-amino-4-(Methylsulfonyl) butyric acid (dl-methionine sulfone, hereafter dl-ABA) single crystals and powder was performed at room temperature. It has been found that this compound indicates the existence of C. O2- and N. H2 radicals after γ-irradiation. While g and hyperfine splitting values for the N. H2 radical were observed, for the C. O2- radical, only the g factor was measured. The EPR spectra have shown that N. H2 radical has two groups each having two distinct sites and C. O2- radical has one site. The principal g and hyperfine values for all sites were analyzed.

  11. Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

    PubMed

    Revtovich, S V; Morozova, E A; Khurs, E N; Zakomirdina, L N; Nikulin, A D; Demidkina, T V; Khomutov, R M

    2011-05-01

    Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and β- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

  12. Using Azido Analogue of S-Adenosyl-L-methionine for Bioorthogonal Profiling of Protein Methylation (BPPM)

    PubMed Central

    Blum, Gil; Islam, Kabirul; Luo, Minkui

    2013-01-01

    Protein methyltransferases (PMTs) utilize S-adenosyl-L-methionine as a cofactor and deliver its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes from transcriptional activation/silencing to cell cycle regulation. With the long-term goal of elucidating the substrates and defining the functions of PMTs, our laboratory recently developed technology based on protein engineering in tandem with SAM analogue cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The following protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analogue; (2) generating the proteome containing the engineered PMT; (3) visualizing the proteome-wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag and finally (4) pulling down the proteome-wide substrates for mass spectrometric analysis. PMID:23667794

  13. Potential role of cysteine and methionine in the protection against hormonal imbalance and mutagenicity induced by furazolidone in female rats.

    PubMed

    Ahmed, Hanaa H; El-Aziem, Sekena H Abd; Abdel-Wahhab, Mosaad A

    2008-01-14

    The use of nitrofurans as veterinary drugs has been banned in the EU since 1993 due to doubts on the safety of the protein-bound residues of these drugs in edible products. Furazolidone (FUZ) is a nitrofuran drug, which has been used for many years as an antibacterial drug in veterinary practice. The aim of the current study is to investigate the role of L-cysteine and L-methionine in the protection against hormonal imbalance and the genotoxicity induced by FUZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis in female rats. Forty female Sprague-Dawley rats were divided into four groups included the untreated control group; a group treated with FUZ (300 mg/kg b.w.); a group treated with a mixture of L-cysteine (300 mg/kg b.w.) and L-methionine (42.8 mg/kg b.w.) and a group treated with FUZ plus the mixture of L-cysteine and L-methionine for 10 days. The results indicated that FUZ induced hormonal disturbances involving thyroid, ovarian and adrenal hormones. Moreover, FUZ increased the micronucleus formation and induced changes in polymorphic band patterns. The combined treatment with FUZ and the mixture of L-cysteine and L-methionine succeeded to prevent or diminish the endocrine disturbance and the clastogenic effects of FUZ. The current study is casting new light on the complex mechanisms underlying the ameliorating action of dietary L-cysteine and L-methionine against FUZ toxicity in experimental animals.

  14. Control of methionine metabolism by the SahR transcriptional regulator in Proteobacteria.

    PubMed

    Novichkov, Pavel S; Li, Xiaoqing; Kuehl, Jennifer V; Deutschbauer, Adam M; Arkin, Adam P; Price, Morgan N; Rodionov, Dmitry A

    2014-01-01

    Sulphur is an essential element in the metabolism. The sulphur-containing amino acid methionine is a metabolic precursor for S-adenosylmethionine (SAM), which serves as a coenzyme for ubiquitous methyltrtansferases. Recycling of organic sulphur compounds, e.g. via the SAM cycle, is an important metabolic process that needs to be tightly regulated. Knowledge about transcriptional regulation of these processes is still limited for many free-living bacteria. We identified a novel transcription factor SahR from the ArsR family that controls the SAM cycle genes in diverse microorganisms from soil and aquatic ecosystems. By using comparative genomics, we predicted SahR-binding DNA motifs and reconstructed SahR regulons in the genomes of 62 Proteobacteria. The conserved core of SahR regulons includes all enzymes required for the SAM cycle: the SAH hydrolase AhcY, the methionine biosynthesis enzymes MetE/MetH and MetF, and the SAM synthetase MetK. By using a combination of experimental techniques, we validated the SahR regulon in the sulphate-reducing Deltaproteobacterium Desulfovibrio alaskensis. SahR functions as a negative regulator that responds to the S-adenosylhomocysteine (SAH). The elevated SAH level in the cell dissociates SahR from its DNA operators and induces the expression of SAM cycle genes. The effector-sensing domain in SahR is related to SAM-dependent methylases that are able to tightly bind SAH. SahR represents a novel type of transcriptional regulators for the control of sulphur amino acid metabolism.

  15. Increased methionine sulfoxide content of apoA-I in type 1 diabetes.

    PubMed

    Brock, Jonathan W C; Jenkins, Alicia J; Lyons, Timothy J; Klein, Richard L; Yim, Eunsil; Lopes-Virella, Maria; Carter, Rickey E; Thorpe, Suzanne R; Baynes, John W

    2008-04-01

    Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.

  16. Association of methionine synthase gene polymorphisms with wool production and quality traits in Chinese Merino population.

    PubMed

    Rong, E G; Yang, H; Zhang, Z W; Wang, Z P; Yan, X H; Li, H; Wang, N

    2015-10-01

    Methionine synthase (MTR) plays a crucial role in maintaining homeostasis of intracellular methionine, folate, and homocysteine, and its activity correlates with DNA methylation in many mammalian tissues. Our previous genomewide association study identified that 1 SNP located in the gene was associated with several wool production and quality traits in Chinese Merino. To confirm the potential involvement of the gene in sheep wool production and quality traits, we performed sheep tissue expression profiling, SNP detection, and association analysis with sheep wool production and quality traits. The semiquantitative reverse transcription PCR analysis showed that the gene was differentially expressed in skin from Merino and Kazak sheep. The sequencing analysis identified a total of 13 SNP in the gene from Chinese Merino sheep. Comparison of the allele frequencies revealed that these 13 identified SNP were significantly different among the 6 tested Chinese Merino strains ( < 0.001). Linkage disequilibrium analysis showed that SNP 3 to 11 were strongly linked in a single haplotype block in the tested population. Association analysis showed that SNP 2 to 11 were significantly associated with the average wool fiber diameter and the fineness SD and that SNP 4 to 11 were significantly associated with the CV of fiber diameter trait ( < 0.05). Single nucleotide polymorphism 2 and SNP 5 to 12 were weakly associated with wool crimp. Similarly, the haplotypes derived from these 13 identified SNP were also significantly associated with the average wool fiber diameter, fineness SD, and the CV of fiber diameter ( < 0.05). Our results suggest that is a candidate gene for sheep wool production and quality traits, and the identified SNP might be used in sheep breeding.

  17. Structure of the angiogenesis inhibitor ovalicin bound to its noncognate target, human Type 1 methionine aminopeptidase

    PubMed Central

    Addlagatta, Anthony; Matthews, Brian W.

    2006-01-01

    Methionine aminopeptidases (MetAPs) remove the initiator methionine during protein biosynthesis. They exist in two isoforms, MetAP1 and MetAP2. The anti-angiogenic compound fumagillin binds tightly to the Type 2 MetAPs but only weakly to Type 1. High-affinity complexes of fumagillin and its relative ovalicin with Type 2 human MetAP have been reported. Here we describe the crystallographic structure of the low-affinity complex between ovalicin and Type 1 human MetAP at 1.1 Å resolution. This provides the first opportunity to compare the structures of ovalicin or fumagillin bound to a Type 1 and a Type 2 MetAP. For both Type 1 and Type 2 human MetAPs the inhibitor makes a covalent adduct with a corresponding histidine. At the same time there are significant differences in the alignment of the inhibitors within the respective active sites. It has been argued that the lower affinity of ovalicin and fumagillin for the Type 1 MetAPs is due to the smaller size of their active sites relative to the Type 2 enzymes. Comparison with the uncomplexed structure of human Type 1 MetAP indicates that there is some truth to this. Several active site residues have to move “outward” by 0.5 Å or so to accommodate the inhibitor. Other residues move “inward.” There are, however, other factors that come into play. In particular, the side chain of His310 rotates by 134° into a different position where (together with Glu128 and Tyr195) it coordinates a metal ion not seen at this site in the native enzyme. PMID:16823043

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

    SciTech Connect

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

    2009-05-28

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

  19. Further studies on the interactions among dietary aluminum, boron, magnesium, and methionine in the rat

    SciTech Connect

    Nielsen, F.H.

    1986-03-01

    An experiment was done to confirm findings that dietary B affects the response of rats to Mg deprivation and/or high dietary Al and that the response is influenced by methionine status. Weanling Sprague-Dawley rats were fed for 49 days a diet based on 70% acid-washed ground corn - 16% casein with the following supplements factorially arranged as variables: B, 0 and 3 ..mu..g/g; Al, 0 and 1000 ..mu..g/g; Mg, 100 and 400 ..mu..g/g; and amino acids, none, 2.5 methionine (Met)/g and 5 mg arginine (Arg)/g. When compared to an earlier experiment, the low Mg was lower, the high Al was higher, and Arg was an addition to exacerbate low Met status. Mg deficiency depressed growth and elevated the spleen wt/body wt, liver wt/body wt, and kidney wt/body wt ratios. The changes were more marked in B-deprived than B-supplemented rats. Also, the differences due to dietary B were most marked when Met was marginal or possibly deficient. High dietary Al depressed growth. The depression was most marked when the diet was Mg-deficient for B-supplemented rats, but was most marked when the diet was Mg-adequate for B-deprived rats. Apparently, the growth depression caused by Mg deficiency in the B-deprived rats prevented any further significant growth depression by high dietary Al. The findings indicate that B might be beneficial in conditions that can cause a hyperparathyroid state in the rat.

  20. Analysis of methionine synthase (rs1805087) gene polymorphism in autism patients in Northern Iran.

    PubMed

    Haghiri, Rosa; Mashayekhi, Farhad; Bidabadi, Elham; Salehi, Zivar

    2016-01-01

    Autism is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social communication. The genetic and environmental factors play roles in the pathogenesis of autism. It was recently shown that the genes involved in the folate/homocysteine pathway may be risk factors for autistic children. One of the genes that may be the risk factor for autism is Methionine synthase (MTR). MTR is responsible for the regeneration of methionine from homocysteine. The aim of this study was to analyze the association of MTR A2756G gene polymorphism (rs1805087) and the risk of autism in a population in northern Iran. The prevalence of MTR A2756G polymorphism was determined in 108 children with autism and 130 controls in northern Iran. Genotypes and allele frequencies were determined in patients and controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The prevalence of genotype frequencies of AA, AG and GG in autistic children were 57.41%, 22.22% and 20.37%, respectively, while in controls were 61.54%, 32.31% and 6.15%, respectively. There was significant difference between the MTR polymorphism distribution in control and patient groups. The prevalence of allele frequencies of A and G in autistic children were 0.69 and 0.31, respectively and in controls were 0.78 and 0.22, respectively (P=0.03). The MTR G allele conferred a 1.6-fold increased risk to autism relative to the A allele (95% CI=1.06-2.41, P=0.02). The present study suggests that the G allele of MTR A2756G polymorphism is associated with an increased risk of autism.

  1. Effect of methionine supplement on physical responses and neurological symptoms in broiler chicks fed grass pea (Lathyrus sativus)-based starter ration.

    PubMed

    Fikre, Asnake; Yami, Alemu; Kuo, Yu-Haey; Ahmed, Seid; Gheysen, Godelieve; Lambein, Fernand

    2010-01-01

    Starter feeding experiments of broiler chicks with raw grass pea (Lathyrus sativus L.) supplemented with different levels of DL-methionine were undertaken for 4 weeks to assess the toxicity of grass pea-based feed and to correlate it with neurological symptoms. Four hundred fifty day-old broiler chicks were divided into two groups and were given formulations containing 35% (ration I) or 98.5% (ration II) grass pea, respectively. Each ration included controls and treatments with added methionine of four different concentrations. Feed intake, weight gain and feed conversion efficiency (FCE) were much higher in ration I than in ration II and these parameters significantly improved by addition of methionine in both rations. Significant increase of neurological signs with higher grass pea intake and significant reduction of acute neurological signs with addition of methionine were observed. Tolerance for grass pea was enhanced with increasing methionine in the diet and with age. Despite a similarity in the initial intake, a significant (p0.05) increase in the final feed intake by the chicks with methionine addition was found in both rations. These results suggest that methionine can improve a grass pea-based diet for broiler chicks and especially can protect young chicks from neurological symptoms.

  2. Impacts of feeding selenium-methionine and chromium-methionine on performance, serum components, antioxidant status, and physiological responses to transportation stress of Baluchi ewe lambs.

    PubMed

    Mousaie, Amir; Valizadeh, Reza; Naserian, Abbas Ali; Heidarpour, Mohammad; Mehrjerdi, Hossein Kazemi

    2014-12-01

    The effects of selenium-methionine (Se-Met) and chromium-methionine (Cr-Met) supplementation on performance and response to transportation stress were studied on 24 Baluchi ewe lambs (18-20 weeks of age) for 9 weeks. The lambs were randomly assigned to four dietary treatments: (1) control; (2) 1.5 mg supplemental Se-Met/kg dry matter (DM) of diet; (3) 0.8 mg supplemental Cr-Met/kg DM of diet; and (4) 1.5 mg Se-Met plus 0.8 mg Cr-Met/kg DM of diet (Se-Cr-Met). At the commencement of week 8, a road transportation stress (TS) was carried out for 30 min. Lambs fed Cr-Met and Se-Cr-Met diets had higher feed intake than the control and Se-Met animals (P < 0.0001). Lambs on Cr-Met diet showed higher average daily gain (ADG) compared to the control group (P = 0.007). Se-Met and Cr-Met supplementation alone or in combination significantly (P < 0.05) reduced feed conversion ratio (FCR). The animals that received Se-Met (P = 0.014), Cr-Met (P = 0.005), and Se-Cr-Met (P = 0.003) supplemented diets had lower glucose concentration than the control. Lambs on Cr-Met had higher blood T3 concentration than control animals (P = 0.040), while Cr-Met (P = 0.039) and Se-Cr-Met (P = 0.032) supplementation increased triiodothyronine (T3) to thyroxin (T4) ratio. Animals fed Se-Met and/or Cr-Met supplements had lower blood malondialdehyde (MDA) in week 9 of the experiment (P < 0.05). Blood ferric-reducing antioxidant power (FRAP) tended to be higher in the Se-Met- and Se-Cr-Met-supplemented groups (P < 0.1).TS reduced feed intake in lambs fed the control diet in week 8 of the experiment (P = 0.003). The lambs given with supplemental Cr-Met exhibited lower glucose concentration before transportation (BT) (P = 0.029) and after transportation (AT) (P = 0.016) compared to the control. Lambs fed Se-Cr-Met had the lowest cortisol concentration BT (P < 0.05). It was concluded that feeding Se-Met and/or Cr-Met supplements could improve

  3. MAT1A variants are associated with hypertension, stroke, and DNA damage and are modulated by vlasma vitamin B6 and folate concentration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elevated plasma homocysteine is a cardiovascular disease (CVD) risk factor. However, the mechanism underlying this relationship is not understood. S-adenosylmethionine synthetase isoform type-1 (MAT1A) is a key enzyme in the metabolism of homocysteine, converting dietary methionine into S-adenosyl m...

  4. Crystal and molecular structure of two geometrically restricted chemotactic tripeptides, analogues of formyl-methionine-leucine-phenylalanine.

    PubMed

    Michel, A G; Lajoie, G; Hassani, C A

    1990-12-01

    The crystal structures of HCO-Met-Leu-Phe-OC(CH3)3, (CH25H39N3O5S), fMLP-OtBu, and HCO-Met psi [CSNH]-Leu-Phe-OCH3, (C22H33N3O4S2), fMS LP-OMe, have been determined by single crystal X-ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP-OtBu are monoclinic, space group P2(1), a = 12.027(4), b = 9.492(3), c = 12.660(4) A, beta = 101.99(3) degrees, Z = 2; those of fMS LP-OMe are orthorhombic, space group P2(1)2(1)2(1), a = 7.130(1), b = 12.097(2), c = 31.060(5) A, Z = 4. The first compounds fMLP-OtBu is the t-butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fMSLP-OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X-ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy-minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C7(eq) conformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C7eq conformations for fMLP is made. The resulting

  5. The effect of condensed tannins in Lotus corniculatus on plasma metabolism of methionine, cystine and inorganic sulphate by sheep.

    PubMed

    Wang, Y; Waghorn, G C; Barry, T N; Shelton, I D

    1994-12-01

    Fresh Lotus corniculatus containing 27 g extractable condensed tannin (CT)/kg dry matter (DM) and 8 g bound CT/kg DM was fed at hourly intervals to sheep held in metabolism cages to study the effects of CT on nutrient digestion and on metabolism of methionine, cystine and inorganic sulphate in plasma. Polyethylene glycol (PEG) was continuously infused into the rumen of half the sheep to remove the effects of CT. Principal measurements in the two groups were plasma irreversible loss (IRL) rate and interconversions of methionine, cystine and inorganic sulphate using 35S labelling. CT in Lotus corniculatus had no effects on the apparent digestion of cellulose and minerals, slightly depressed DM, organic matter and hemicellulose digestion and markedly reduced the apparent digestion of N (P < 0.01). The concentration of NH3 and molar proportions of iso-butyric acid, iso-valeric acid and n-valeric acid in rumen fluid were markedly increased by the PEG infusion (P < 0.01), whereas total volatile fatty acid concentration and molar proportions of acetic acid, propionic acid and n-butyric acid were not affected. PEG infusion temporarily increased rumen protozoa numbers. CT greatly increased the IRL of plasma cystine (13.1 v. 7.0 mumol/min; P < 0.05) and reduced IRL of plasma inorganic sulphate (36.8 v. 48.1 mumol/min; P < 0.01) but had no effect on methionine IRL. CT increased transulphuration of methionine to cystine (4.37 v. 1.24 mumol/min; P < 0.05), increased cystine entering the plasma from whole-body protein turnover plus absorption from the small intestine (9.34 v. 5.75 mumol/min; P < 0.05) and increased cystine flux to body synthetic reactions (11.89 v. 5.41 mumol/min; P < 0.05). CT had no effect on the proportion of methionine total flux transferred to sulphate (0.05 v. 0.06; P < 0.05), reduced the proportion of methionine flux transferred to body synthetic reactions (0.68 v. 0.86) and markedly reduced the proportion of cystine flux transferred to sulphate (0.09 v

  6. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

    PubMed Central

    2015-01-01

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  7. Source of carbohydrate and metabolizable lysine and methionine in the diet of recently weaned dairy calves on digestion and growth.

    PubMed

    Hill, T M; Quigley, J D; Bateman, H G; Aldrich, J M; Schlotterbeck, R L

    2016-04-01

    Two 56-d trials with weaned Holstein dairy calves (initially 72 ± 1.8 kg of body weight, 58 to 60 d of age) fed 95% concentrate and 5% chopped grass hay diets were conducted. Each trial used 96 calves (4 calves/pen). During 15 of the last 21 d of the first trial and 10 of 14 d of the second and third week of the second trial, fecal samples were taken to estimate digestibility using acid-insoluble ash as an internal marker. Digestibility estimates along with 56-d average daily gain (ADG), hip width change, body condition score, and fecal score were analyzed with pen as the experimental unit. In trial 1, a textured diet (19% crude protein) with high starch [52% starch, 13% neutral detergent fiber (NDF)] based on whole corn and oats or a pelleted low-starch (20% starch, 35% NDF), high-digestible fiber diet were used. Within starch level, diets were formulated from supplemental soybean meal or soybean meal with blood meal and Alimet (Novus International Inc., St. Charles, MO) to provide 2 metabolizable protein levels (1 and 1.07% metabolizable lysine plus methionine). The 4 treatments were analyzed as a completely randomized design with a 2 by 2 factorial arrangement (6 pens/diet). In trial 2, all pelleted diets (19% crude protein) were fed. Diets were based on soybean hulls, wheat middlings, or corn, which contained increasing concentrations of starch (13, 27, and 42% starch and 42, 23, and 16% NDF, respectively; 8 pens/diet). Contrast statements were constructed to separate differences in the means (soybean hulls plus wheat middlings vs. corn; soybean hulls vs. wheat middlings). In trial 1, intake of organic matter (OM) did not differ. Digestibility of OM was greater in calves fed high- versus low starch-diets. Digestibility of NDF and starch were less in calves fed the high- versus low-starch diets. Calf ADG and hip width change were greater for high- versus low-starch diets. Source of protein did not influence digestibility or ADG. In trial 2, intake of OM was not

  8. Influence of methionine supplementation of growing diets enriched with lysine on feedlot performance and characteristics of digestion in Holstein steer calves

    PubMed Central

    Torrentera, Noemí; Carrasco, Ramsés; Salinas-Chavira, Jaime; Plascencia, Alejandro; Zinn, Richard A.

    2017-01-01

    Objective Two trials were conducted in order to examine the effects of level of supplemental methionine on productive performance, dietary energetic, plasma amino acid concentration, and digestive function. Methods Dietary treatments consisted of a steam-flaked corn-based diet containing urea as the only source of supplemental nitrogen supplemented with no supplemental amino acid (control), or control plus 1.01% lysine and 0.032%, 0.064%, 0.096%, or 0.128% methionine. In Trial 1, 150 Holstein steer calves (127±4.9 kg) were utilized to evaluate the influence of treatments on growth-performance, dietary energetic, plasma amino acid concentration during the first 112 days of growing period. During the initial 56-d period calves received the 5 experimental diets. During the subsequent 56-d period all calves were fed the control diet. Results During the initial 56-d period, methionine supplementation increased (linear effect, p<0.01) plasma methionine. In the presence of supplemental lysine, increases on level of methionine in diet did not affect average daily gain. However, increased gain efficiency (quadratic effect, p = 0.03) and estimated dietary net energy (NE; linear effect, p = 0.05). Estimated metabolizable methionine supply was closely associated (R2 = 0.95) with efficiency NE utilization for maintenance and gain. During the subsequent 56-d period, when all calves received the control diet (no amino acid supplementation), plasma amino acid concentrations and growth performance was not different among groups. However, the effects of methionine supplementation during the initial 56-period carried over, so that following a 56-d withdrawal of supplementation, the overall 112-d effects on gain efficiency (quadratic effect, p = 0.05) dietary NE (linear effect, p≤0.05) remained appreciable. In Trial 2, 5 cannulated Holstein steers were used to evaluate treatment effects on characteristics of digestion and amino acid supply to the small intestine. There were no

  9. Pre-steady-state kinetic and structural analysis of interaction of methionine γ-lyase from Citrobacter freundii with inhibitors.

    PubMed

    Kuznetsov, Nikita A; Faleev, Nicolai G; Kuznetsova, Alexandra A; Morozova, Elena A; Revtovich, Svetlana V; Anufrieva, Natalya V; Nikulin, Alexei D; Fedorova, Olga S; Demidkina, Tatyana V

    2015-01-02

    Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.

  10. A comprehensive picture of non-site specific oxidation of methionine residues by peroxides in protein pharmaceuticals.

    PubMed

    Chu, Jhih-Wei; Yin, Jin; Brooks, Bernard R; Wang, Daniel I C; Ricci, Margaret Speed; Brems, David N; Trout, Bernhardt L

    2004-12-01

    In this article, a comprehensive picture of the oxidation of protein pharmaceuticals by peroxides is developed based on our earlier computational and experimental studies. We propose a new mechanism, the water-mediated mechanism, for the oxidation of methionine residues, and it has been shown to satisfy all available experimental data including new data reported here. Based on the water-mediated mechanism, we found a structural property, average 2-shell water coordination number, that correlates well to the relative rates of oxidation of methionine groups. We used this to study the oxidation of granulocyte colony-stimulating factor (G-CSF) and 1-34 human parathyroid hormone hPTH(1-34). We believe that this comprehensive picture should aid researchers in the pharmaceutical sciences to develop solvent formulations for therapeutic proteins in a more rational way.

  11. Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.

    PubMed

    Singh, Satish K; Roberts, Sue A; McDevitt, Sylvia F; Weichsel, Andrzej; Wildner, Guenter F; Grass, Gregor B; Rensing, Christopher; Montfort, William R

    2011-10-28

    The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Å with the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.

  12. Effects of reducing dietary protein, methionine, choline, folic acid, and vitamin B12 during the late stages of the egg production cycle on performance and eggshell quality.

    PubMed

    Keshavarz, K

    2003-09-01

    A series of four experiments was conducted to determine whether-shell quality during the late stages of egg production can be improved by using diets that are effective in reducing egg size. The experiments involved dietary manipulation of protein, methionine, choline, folic acid, and vitamin B12. In experiment 1, reducing dietary protein in combination of reducing the dietary methionine and choline or this diet without supplemental folic acid and vitamin B12 resulted in reduced egg weight and improved shell quality. However, egg production also was drastically reduced. In experiment 2, reducing the dietary level of methionine, without adding supplemental choline, folic acid, and vitamin B12 reduced egg size and improved shell quality, but egg production was reduced as well. In this experiment reducing the dietary methionine without supplemental folic acid and vitamin B12 reduced egg size and improved shell quality with no adverse effect on egg production. In experiment 3, reducing the dietary level of methionine and choline or reducing the dietary level of choline, folic acid, and vitamin B12 reduced egg size and improved shell quality without adverse effects on egg production. On the other hand, reducing dietary methionine, folic acid, vitamin B12, and supplemental choline reduced egg weight and improved shell quality but lowered egg production. In experiment 4, reducing dietary methionine together with reducing choline and vitamin B12 reduced egg size and improved shell quality with no adverse effect on egg production. The results of this series of experiments generally indicate that certain manipulations of the combination of methionine, choline, folic acid, and vitamin B12 have the potential to reduce egg weight and improve shell quality without affecting egg production during the latter stages of the egg production cycle.

  13. Comparative effects of zinc-nano complexes, zinc-sulphate and zinc-methionine on performance in broiler chickens.

    PubMed

    Mohammadi, V; Ghazanfari, S; Mohammadi-Sangcheshmeh, A; Nazaran, M H

    2015-01-01

    Micronutrients, especially zinc, have an important role in normal metabolism and growth of broilers. Using novel technologies helps to synthesise novel zinc complexes to deliver this micronutrient more efficiently. In the present study, the effects of different zinc complexes and nano complexes on broiler performance were compared. Broilers in 6 groups were given basal diet (without zinc) and basal diet supplemented with zinc-sulphate, zinc-methionine, zinc-nano-sulphate, zinc-nano-methionine and zinc-nano-max (that was synthesised based on nanochelating technology) at a concentration of 80 mg/kg of diet. At 1-42 d of age, dietary zinc-nano-sulphate supplementation decreased weight gain and feed intake. However, feed conversion ratio was not influenced by treatments. Carcass yield (%) of birds in the zinc-nano-sulphate and control groups were dramatically reduced at 42 d of age and abdominal fat (%) increased in these groups. Relative to the control group, the antibody titre, spleen and bursa of Fabricius (%) were significantly higher in groups supplemented with zinc. Heterophil (%) was also significantly higher in the zinc-nano-methionine group in blood on d 42 compared to the control, zinc-sulphate and zinc-nano-sulphate. Compared to the controls, the mean malondialdehyde content in thigh tissue was significantly reduced in groups supplemented with zinc at the time 0, 50, 100 and 150 min after oxidation. Tibia zinc concentration in nanoparticle zinc samples was significantly higher relative to the control and zinc-sulphate groups. Taken together, our data indicate that delivery of zinc in the structure of zinc-nano-methionine and zinc-nano-max at concentrations of 80 mg/kg of diet improves growth performance. However, dietary zinc-nano-sulphate decreased growth performance in broilers.

  14. Correlation of microvascular fractal dimension with positron emission tomography [(11)C]-methionine uptake in glioblastoma multiforme: preliminary findings.

    PubMed

    Di Ieva, Antonio; Grizzi, Fabio; Tschabitscher, Manfred; Colombo, Piergiuseppe; Casali, Massimiliano; Simonelli, Matteo; Widhalm, Georg; Muzzio, Pier Carlo; Matula, Christian; Chiti, Arturo; Rodriguez y Baena, Riccardo

    2010-09-01

    Neuroradiological and metabolic imaging is a fundamental diagnostic procedure in the assessment of patients with primary and metastatic brain tumors. The correlation between objective parameters capable of quantifying the neoplastic angioarchitecture and imaging data may improve our understanding of the underlying physiopathology and make it possible to evaluate treatment efficacy in brain tumors. Only a few studies have so far correlated the quantitative parameters measuring the neovascularity of brain tumors with the metabolic profiles measured by means of amino acid uptake in positron emission tomography (PET) scans. Fractal geometry offers new mathematical tools for the description and quantification of complex anatomical systems, including microvascularity. In this study, we evaluated the microvascular network complexity of six cases of human glioblastoma multiforme quantifying the surface fractal dimension on CD34 immunostained specimens. The microvascular fractal dimension was estimated by applying the box-counting algorithm. As the fractal dimension depends on the density, size and shape of the vessels, and their distribution pattern, we defined it as an index of the whole complexity of microvascular architecture and compared it with the uptake of (11)C-methionine (MET) assessed by PET. The different fractal dimension values observed showed that the same histological category of brain tumor had different microvascular network architectures. Fractal dimension ranged between 1.19 and 1.77 (mean: 1.415+/-0.225), and the uptake of (11)C-methionine ranged between 1.30 and 5.30. A statistically significant direct correlation between the microvascular fractal dimension and the uptake of (11)C-methionine (p=0.02) was found. Our preliminary findings indicate that that vascularity (estimated on the histologic specimens by means of the fractal dimension) and (11)C-methionine uptake (assessed by PET) closely correlate in glioblastoma multiforme and that microvascular

  15. Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.).

    PubMed

    Zeh, Michaela; Leggewie, Georg; Hoefgen, Rainer; Hesse, Holger

    2002-02-01

    A potato cDNA clone, StMS1, that encodes a methionine synthase was isolated. This protein was identified on the basis of both structural and functional evidence. The predicted sequence of the protein encoded by StMS1 shows a high degree of similarity to methionine synthases from other organisms and the expression of StMS1 in bacterial mutant strains restored the mutant's ability to synthesize methionine. Genomic organization and expression analyses suggest that StMS1 is a low-copy gene and is differentially expressed in potato organs. StMS1 expression was found in all tissues, but at elevated levels in flowers, basal levels in sink and source leaves, roots and stolons, and low levels in stems and tubers. RNA expression data were confirmed by western blot analysis except that the protein content in leaves was less than expected from the RNA data. Western blot analysis of subcellular fractions revealed that the protein is located in the cytosol. However, the changing pattern of gene expression during the day/night period implied a light-dependent control of MS transcription normally seen for enzymes localized in plastids. The expression of MS was shown to be light-inducible with its highest expression at midday. These RNA data were not confirmed at the protein level since protein content levels remained constant over the whole day. Feeding experiments of detached leaves revealed that sucrose or sucrose-derived products are responsible for StMS1 induction. This induction can be blocked by treatment with DCMU during the light period. Western analysis revealed that the amount of StMS1 is not affected by either treatment. This experiment confirmed the presence of a day/night rhythm. Methionine synthase expression is regulated by photoassimilates but this seems not to detectably alter protein levels.

  16. Folate, vitamin B(6) , vitamin B(12) , methionine and alcohol intake in relation to ovarian cancer risk.

    PubMed

    Harris, Holly R; Cramer, Daniel W; Vitonis, Allison F; DePari, Mary; Terry, Kathryn L

    2012-08-15

    Folate, methionine, vitamin B(6) and vitamin B(12) may influence carcinogenesis due to their roles in the one-carbon metabolism pathway, which is critical for DNA synthesis, methylation and repair. Low intake of these nutrients has been associated with an increased risk of breast, colon and endometrial cancers. Previous studies that have examined the relation between these nutrients and ovarian cancer risk have been inconsistent and have had limited power to examine the relation by histologic subtype. We investigated the association between folate, methionine, vitamin B(6) , vitamin B(12) and alcohol among 1910 women with ovarian cancer and 1989 controls from a case-control study conducted in eastern Massachusetts and New Hampshire from 1992 to 2008. Diet was assessed via food frequency questionnaire. Participants were asked to recall diet one-year before diagnosis or interview. Logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (95% CIs). We also examined whether the associations varied by ovarian cancer histologies using polytomous logistic regression. We observed an inverse association between dietary vitamin B(6) (covariate-adjusted OR = 0.76, 95% CI 0.64-0.92; p(trend) = 0.002) and methionine intake (covariate-adjusted OR = 0.72, 95% CI = 0.60-0.87; p(trend) < 0.001) and ovarian cancer risk comparing the highest to lowest quartile. The association with dietary vitamin B(6) was strongest for serous borderline (covariate-adjusted OR = 0.49, 95% CI = 0.32-0.77; p(trend) = 0.001) and serous invasive (covariate-adjusted OR = 0.74, 95% CI = 0.58-0.94; p(trend) = 0.012) subtypes. Overall, we observed no significant association between folate and ovarian cancer risk. One-carbon metabolism related nutrients, especially vitamin B(6) and methionine, may lower ovarian cancer risk.

  17. D-Methionine attenuated cisplatin-induced vestibulotoxicity through altering ATPase activities and oxidative stress in guinea pigs.

    PubMed

    Cheng, Po-Wen; Liu, Shing-Hwa; Young, Yi-Ho; Lin-Shiau, Shoei-Yn

    2006-09-01

    Cisplatin has been used as a chemotherapeutic agent to treat many kinds of malignancies. Its damage to the vestibulo-ocular reflex (VOR) system has been reported. However, the underlying biochemical change in the inner ear or central vestibular nervous system is not fully understood. In this study, we attempted to examine whether cisplatin-induced vestibulotoxicity and D-methionine protection were correlated with the changes of ATPase activities and oxidative stress of ampullary tissue of vestibules as well as cerebellar cortex (the inhibitory center of VOR system) of guinea pigs. By means of a caloric test coupled with electronystagmographic recordings, we found that cisplatin exposure caused a dose-dependent (1, 3, or 5 mg/kg) vestibular dysfunction as revealed by a decrease of slow phase velocity (SPV). In addition, cisplatin significantly inhibited the Na(+), K(+)-ATPase and Ca(2+)-ATPase activities in the ampullary tissue with a good dose-response relationship but not those of cerebellar cortex. Regression analysis indicated that a decrease of SPV was well correlated with the reduction of Na(+), K(+)-ATPase and Ca(2+)-ATPase activities of the ampullary tissue. D-Methionine (300 mg/kg) reduced both abnormalities of SPV and ATPase activities in a correlated manner. Moreover, cisplatin exposure led to a significant dose-dependent increase of lipid peroxidation and nitric oxide concentrations of the vestibules, which could be significantly suppressed by D-methionine. However, cisplatin did not alter the levels of lipid peroxidation and nitric oxide of the cerebellum. In conclusion, cisplatin inhibited ATPase activities and increased oxidative stress in guinea pig vestibular labyrinths. D-Methionine attenuated cisplatin-induced vestibulotoxicity associated with ionic disturbance through its antioxidative property.

  18. D-Methionine attenuated cisplatin-induced vestibulotoxicity through altering ATPase activities and oxidative stress in guinea pigs

    SciTech Connect

    Cheng, P.-W.; Liu, S.-H.; Young, Y.-H.; Lin-Shiau, Shoei-Yn . E-mail: syl@ha.mc.ntu.edu.tw

    2006-09-01

    Cisplatin has been used as a chemotherapeutic agent to treat many kinds of malignancies. Its damage to the vestibulo-ocular reflex (VOR) system has been reported. However, the underlying biochemical change in the inner ear or central vestibular nervous system is not fully understood. In this study, we attempted to examine whether cisplatin-induced vestibulotoxicity and D-methionine protection were correlated with the changes of ATPase activities and oxidative stress of ampullary tissue of vestibules as well as cerebellar cortex (the inhibitory center of VOR system) of guinea pigs. By means of a caloric test coupled with electronystagmographic recordings, we found that cisplatin exposure caused a dose-dependent (1, 3, or 5 mg/kg) vestibular dysfunction as revealed by a decrease of slow phase velocity (SPV). In addition, cisplatin significantly inhibited the Na{sup +}, K{sup +}-ATPase and Ca{sup 2+}-ATPase activities in the ampullary tissue with a good dose-response relationship but not those of cerebellar cortex. Regression analysis indicated that a decrease of SPV was well correlated with the reduction of Na{sup +}, K{sup +}-ATPase and Ca{sup 2+}-ATPase activities of the ampullary tissue. D-Methionine (300 mg/kg) reduced both abnormalities of SPV and ATPase activities in a correlated manner. Moreover, cisplatin exposure led to a significant dose-dependent increase of lipid peroxidation and nitric oxide concentrations of the vestibules, which could be significantly suppressed by D-methionine. However, cisplatin did not alter the levels of lipid peroxidation and nitric oxide of the cerebellum. In conclusion, cisplatin inhibited ATPase activities and increased oxidative stress in guinea pig vestibular labyrinths. D-Methionine attenuated cisplatin-induced vestibulotoxicity associated with ionic disturbance through its antioxidative property.

  19. Simultaneous 11C-Methionine Positron Emission Tomography/Magnetic Resonance Imaging of Suspected Primary Brain Tumors

    PubMed Central

    Deuschl, Cornelius; Goericke, Sophia; Grueneisen, Johannes; Sawicki, Lino Morris; Goebel, Juliane; El Hindy, Nicolai; Wrede, Karsten; Binse, Ina; Poeppel, Thorsten; Quick, Harald; Forsting, Michael; Hense, Joerg; Umutlu, Lale; Schlamann, Marc

    2016-01-01

    Introduction The objective of this study was to assess the diagnostic value of integrated 11C- methionine PET/MRI for suspected primary brain tumors, in comparison to MRI alone. Material and Methods Forty-eight consecutive patients with suspected primary brain tumor were prospectively enrolled for an integrated 11C-methionine PET/MRI. Two neuro-radiologists separately evaluated the MRI alone and the integrated PET/MRI data sets regarding most likely diagnosis and diagnostic confidence on a 5-point scale. Reference standard was histopathology or follow-up imaging. Results Fifty-one suspicious lesions were detected: 16 high-grade glioma and 25 low-grade glioma. Ten non-malignant cerebral lesions were described by the reference standard. MRI alone and integrated PET/MRI each correctly classified 42 of the 51 lesions (82.4%) as neoplastic lesions (WHO grade II, III and IV) or non-malignant lesions (infectious and neoplastic lesions). Diagnostic confidence for all lesions, low-grade astrocytoma and high-grade astrocytoma (3.7 vs. 4.2, 3,1 vs. 3.8, 4.0 vs. 4,7) were significantly (p < 0.05) better with integrated PET/MRI than in MRI alone. Conclusions The present study demonstrates the high potential of integrated 11C-methionine-PET/MRI for the assessment of suspected primary brain tumors. Although integrated methionine PET/MRI does not lead to an improvement of correct diagnoses, diagnostic confidence is significantly improved. PMID:27907162

  20. New Enzyme Prodrug and Methionine-Depletion Combination Therapy of Breast Cancer Designed for Effective Delivery to the Tumor

    DTIC Science & Technology

    2010-10-01

    AD_ ________________ Award Number: W81XWH-08-1-0722 TITLE: New Enzyme Prodrug and...14 Sept 2010 4. TITLE AND SUBTITLE New Enzyme Prodrug and Methionine-Depletion Combination Therapy of 5a. CONTRACT NUMBER Breast...to human endothelial cells, MCF-7 breast cancer cells, and MDA-MB-231 breast cancer cells grown in vitro. Tests with the enzyme prodrug over a

  1. Interactive effects of sex, social environment, dietary restriction, and methionine on survival and reproduction in fruit flies.

    PubMed

    Zajitschek, Felix; Zajitschek, Susanne R K; Friberg, Urban; Maklakov, Alexei A

    2013-08-01

    For the evolution of life histories, the trade-off between survival and reproduction is fundamental. Because sexes optimize fitness in different ways, this trade-off is expected to be resolved differently by males and females. Consequently, the sexes are predicted to respond differently to changes in resource availability. In fruit flies, research on dietary restriction has focused largely on females maintained in the absence of males, thereby neglecting sexual interactions that affect reproductive behavior of both sexes under more natural conditions. Here, we tested for the interactive effects of diet (40, 60, 100, and 300 % of standard yeast concentrations) and social environment (separate-sex vs. mixed-sex groups) on male and female Drosophila melanogaster life histories. Additionally, we evaluated the essential amino acid methionine as an agent that can uncouple the survival-reproduction trade-off. We show sex differences in the effect of social environment on survival patterns, but not on reproductive fitness. In females, yeast had a positive effect on reproduction and a negative effect on survival. In males, yeast had a negative effect on reproduction and the effect on survival depended on the social environment. Methionine reduced survival, but had no effect on reproduction. Our findings highlight the need to include both sexes and to vary social environments in research programs aimed at lifespan extension and call for further evaluation of the fecundity-restoring effect of methionine.

  2. Methionine sulfoxide reductase A (MsrA) contributes to Salmonella Typhimurium survival against oxidative attack of neutrophils.

    PubMed

    Trivedi, Raj Narayan; Agarwal, Pranjali; Kumawat, Manoj; Pesingi, Pavan Kumar; Gupta, Vivek Kumar; Goswami, Tapas Kumar; Mahawar, Manish

    2015-12-01

    Salmonella Typhimurium (ST) must evade neutrophil assault for infection establishment in the host. Myeloperoxidase generated HOCl is the key antimicrobial agent produced by the neutrophils; and methionine (Met) residues are the primary targets of this oxidant. Oxidation of Mets leads to methionine sulfoxide (Met-SO) formation and consequently compromises the protein function(s). Methionine sulfoxide reductase A (MsrA) reductively repairs Met-SO to Mets. In this manner, MsrA maintains the function(s) of key proteins which are important for virulence of ST and enhance the survival of this bacterium under oxidative stress. We constructed msrA gene deletion strain (ΔmsrA). The primers located in the flanking regions to ΔmsrA gene amplified 850 and 300 bp amplicons in ST and ΔmsrA strains, respectively. The ΔmsrA strain grew normally in in vitro broth culture. However, ΔmsrA strain showed high susceptibility (p<0.001) to very low concentrations of HOCl which was restored (at least in part) by plasmid based complementation. ΔmsrA strain was hypersensitive (than ST) to the granules isolated from neutrophils. Further, the ΔmsrA strain was significantly (p<0.05) more susceptible to neutrophil mediated killing.

  3. Methionine synthase: high-resolution mapping of the human gene and evaluation as a candidate locus for neural tube defects.

    PubMed

    Brody, L C; Baker, P J; Chines, P S; Musick, A; Molloy, A M; Swanson, D A; Kirke, P N; Ghosh, S; Scott, J M; Mills, J L

    1999-08-01

    Periconceptual folate supplementation has been found to prevent the occurrence of many neural tube defects (NTDs). Consequently, genetic variation in folate metabolism genes is expected to contribute to the risk for neural tube defects. Methionine synthase catalyzes the vitamin B(12)-dependent conversion of homocysteine and 5-methyltetrahydrofolate to methionine and tetrahydrofolate. The observation that homocysteine and vitamin B(12) levels are independent predictors of NTD risk suggested that methionine synthase could be a candidate gene for NTDs. To assess the role of the MS gene in NTDs, we performed high-resolution physical mapping of the MS locus, isolated highly polymorphic markers linked to the MS gene, and tested for an association between specific MS alleles and NTDs. We mapped the MS gene to a position between 909 and 913 cR(10000) on chromosome 1 by radiation hybrid mapping. Polymorphic markers D1S1567 and D1S1568 map to locations no more than 900 and 194 kb from the MS gene, respectively. The segregation of these polymorphic markers was measured in 85 Irish NTD families. No allele of either marker showed a significant association with NTDs using the transmission disequilibrium test. A lack of association was also observed for the D1919G missense mutation within the gene. Our results suggest that inherited variation in the MS gene does not contribute to NTD risk in this population.

  4. Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants 1

    PubMed Central

    De Clercq, Ann; Vandewiele, Martine; Van Damme, Jozef; Guerche, Philippe; Van Montagu, Marc; Vandekerckhove, Joël; Krebbers, Enno

    1990-01-01

    We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein. Images Figure 3 Figure 4 PMID:16667878

  5. The effect of methionine, thiouracil, dienestrol diacetate and thyroprotein on the development and prevention of fatty liver in pullets.

    PubMed

    Roberson, R H; Trujillo, T

    1975-05-01

    The effect of two levels each of methionine (0.0 and 0.07 percent), thiouracil (0.0 and 0.05 percent), dienestrol diacetate (0.0 and 0.007 percent), and thyroactive casein (0.0 and 0.0125 percent) on the performancy, organ changes, and liver composition in 640 pullets of two strains was studied in a 24 factorial arrangement of treatments. Egg production, egg characteristics, feed conversion, organ weights, and liver composition were parameters measured. Supplemental methionine increased the phosphorus content of liver fat in strain A, but other parameters in the two strains were mot affected by the increase in dietary methionine. The thiouracil increased weight grains, gram of fat per total liver, percent of liver fat, thyroid weight, and heart weight but decreased the phosphorus content of liver fat. Nine typical cases of fatty liver syndrome with large liver hematomas occurred in the thiouracil treated birds and one case occurred in an untreated pullet. Dienestrol diacetate did not affect egg production, egg characteristics, organ weights, and liver composition in the two strains. Thyroprotein decreased weight gain, abdominal fat, liver weight. liver fat, thyroid weight, and percent red cells, but decreased percent blood sports in eggs and adjusted weights of the kidney and heart in both strains.

  6. Molecular characterization and expression profile of methionine sulfoxide reductase gene family in maize (Zea mays) under abiotic stresses.

    PubMed

    Zhu, Jiantang; Ding, Pengcheng; Li, Qingqing; Gao, YanKun; Chen, Fanguo; Xia, Guangmin

    2015-05-15

    Methionine (Met) oxidation to methionine sulfoxide (MetSO) is a common form of damage caused by reactive oxygen species (ROS) accumulation via various environmental stresses. Methionine sulfoxide reductase (MSR) repairs oxidized Met and protects organisms from oxidative damage. Two types of MSR, A and B, have been identified based on substrate stereo specificity; they share no sequence similarity. In the present study, we characterized six genes encoding the putative MSR from two public databases. We compared them with MSRs from 6 species, and evaluated molecular characterization, phylogenetic analysis, tertiary structure and conserved motifs. On the basis of in silico and the qRT-PCR experimental data, we analyzed cDNA sequences and expression patterns of ZmMSR genes in different organs in maize. We found that ZmMSR genes were induced by polyethylene glycol (PEG) and NaCl, both known to generate oxidative stress. The results show that MSRs are conserved in different species, suggesting that MSRs across different species share common mechanisms related to diverse defense responses.

  7. Cobalamin-independent Methionine Synthase Distribution and Influence on Vitamin B12 Growth Requirements in Marine Diatoms.

    PubMed

    Ellis, Kelsey A; Cohen, Natalie R; Moreno, Carly; Marchetti, Adrian

    2017-02-01

    The requirement for cobalamin (vitamin B12) in microalgae is primarily a function of the type of methionine synthase present within their gene repertoires. Our 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 genera 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. Pseudo-nitzschia 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 regions such as the Southern Ocean where B12 concentrations may be consistently low. Our findings demonstrate the important role vitamins can play in diatom community dynamics within areas where vitamin supply may be variable and limiting.

  8. Heterologous production of methionine-gamma-lyase from Brevibacterium linens in Lactococcus lactis and formation of volatile sulfur compounds.

    PubMed

    Hanniffy, Sean B; Philo, Mark; Peláez, Carmen; Gasson, Michael J; Requena, Teresa; Martínez-Cuesta, M C

    2009-04-01

    The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-gamma-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.

  9. Characterization of a methionine sulfoxide reductase B from tomato (Solanum lycopersicum), and its protecting role in Saccharomyces cerevisiae.

    PubMed

    Dai, Changbo; Liu, Likun; Wang, Myeong Hyeon

    2013-01-01

    In the present study, we isolated a methionine sulfoxide reductase B gene, termed SlMSRB1, from tomato (Solanum lycopersicum). In the organ-specific analysis, high expression levels of SlMSRB1 were detected in red mature fruits, leaves and flowers while low transcriptional levels of SlMSRB1 mRNA were observed in stems and roots. In the green fluorescence analysis of SlMSRB1- overexpressed Arabidopsis, signal corresponding to SlMSRB1 was merely detected in chloroplast, suggesting that tomato MSRB1 is a chloroplastial localization protein. Substrate specificity analysis of recombinant SlMSRB1 showed that the enzyme was only targeted to the R epimer of methionine sulfoxide (MetSO) and was able to convert both free and protein-bound MetSO back to methionine in the presence of dithithreitol (DTT). In addition, SlMSRB1 exhibited no activity in thioredoxin dependent system or the substitution of cysteine at position 181 in the DTT-dependent reduction system. Finally, overexpression of SlMSRB1 in yeast revealed that the SlMSRB1 gene might play a critical role in protecting Saccharomyces cerevisiae against oxidative stress.

  10. Overexpression of methionine sulfoxide reductases A and B2 protects MOLT-4 cells against zinc-induced oxidative stress.

    PubMed

    Cabreiro, Filipe; Picot, Cĕdric R; Perichon, Martine; Friguet, Bertrand; Petropoulos, Isabelle

    2009-02-01

    Among the amino acids, methionine is the most susceptible to oxidation, and methionine sulfoxide can be catalytically reduced within proteins by methionine sulfoxide reductase A (MsrA) and B (MsrB). As one of the very few repair systems for oxidized proteins, MsrA and MsrB enzymes play a major role in protein homeostasis during aging and have also been involved in cellular defenses against oxidative stress, by scavenging reactive oxygen species. To elucidate the role of zinc on the Msr system, the effects of zinc treatment on control and stably overexpressing MsrA and MsrB2 MOLT-4 leukemia cells have been analyzed. Here we show that zinc treatment has a pro-antioxidant effect in MOLT-4 cells by inducing the transcription of metallothioneins and positively modulating the activity of the Msr enzymes. In contrast, due to its pro-oxidant effect, zinc also led to increased cell death, reactive oxygen species production, and protein damage. Our results indicate that overexpression of the Msr enzymes, due to their antioxidant properties, counteracts the pro-oxidant effects of zinc treatment, which lead to a cellular protection against protein oxidative damage and cell death, by reducing the production of reactive oxygen species.

  11. (13)C-metabolic flux analysis in S-adenosyl-L-methionine production by Saccharomyces cerevisiae.

    PubMed

    Hayakawa, Kenshi; Kajihata, Shuichi; Matsuda, Fumio; Shimizu, Hiroshi

    2015-11-01

    S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production.

  12. Influence of cysteine and methionine availability on protein peroxide scavenging activity and phenolic stability in emulsions.

    PubMed

    Zhou, Lisa; Elias, Ryan J

    2014-03-01

    Plant phenolics are secondary metabolites that have been shown to confer beneficial health effects in humans. However, many of these compounds undergo metal-catalysed oxidation reactions, leading to the generation of hydrogen peroxide (H2O2) and other reactive oxygen species that may negatively impact product stability. In proteins, methionine (Met) and cysteine (Cys) are capable of reacting directly with peroxides. Thus, the dairy proteins, casein (CAS) and β-lactoglobulin (BLG), were examined for their ability to scavenge H2O2 (400μM) and influence (-)-epigallocatechin-3-gallate (EGCG) oxidation (400μM) in Tween- or sodium dodecyl sulphate (SDS)-stabilised hexadecane emulsions. To examine the effect that the accessibility of these amino acids have on their peroxide scavenging activities, proteins were pre-treated with tert-butyl hydroperoxide (TBHP), a bulky peroxide, to oxidise only solvent accessible Met residues or H2O2, the smallest peroxide, to oxidise buried Met residues. In CAS treatments, higher Met content yielded greater peroxide scavenging activity and EGCG stability. CAS treatments also showed significantly higher peroxide scavenging activity compared to the corresponding BLG treatment. However, BLG peroxide scavenging activity was greatly enhanced in SDS-stabilised emulsions due to protein denaturation and subsequent exposure of previously buried Cys residues.

  13. Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism

    PubMed Central

    Romine, Margaret F.; Rodionov, Dmitry A.; Maezato, Yukari; Anderson, Lindsey N.; Nandhikonda, Premchendar; Rodionova, Irina A.; Carre, Alexandre; Li, Xiaoqing; Xu, Chengdong; Clauss, Therese R. W.; Metz, Thomas O.; Wright, Aaron T.

    2017-01-01

    Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12. Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism. PMID:28137868

  14. Methionine Sulfoxide Reductases Protect against Oxidative Stress in Staphylococcus aureus Encountering Exogenous Oxidants and Human Neutrophils

    PubMed Central

    Pang, Yun Yun; Schwartz, Jamie; Bloomberg, Sarah; Boyd, Jeffrey M; Horswill, Alexander R.; Nauseef, William M.

    2013-01-01

    To establish infection successfully, S. aureus must evade clearance by polymorphonuclear neutrophils (PMN). We studied the expression and regulation of the methionine sulfoxide reductases (Msr) that are involved in the repair of oxidized staphylococcal proteins and investigated their influence over the fate of S. aureus exposed to oxidants or PMN. We evaluated a mutant deficient in msrA1 and msrB for susceptibility to hydrogen peroxide, hypochlorous acid and PMN. The expression of msrA1 in wild-type bacteria ingested by human PMN was assessed by real-time PCR. The regulation of msr was studied by screening a library of two-component regulatory system (TCS) mutants for altered msr responses. Relative to the wild-type, bacteria deficient in Msr were more susceptible to oxidants and to PMN. Upregulation of staphylococcal msrA1 occurred within the phagosomes of normal PMN and PMN deficient in NADPH oxidase activity. Furthermore, PMN granule-rich extract stimulated the upregulation of msrA1. Modulation of msrA1 within PMN was shown to be partly dependent on the VraSR TCS. Msr contributes to staphylococcal responses to oxidative attack and PMN. Our study highlights a novel interaction between the oxidative protein repair pathway and the VraSR TCS that is involved in cell wall homeostasis. PMID:24247266

  15. Methionine aminopeptidase 2 is a key regulator of apoptotic like cell death in Leishmania donovani.

    PubMed

    Kumar, Ritesh; Tiwari, Kartikeya; Dubey, Vikash Kumar

    2017-12-01

    We investigate the role of methionine aminopeptidase 2 (MAP2) in miltefosine induced programmed cell death (PCD) in promastigote form of L. donovani. We report that TNP-470, an inhibitor of MAP2, inhibits programmed cell death in miltefosine treated promastigotes. It inhibits the biochemical features of metazoan apoptosis, including caspase3/7 protease like activity, oligonucleosomal DNA fragmentation, collapse of mitochondrial transmembrane potential, and increase in cytosolic pool of calcium ions but did not prevent the cell death and phosphatidyl serine externalization. The data suggests that the MAP2 is involved in the regulation of PCD in parasite. Moreover, TNP-470 shows the leishmanicidal activity (IC50 = 15 µM) and in vitro inhibition of LdMAP2 activity (K i  = 13.5 nM). Further studies on MAP2 and identification of death signaling pathways provide valuable information that could be exploited to understand the role of non caspase proteases in PCD of L. donovani.

  16. Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation.

    PubMed

    Srikanth, R; Wilson, Jonathan; Bridgewater, Juma D; Numbers, Jason R; Lim, Jihyeon; Olbris, Mark R; Kettani, Ali; Vachet, Richard W

    2007-08-01

    Oxidative modifications to the side chains of sulfur-containing amino acids often limit the number of product ions formed during collision-induced dissociation (CID) and thus make it difficult to obtain sequence information for oxidized peptides. In this work, we demonstrate that electron-transfer dissociation (ETD) can be used to improve the sequence information obtained from peptides with oxidized cysteine and methionine residues. In contrast to CID, ETD is found to be much less sensitive to the side-chain chemistry, enabling extensive sequence information to be obtained in cases where CID fails to provide this information. These results indicate that ETD is a valuable technique for studying oxidatively modified peptides and proteins. In addition, we report a unique and very abundant product ion that is formed in the CID spectra of peptides having N-terminal cysteine sulfinic acid residues. The mechanism for this unique dissociation pathway involves a six-membered cyclic intermediate and leads to the facile loss of NH(3) and SO(2), which corresponds to a mass loss of 81 Da. While the facile nature of this dissociation pathway limits the sequence information present in CID spectra of peptides with N-terminal cysteine sulfinic acid residues, extensive sequence information for these peptides can be obtained with ETD.

  17. Dietary methionine restriction in mice elicits an adaptive cardiovascular response to hyperhomocysteinemia.

    PubMed

    Ables, Gene P; Ouattara, Amadou; Hampton, Thomas G; Cooke, Diana; Perodin, Frantz; Augie, Ines; Orentreich, David S

    2015-03-06

    Dietary methionine restriction (MR) in rodents increased lifespan despite higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia, which are symptoms associated with increased risk for cardiovascular disease. We investigated this paradoxical effect of MR on cardiac function using young, old, and apolipoprotein E-deficient (ApoE-KO) mice. Indeed, MR animals exhibited higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia with a molecular pattern consistent with cardiac stress while maintaining the integrity of cardiac structure. Baseline cardiac function, which was measured by non-invasive electrocardiography (ECG), showed that young MR mice had prolonged QRS intervals compared with control-fed (CF) mice, whereas old and ApoE-KO mice showed similar results for both groups. Following β-adrenergic challenge, responses of MR mice were either similar or attenuated compared with CF mice. Cardiac contractility, which was measured by isolated heart retrograde perfusion, was similar in both groups of old mice. Finally, the MR diet induced secretion of cardioprotective hormones, adiponectin and fibroblast growth factor 21 (FGF21), in MR mice with concomitant alterations in cardiac metabolic molecular signatures. Our findings demonstrate that MR diet does not alter cardiac function in mice despite the presence of hyperhomocysteinemia because of the adaptive responses of increased adiponectin and FGF21 levels.

  18. Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK).

    PubMed

    Li, Weiwei; Chen, Wenna; Herberman, Ronald B; Plotnikoff, Nicolas P; Youkilis, Gene; Griffin, Noreen; Wang, Enhua; Lu, Changlong; Shan, Fengping

    2014-03-28

    The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone.

  19. Inhibition of the methionine aminopeptidase 2 enzyme for the treatment of obesity

    PubMed Central

    Joharapurkar, Amit A; Dhanesha, Nirav A; Jain, Mukul R

    2014-01-01

    Worldwide prevalence of obesity has nearly doubled since 1980. Obesity is the result of interactions among the environmental factors, genetic predisposition, and human behavior. Even modest weight reduction in obese patients provides beneficial health outcomes. For effective weight reduction, a drug should either increase energy expenditure or decrease energy intake without causing serious adverse effects. To overcome lack of efficacy and central nervous system related side effects, exploitation of the peripheral mechanism of anti-obesity action is needed. Inhibition of pathological angiogenesis in adipose tissue is one such peripheral mechanism that has attracted the attention of researchers in this area. Although originally developed as anti-cancer agents, methionine aminopeptidase (MetAP2) inhibitors induce significant and sustained weight reduction. Here, we review preclinical and clinical pharmacology of MetAP2 inhibitors. Beloranib is a prototype MetAP2 inhibitor, and currently in advanced clinical trials for the treatment of obesity. Clinical data of beloranib indicate that MetAP2 inhibitors could be a future treatment option for weight reduction without serious adverse effects. Further clinical data from Phase III trials will add to our growing knowledge of MetAP2 inhibitor potential for anti-obesity therapy. PMID:24611021

  20. H2S protects against methionine-induced oxidative stress in brain endothelial cells.

    PubMed

    Tyagi, Neetu; Moshal, Karni S; Sen, Utpal; Vacek, Thomas P; Kumar, Munish; Hughes, William M; Kundu, Soumi; Tyagi, Suresh C

    2009-01-01

    Homocysteine (Hcy) causes cerebrovascular dysfunction by inducing oxidative stress. However, to date, there are no strategies to prevent Hcy-induced oxidative damage. Hcy is an H2S precursor formed from methionine (Met) metabolism. We aimed to investigate whether H2S ameliorated Met-induced oxidative stress in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to Met treatment in the presence or absence of NaHS (donor of H2S). Met-induced cell toxicity increased the levels of free radicals in a concentration-dependent manner. Met increased NADPH-oxidase-4 (NOX-4) expression and mitigated thioredxion-1(Trx-1) expression. Pretreatment of bEnd3 with NaHS (0.05 mM) attenuated the production of free radicals in the presence of Met and protected the cells from oxidative damage. Furthermore, NaHS enhanced inhibitory effects of apocynin, N-acetyl-l-cysteine (NAC), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), Nomega-nitro-l-arginine methyl ester (L-NAME) on ROS production and redox enzymes levels induced by Met. In conclusion, the administration of H2S protected the cells from oxidative stress induced by hyperhomocysteinemia (HHcy), which suggested that NaHS/H2S may have therapeutic potential against Met-induced oxidative stress.

  1. Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism.

    PubMed

    Romine, Margaret F; Rodionov, Dmitry A; Maezato, Yukari; Anderson, Lindsey N; Nandhikonda, Premchendar; Rodionova, Irina A; Carre, Alexandre; Li, Xiaoqing; Xu, Chengdong; Clauss, Therese R W; Kim, Young-Mo; Metz, Thomas O; Wright, Aaron T

    2017-02-14

    Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12 Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

  2. Mesoporous silica nanoparticles functionalized with folic acid/methionine for active targeted delivery of docetaxel

    PubMed Central

    Khosravian, Pegah; Shafiee Ardestani, Mehdi; Khoobi, Mehdi; Ostad, Seyed Naser; Dorkoosh, Farid Abedin; Akbari Javar, Hamid; Amanlou, Massoud

    2016-01-01

    Mesoporous silica nanoparticles (MSNs) are known as carriers with high loading capacity and large functionalizable surface area for target-directed delivery. In this study, a series of docetaxel-loaded folic acid- or methionine-functionalized mesoporous silica nanoparticles (DTX/MSN-FA or DTX/MSN-Met) with large pores and amine groups at inner pore surface properties were prepared. The results showed that the MSNs were successfully synthesized, having good pay load and pH-sensitive drug release kinetics. The cellular investigation on MCF-7 cells showed better performance of cytotoxicity and cell apoptosis and an increase in cellular uptake of targeted nanoparticles. In vivo fluorescent imaging on healthy BALB/c mice proved that bare MSN-NH2 are mostly accumulated in the liver but MSN-FA or MSN-Met are more concentrated in the kidney. Importantly, ex vivo fluorescent images of tumor-induced BALB/c mice organs revealed the ability of MSN-FA to reach the tumor tissues. In conclusion, DTX/MSNs exhibited a good anticancer activity and enhanced the possibility of targeted drug delivery for breast cancer. PMID:27980423

  3. Chiral effects in amino acid adsorption on Au(111): A comparison of cysteine, homocysteine and methionine

    NASA Astrophysics Data System (ADS)

    Popa, Tatiana; Ting, Elvis C. M.; Paci, Irina

    2014-11-01

    A combined classical/quantum methodology is used to examine chiral effects upon adsorption of three sulfur-containing amino acids on the Au(111) surface: cysteine, homocysteine and methionine. Parallel tempering Monte Carlo simulations were employed to broadly examine the configurational space of monomers, dimers and trimers of the molecules on the gold surface. Density functional theory was applied to promising structural targets in order to incorporate higher order electronic structure effects in a study of relative stabilities of the various molecular states upon adsorption. As the precursors of chiral structure formation, like and unlike dimers were investigated at some length, with consideration given to the mode of sorption (chemisorption of physisorption) and the existence of zwitterionic states. We found that neutral (non-zwitterionic) molecules adsorbed weakly on the highly-coordinated Au(111) surfaces. As a consequence, pair configurations in dimers were insufficiently constrained to lead to differential stabilities of homochiral and heterochiral dimers. Whereas neutral molecule interactions were non-discriminating, strong chiral discrimination was found in zwitterionic amino acids. The zwitterionic forms of the larger molecules equilibrated closer to the surface, and the stronger molecule-molecule and molecule-surface interactions were such that homochiral dimers were stable whereas heterochiral dimers were not.

  4. Specific lipidome signatures in central nervous system from methionine-restricted mice.

    PubMed

    Jové, Mariona; Ayala, Victòria; Ramírez-Núñez, Omar; Naudí, Alba; Cabré, Rosanna; Spickett, Corinne M; Portero-Otín, Manuel; Pamplona, Reinald

    2013-06-07

    Membrane lipid composition is an important correlate of the rate of aging of animals. Dietary methionine restriction (MetR) increases lifespan in rodents. The underlying mechanisms have not been elucidated but could include changes in tissue lipidomes. In this work, we demonstrate that 80% MetR in mice induces marked changes in the brain, spinal cord, and liver lipidomes. Further, at least 50% of the lipids changed are common in the brain and spinal cord but not in the liver, suggesting a nervous system-specific lipidomic profile of MetR. The differentially expressed lipids includes (a) specific phospholipid species, which could reflect adaptive membrane responses, (b) sphingolipids, which could lead to changes in ceramide signaling pathways, and (c) the physiologically redox-relevant ubiquinone 9, indicating adaptations in phase II antioxidant response metabolism. In addition, specific oxidation products derived from cholesterol, phosphatidylcholine, and phosphatidylethanolamine were significantly decreased in the brain, spinal cord, and liver from MetR mice. These results demonstrate the importance of adaptive responses of membrane lipids leading to increased stress resistance as a major mechanistic contributor to the lowered rate of aging in MetR mice.

  5. Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol

    PubMed Central

    Jia, Kai-Zhi; Zhang, Quan; Sun, Lin-Yang; Xu, Yang-Hua; Li, Hong-Mei; Tang, Ya-Jie

    2016-01-01

    Eukaryote-derived methioninase, catalyzing the one-step degradation of methionine (Met) to methanethiol (MTL), has received much attention for its low immunogenic potential and use as a therapeutic agent against Met-dependent tumors. Although biological and chemical degradation pathways for Met-MTL conversion are proposed, the concrete molecular mechanism for Met-MTL conversion in eukaryotes is still unclear. Previous studies demonstrated that α-keto-methylthiobutyric acid (KMBA), the intermediate for Met-MTL conversion, was located extracellularly and the demethiolase STR3 possessed no activities towards Met, which rule out the possibility of intracellular Met-MTL conversion pathway inside eukaryotes. We report here that degradation of Met resulted in intracellular accumulation of KMBA in Clonostachys rosea. Addition of Met to culture media led to the production of MTL and downregulation of STR3, while incubation of Met with surrogate substrate α-ketoglutaric acid enhanced the synthesis of MTL and triggered the upregulation of STR3. Subsequent biochemical analysis with recombinant STR3 showed that STR3 directly converted both Met and its transamination product KMBA to MTL. These results indicated that STR3 as rate-limiting enzyme degrades Met and KMBA into MTL. Our findings suggest STR3 is a potential target for therapeutic agents against Met-dependent tumors and aging. PMID:26902928

  6. 7-Carboxy-7-deazaguanine Synthase: A Radical S-Adenosyl-l-methionine Enzyme with Polar Tendencies

    PubMed Central

    2017-01-01

    Radical S-adenosyl-l-methionine (SAM) enzymes are widely distributed and catalyze diverse reactions. SAM binds to the unique iron atom of a site-differentiated [4Fe-4S] cluster and is reductively cleaved to generate a 5′-deoxyadenosyl radical, which initiates turnover. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes a key step in the biosynthesis of 7-deazapurine containing natural products. 6-Carboxypterin (6-CP), an oxidized analogue of the natural substrate 6-carboxy-5,6,7,8-tetrahydropterin (CPH4), is shown to be an alternate substrate for CDG synthase. Under reducing conditions that would promote the reductive cleavage of SAM, 6-CP is turned over to 6-deoxyadenosylpterin (6-dAP), presumably by radical addition of the 5′-deoxyadenosine followed by oxidative decarboxylation to the product. By contrast, in the absence of the strong reductant, dithionite, the carboxylate of 6-CP is esterified to generate 6-carboxypterin-5′-deoxyadenosyl ester (6-CP-dAdo ester). Structural studies with 6-CP and SAM also reveal electron density consistent with the ester product being formed in crystallo. The differential reactivity of 6-CP under reducing and nonreducing conditions highlights the ability of radical SAM enzymes to carry out both polar and radical transformations in the same active site. PMID:28045519

  7. S-adenosyl-L-methionine decarboxylase activity in the rat epididymis: ontogeny and androgenic control.

    PubMed

    de las Heras, M A; Calandra, R S

    1991-01-01

    The authors describe the occurrence of high levels of S-adenosyl-L-methionine decarboxylase (SAMDC) activity in the rat epididymis, and its ontogeny and androgenic control. As early as 15 days of age, SAMDC activity exists, although a peak of activity is observed at 25 days. Bilateral orchidectomy resulted in a decline of epididymal SAMDC activity. However, an androgen-independent fraction, accounting for 34% of total activity, appears to exist in the epididymis. In 45-day-old orchidectomized rats, SAMDC activity was stimulated by testosterone treatment in a dose-dependent manner. However, treatment of 45-day-old intact animals with a high dose of the androgen failed to modify SAMDC activity, indicating that, at this age, the enzyme is maximally stimulated by endogenous androgens. The observed effect of testosterone on castrated rats was completely abolished by concomitant treatment with the antiandrogen flutamide. This compound was ineffective on the androgen-insensitive fraction. To assess the contribution of circulating and luminal androgens to the maintenance of epididymal SAMDC, rats were unilaterally orchidectomized and activity was determined in both epididymides after 7 days. The SAMDC activity was identical in epididymides from both sides, suggesting circulating androgens suffice to maintain normal levels of activity. It was concluded that androgens regulate epididymal SAMDC activity, although an androgen-independent fraction appears to exist.

  8. Purification and characterization of the Streptococcus salivarius methionine aminopeptidase (MetAP).

    PubMed

    Boufous, El Houssine; Vadeboncoeur, Christian

    2003-10-01

    Streptococcus salivarius methionine aminopeptidase (MetAP) was purified from a recombinant Escherichia coli strain containing the S. salivarius map gene, which codes for MetAP. S. salivarius map coded for a protein of 286 amino acids with a calculated molecular mass of 31,723 Da and a pI of 4.6. The native enzyme eluted from a Superdex column as a protein with a molecular mass of 30.6 kDa and cleaved N-terminal Met of peptide only when the penultimate amino acid was Gly, Ala, Ser, Val, Pro, or Thr. The enzyme was more active against tetrapeptides than tripeptides and did not recognize dipeptides. It required the presence of a metal cation for activity, with a preference for Co(2+) over Mn(2+). S. salivarius MetAP has a pH optimum of 8.0 and an optimal temperature at 50 degrees C. The S. salivarius protein had an extra sequence of 24 amino acids between two conserved aspartate residues involved in the coordination of the metal ion. A similar extra sequence is present in MetAP from other streptococci and from Lactococcus lactis, but not from other bacteria or eukaryotes.

  9. Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470

    SciTech Connect

    Alvarado, J.; Nemkal, A; Sauder, J; Russell, M; Akiyoshi, D; Shi, W; Almo, S; Weiss, L

    2009-01-01

    Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity in vitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

  10. Two highly homologous methionine sulfoxide reductase A from tomato (Solanum lycopersicum), exhibit distinct catalytic properties.

    PubMed

    Dai, Changbo; Han, Woong; Wang, Myeong-Hyeon

    2012-04-01

    E4, which is a fruit-ripening gene that is strongly induced by ethylene, has been reported to be a member of the methionine sulfoxide reductase A (MSRA) gene. In the present study, we determined for the first time the enzymatic activity and delineated the catalytic mechanism of the E4 protein via site-directed mutagenesis. The disulfide intermolecular cross-linking, kinetics parameter, thiol content titration analysis of wild-type and mutated E4 proteins revealed that the cysteine at position 37 (Cys-37) was the key catalytic residue, and Cys-194, but not Cys-180 served as the first recycling Cys in the thioredoxin (Trx)-dependent regeneration system. In addition, the SlMSRA2 protein, which was encoded by another MSRA gene, shared high similarity with the E4 protein and was truncated at the C-terminus. The wild-type and mutated SlMSRA2 enzymes had similar activities compared to the E4 protein using DTT as a reductant, but showed extremely low activities in the Trx-dependent reduction system. Our results indicated that E4 and SlMSRA2 proteins might exhibit distinct catalytic mechanisms.

  11. Crystal structure of dengue virus methyltransferase without S-adenosyl-L-methionine.

    PubMed

    Noble, Christian G; Li, Shi-Hua; Dong, Hongping; Chew, Sock Hui; Shi, Pei-Yong

    2014-11-01

    Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2'-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket.

  12. Incorporation of radiolabeled polyamines and methionine into turnip yellow mosaic virus in protoplasts from infected plants

    SciTech Connect

    Balint, R.; Cohen, S.S.

    1985-07-15

    Turnip yellow mosaic virus contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By 7 days after inoculation, a majority of protoplasts isolated from newly emerging leaves stain with fluorescent antibody to the virus. (/sup 14/C)Spermidine (10 microM) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hr. However, after an initial rise, the spermidine content of the cell returned to its original level, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous (/sup 14/C)spermidine was also readily incorporated into the ribonucleoprotein component(s) of the virus, where it appeared as both spermidine and spermine. The specific radioactivities of the viral polyamines were approximately twice those of spermidine and spermine extracted from the whole cell. Radioactivity from (2-/sup 14/C)methionine was readily incorporated into the protein, spermidine, and spermine of the virus. Again, the specific activities of these amines were substantially higher in the virus than in the whole cell. Thus, newly formed virus contained predominantly newly synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virus.

  13. Does the Naked Neck Meat Type Chicken Yield Lower Methionine Requirement Data?

    PubMed

    Khan, Daulat R; Wecke, Christian; Liebert, Frank

    2015-03-25

    Methionine (Met) requirement studies with homozygous (Na/Na) and heterozygous (Na/na) naked neck meat type chicken utilized 144 birds of average weight (50% each genotype and sex) within two N balance experiments involving both the starter (d10-20) and grower period (d25-35). The birds were randomly allotted to five experimental diets with graded protein supply and Met as the limiting amino acid. The proportion of native feed protein sources (soy protein concentrate, maize, wheat, fishmeal and wheat gluten) was kept constant to ensure a uniform protein quality in all diets. The Met requirement depending on genotype, sex, age period and growth performance (protein deposition) was estimated using a non-linear modeling procedure of N utilization in monogastric animals. On average, 0.47% (Na/Na) and 0.45% (Na/na) dietary Met was established as adequate in the starter diet, as well as 0.37% (Na/Na) and 0.36% (Na/na) Met in the grower diet for both of the sexes. In conclusion, the Met requirement of the naked neck chicken is not significantly different from its normally-feathered counterparts. In addition, the low feather production was not reflected by reduced requirement for Met in naked neck birds. However, these conclusions are valid only at the given Met:Cys ratio (1:1) in the experimental diets.

  14. Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants1

    PubMed Central

    Zeh, Michaela; Casazza, Anna Paola; Kreft, Oliver; Roessner, Ute; Bieberich, Katrin; Willmitzer, Lothar; Hoefgen, Rainer; Hesse, Holger

    2001-01-01

    Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality. PMID:11706163

  15. NIa-pro of Papaya ringspot virus interacts with papaya methionine sulfoxide reductase B1.

    PubMed

    Gao, Le; Shen, Wentao; Yan, Pu; Tuo, Decai; Li, Xiaoying; Zhou, Peng

    2012-12-05

    A chloroplast-localized papaya methionine sulfoxide reductase B1 (PaMsrB1) interacting with Papaya ringspot virus (PRSV) NIa-Pro was identified using a Sos recruitment two-hybrid system (SRS). SRS analysis of several deletion mutants of PRSV NIa-Pro and PaMsrB1 demonstrated that the C-terminal (residues 133-239) fragment of PRSV NIa-Pro and residues 112-175 of PaMsrB1 were necessary for this interaction between PRSV NIa-Pro and PaMsrB1. MsrB1 can repair Met-oxidized proteins damaged by reactive oxygen species (ROS). We confirmed that PRSV infection leads to ROS accumulation and a slight upregulation of level PaMsrB1 mRNA in papaya. This interaction between PaMsrB1 with PRSV NIa-Pro may disturb the import of PaMsrB1 into the chloroplasts. These results suggest that this specific interaction could interfere with PaMsrB1 into the chloroplasts to scavenge ROS caused by PRSV infection. This may be a novel mechanism of PRSV towards the host defense.

  16. Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine

    PubMed Central

    Alkatib, Sibah; Fleischmann, Tobias T.; Scharff, Lars B.; Bock, Ralph

    2012-01-01

    The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons. PMID:22553362

  17. Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol.

    PubMed

    Jia, Kai-Zhi; Zhang, Quan; Sun, Lin-Yang; Xu, Yang-Hua; Li, Hong-Mei; Tang, Ya-Jie

    2016-02-23

    Eukaryote-derived methioninase, catalyzing the one-step degradation of methionine (Met) to methanethiol (MTL), has received much attention for its low immunogenic potential and use as a therapeutic agent against Met-dependent tumors. Although biological and chemical degradation pathways for Met-MTL conversion are proposed, the concrete molecular mechanism for Met-MTL conversion in eukaryotes is still unclear. Previous studies demonstrated that α-keto-methylthiobutyric acid (KMBA), the intermediate for Met-MTL conversion, was located extracellularly and the demethiolase STR3 possessed no activities towards Met, which rule out the possibility of intracellular Met-MTL conversion pathway inside eukaryotes. We report here that degradation of Met resulted in intracellular accumulation of KMBA in Clonostachys rosea. Addition of Met to culture media led to the production of MTL and downregulation of STR3, while incubation of Met with surrogate substrate α-ketoglutaric acid enhanced the synthesis of MTL and triggered the upregulation of STR3. Subsequent biochemical analysis with recombinant STR3 showed that STR3 directly converted both Met and its transamination product KMBA to MTL. These results indicated that STR3 as rate-limiting enzyme degrades Met and KMBA into MTL. Our findings suggest STR3 is a potential target for therapeutic agents against Met-dependent tumors and aging.

  18. Cellular and Molecular Remodeling of Inguinal Adipose Tissue Mitochondria by Dietary Methionine Restriction

    PubMed Central

    Patil, Yuvraj N.; Dille, Kelly N.; Burk, David H.; Cortez, Cory C.; Gettys, Thomas W.

    2015-01-01

    Dietary methionine restriction (MR) produces a coordinated series of biochemical and physiological responses that improve biomarkers of metabolic health, increase energy expenditure, limit fat accretion, and improve overall insulin sensitivity. Inguinal white adipose tissue (IWAT) is a primary target and site of action where the diet initiates transcriptional programs linked to enhancing both synthesis and oxidation of lipid. Using a combination of ex vivo approaches to assess dietary effects on cell morphology and function, we report that dietary MR produced a 4-fold increase in multilocular, UCP1-expressing cells within this depot in conjunction with significant increases in mitochondrial content, size, and cristae density. Dietary MR increased expression of multiple enzymes within the citric acid cycle, as well as respiratory complexes I, II and III. The physiological significance of these responses, evaluated in isolated mitochondria by high resolution respirometry, was a significant increase in respiratory capacity measured using multiple substrates. The morphological, transcriptional, and biochemical remodeling of IWAT mitochondria enhances the synthetic and oxidative capacity of this tissue, and collectively underlie its expanded role as a significant contributor to the overall increase in metabolic flexibility and uncoupled respiration produced by the diet. PMID:26278039

  19. Hypoglycemic activity and acute oral toxicity of chromium methionine complexes in mice.

    PubMed

    Tang, Hai-yan; Xiao, Qing-gui; Xu, Hong-bin; Zhang, Yi

    2015-01-01

    The hypoglycemic activity of chromium methionine (CrMet) in alloxan-induced diabetic (AID) mice was investigated and compared with those of chromium trichloride hexahydrate (CrCl3·6H2O) and chromium nicotinate (CrNic) through a 15-day feeding experiment. The acute oral toxicity of CrMet was also investigated in ICR (Institute for Cancer Research) mice by a single oral gavage. The anti-diabetic activity of CrMet was explored in detail from the aspects of body weight (BW), blood glucose, triglyceride, total cholesterol, liver glycogen levels, aspartate transaminase (AST) and alanine transaminase (ALT) levels. The obtained results showed that CrMet had beneficial effects on glucose and lipid metabolism, and might possess hepatoprotective efficacy for diabetes. Daily treatment with 500 and 1000μg Cr/kg BW of CrMet in AID mice for 15 days indicated that this low-molecular-weight organic chromium complex had better bioavailability and more beneficial effects on diabetics than CrCl3·6H2O. CrMet also had advantage over CrNic in the control of AST and ALT activities. Acute toxicity studies revealed that CrMet had low toxicity potential and relatively high safety margins in mice with the LD50 value higher than 10.0g/kg BW. These findings suggest that CrMet might be of potential value in the therapy and protection of diabetes.

  20. Effect of copper- and zinc-methionine supplementation on bioavailability, mineral status and tissue concentrations of copper and zinc in ewes.

    PubMed

    Pal, D T; Gowda, N K S; Prasad, C S; Amarnath, R; Bharadwaj, U; Suresh Babu, G; Sampath, K T

    2010-04-01

    The effect of feeding Cu- and Zn-methionine to ewes was studied in a 240d feeding trial. The plasma and tissue Cu and Zn concentrations and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity were employed to assess the relative bioavailability from Cu- and Zn-methionine. The macro and micronutrient intake, utilization, plasma mineral status, tissue accumulation of Cu and Zn as well as wool concentration of Cu and Zn were studied in ewes (n=12) fed a corn-soybean meal based basal diet with 50% more Cu and Zn supplementation over the basal diet either from Cu- and Zn-sulfate (Cu-Sulf+Zn-Sulf group) or Cu- and Zn-methionine (Cu-Meth+Zn-Meth group). The average daily feed intake and body weight gain of ewes did not differ due to dietary supplementation of Cu- and Zn-methionine. However, dry matter intake was comparatively lower and thus resulted in better feed: gain in Cu- and Zn-methionine group as compared to ewes fed Cu- and Zn-sulfate. Supplementation of Cu and Zn over the basal diet either from methionine-chelated or sulfate sources resulted in increased plasma Cu and Zn as well as Cu/Zn-SOD activity on d-30, which indicated a positive correlation between plasma Cu and Zn and Cu/Zn-SOD activity. The gut absorption, liver concentrations of Cu and Zn, and liver Cu/Zn-SOD activity were significantly (P<0.01) higher in ewes supplemented with Cu- and Zn-methionine compared to Cu- and Zn-sulfate. Periodical analysis of wool samples indicated no significant difference in Cu and Zn content between Cu-and Zn-methionine and Cu- and Zn-sulfate groups. Feeding of Cu and Zn from methionine-chelated source resulted in reduced (P<0.01) excretion of Cu and Zn in feces indicating their better utilization, and this will have positive implication on environment. The gut absorption values, plasma and liver tissue concentrations of Cu and Zn supported the hypothesis that Cu- and Zn-methionine supplements have better bioavailability compared to Cu- and Zn-sulfate and Cu- and Zn

  1. Mechanistic Diversity in the RuBisCO Superfamily: The Enolase in the Methionine

    SciTech Connect

    Imker,H.; Fedorov, A.; Fedorov, E.; Almo, S.; Gerlt, J.

    2007-01-01

    D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ('enolization') of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this 'enolase' is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO- and 'enolase'-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg{sup 2+}, we sought to establish structure-function relationships for the 'enolase' reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the 'enolases' from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the 'enolase'-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated 'enolase' from G. kaustophilus (carboxylated on Lys 173) liganded with Mg{sup 2+} and 2

  2. Methionine+cystine requirement of broiler chickens fed low-density diets under tropical conditions.

    PubMed

    Aftab, Usama; Ashraf, Muhammad

    2009-03-01

    Two experiments were conducted to determine the M+C requirement of straight-run broiler chickens (Hubbard x Hubbard) during the period 4-21 (Exp. 1) and 21-40 (Exp. 2) days of age. Experiments were conducted during summer months (July-August) in open-sided houses, thus exposing chicks to chronic heat stress. Daily min-max temperature averaged 26-37C (Exp. 1) and 23-36C (Exp. 2). M+C deficient basal diets were formulated to contain low-nutrient-density, i.e., 2750 kcal per kg ME, 20.1% CP (Exp. 1), and 2780 kcal per kg ME, 17.0% CP (Exp. 2). Diets were supplemented with DL-methionine to provide total M+C level ranging from 0.64 to 0.89 % (six increments) and 0.54 to 0.79% (six increments), respectively in experiment 1 and 2. Requirements (0.95 of the maximum quadratic response) were found to be 0.77 and 0.75% total M+C, respectively for gain and feed efficiency, during 4-21 days; and 0.67% total M+C for both gain and feed efficiency during 21-40 days of age. Calculated on the digestible M+C basis, the estimates were 0.67 and 0.65% respectively for gain and feed efficiency during 4-21 days of age; and 0.60% for gain and feed efficiency during 21-40 days of age.

  3. Divergent evolution of an atypical S-adenosyl-l-methionine-dependent monooxygenase involved in anthracycline biosynthesis.

    PubMed

    Grocholski, Thadée; Dinis, Pedro; Niiranen, Laila; Niemi, Jarmo; Metsä-Ketelä, Mikko

    2015-08-11

    Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-L-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-L-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

  4. S-adenosyl-methionine-dependent methyltransferases: highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological applications.

    PubMed

    Struck, Anna-Winona; Thompson, Mark L; Wong, Lu Shin; Micklefield, Jason

    2012-12-21

    S-adenosyl methionine (SAM) is a universal biological cofactor that is found in all branches of life where it plays a critical role in the transfer of methyl groups to various biomolecules, including DNA, proteins and small-molecule secondary metabolites. The methylation process thus has important implications in various disease processes and applications in industrial chemical processing. This methyl transfer is catalysed by SAM-dependent methyltransferases (MTases), which are by far the largest groups of SAM-dependent enzymes. A significant amount is now known regarding the structural biology and enzymology of these enzymes, and, consequently, there is now significant scope for the development of new MTases and SAM analogues for applications from biomolecular imaging to biocatalytic industrial processes. This review will focus on current efforts in the manipulation of class I and V SAM-dependent MTases and the use of synthetic SAM analogues, which together offer the best prospects for rational redesign towards biotechnological applications. Firstly, metabolic engineering of organisms incorporating small-molecule MTases is discussed; this can be applied in a variety of areas from the industrial bioprocessing of flavourants and antibiotics to frontier research in biofuel production and bioremediation. Secondly, the application of MTases in combination with SAM analogues is reviewed; this allows the tagging of proteins and oligonucleotides with moieties other than the methyl group. Such tagging allows the isolation of the tagged biomolecule and aids its visualisation by a range of analytical methods. The review then summarises the potential advantages of MTase-mediated chemistry and offers some future perspectives on downstream applications.

  5. Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

    SciTech Connect

    Som, S.; Friedman, S. )

    1990-03-15

    Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either (methyl-3H)AdoMet or (35S)AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of (3H)AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported. The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.

  6. Methionine synthase and thymidylate synthase gene polymorphisms and colorectal adenoma risk: the self defense forces study.

    PubMed

    Yoshimitsu, Shinichiro; Morita, Makiko; Hamachi, Tadamichi; Tabata, Shinji; Abe, Hiroshi; Tajima, Osamu; Uezono, Kousaku; Ohnaka, Keizo; Kono, Suminori

    2012-10-01

    Folate-mediated one-carbon metabolism has been implicated in colorectal carcinogenesis. We investigated associations of functional genetic polymorphisms of methionine synthase (MTR), MTR reductase (MTRR), and thymidylate synthase (TS) with colorectal adenomas. The study subjects were 455 cases of colorectal adenomas and 1052 controls with no polyp at colonoscopy. Genotypes were determined for MTR A2756G, MTRR A66G and two polymorphisms in the TS gene, 28-bp tandem repeat polymorphism in the promoter enhancer region (TSER) and 6-bp deletion polymorphism at position 1494 in the 3' untranslated region (TS 1494del6). We also examined the alcohol-genotype and gene-gene interactions on adenoma risk. The GG genotype of MTR A2756G was associated with an increased risk of colorectal adenomas; odds ratios for AG and GG versus AA genotype were 0.99 (95% confidence interval 0.78-1.26) and 1.72 (1.04-2.82), respectively. The increase in the risk associated with MTR 2756GG genotype was evident in men with high alcohol consumption (≥30 mL/d), but not in those with low alcohol consumption (interaction P = 0.03). Men who were homozygous for the TSER double-repeat allele had a slightly decreased risk of colorectal adenomas as compared with those homozygous for the TSER triple-repeat allele. Neither MTRR A66G nor TS 1494del6 was associated with colorectal adenomas. There was no measurable interaction either between MTR A2756G and MTRR A66G or between TSER and TS 1494del6. MTR A2756G appears to be associated with colorectal adenoma risk differently according to alcohol consumption. The MTR-catalyzed reaction may play an important role in the development of colorectal adenomas.

  7. Effects of methionine synthase and methylenetetrahydrofolate reductase gene polymorphisms on markers of one-carbon metabolism.

    PubMed

    Ho, Vikki; Massey, Thomas E; King, Will D

    2013-11-01

    Genetic and nutritional factors play a role in determining the functionality of the one-carbon (1C) metabolism cycle, a network of biochemical reactions critical to intracellular processes. Genes encoding enzymes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) may determine biomarkers of the cycle including homocysteine (HCY), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). MTHFR C677T is an established genetic determinant of HCY but less is known of its effect on SAM and SAH. Conversely, the relationship between MTR A2756G and HCY remains inconclusive, and its effect on SAM and SAH has only been previously investigated in a female-specific population. Folate and vitamin B12 are essential substrate and cofactor of 1C metabolism; thus, consideration of gene-nutrient interactions may clarify the role of genetic determinants of HCY, SAM and SAH. This cross-sectional study included 570 healthy volunteers from Kingston, Ontario, Ottawa, Ontario and Halifax, Nova Scotia, Canada. Least squares regression was used to examine the effects of MTR and MTHFR polymorphisms on plasma HCY, SAM and SAH concentrations; gene-gene and gene-nutrient interactions were considered with the inclusion of cross-products in the model. Main effects of MTR and MTHFR polymorphisms on HCY concentrations were observed; however, no gene-gene or gene-nutrient interactions were found. No association was observed for SAM. For SAH, interactions between MTR and MTHFR polymorphisms, and MTHFR polymorphism and serum folate were found. The findings of this research provide evidence that HCY and SAH, biomarkers of 1C metabolism, are influenced by genetic and nutritional factors and their interactions.

  8. 11C-Methionine-PET in Multiple Myeloma: Correlation with Clinical Parameters and Bone Marrow Involvement.

    PubMed

    Lapa, Constantin; Knop, Stefan; Schreder, Martin; Rudelius, Martina; Knott, Markus; Jörg, Gerhard; Samnick, Samuel; Herrmann, Ken; Buck, Andreas K; Einsele, Hermann; Lückerath, Katharina

    2016-01-01

    Multiple myeloma (MM) remains an essentially incurable hematologic malignancy originating from clonal plasma cells. This study evaluated the usefulness of the radiotracers (11)C-methionine (MET) and (18)F-2`-deoxy-2`-fluorodeoxyglucose (FDG) for staging and re-staging in MM. 43 patients with MM underwent both MET- and FDG-PET/CT for staging or re-staging within 3±2 days. Scans were compared on a patient and on a lesion basis. Tracer uptake was correlated with the degree of bone marrow (BM) involvement and standard clinical parameters of disease activity. Additionally, BM samples were stained for L-type amino acid transporter 1 (LAT1) expression in 15 patients. MET-PET detected focal lesions (FL) in 39/43 subjects (90.7%), whereas 10 patients were missed in FDG-PET/CT (detection rate, 33/43; 76.7%; p<0.05). MET depicted more FL in 28/43 patients (65.1%; p<0.001), whereas in the remainder (34.9%, n=15) both tracers yielded comparable results. LAT1 was highly expressed on the cell surface of myeloma cells. Both FDG and MET uptake correlated significantly with biopsy-proven BM involvement (p<0.001), with MET demonstrating a stronger correlation (SUVmean, r=0.9 vs r=0.6; SUVmax, r=0.88 vs r=0.58). Abnormal beta-2-microglobulin and free light chain levels correlated with the presence of focal intramedullary lesions detected in MET- or FDG-PET/CT (MET, p=0.006 and p=0.01, respectively; FDG, p=0.02 and p=0.01). MET appears to be superior to FDG for staging and re-staging of both intra- and extramedullary MM lesions. Tracer uptake correlates with BM involvement, β2m and FLC levels and appears to be a more accurate marker of tumor burden and disease activity.

  9. 11C-Methionine-PET in Multiple Myeloma: Correlation with Clinical Parameters and Bone Marrow Involvement

    PubMed Central

    Lapa, Constantin; Knop, Stefan; Schreder, Martin; Rudelius, Martina; Knott, Markus; Jörg, Gerhard; Samnick, Samuel; Herrmann, Ken; Buck, Andreas K.; Einsele, Hermann; Lückerath, Katharina

    2016-01-01

    Multiple myeloma (MM) remains an essentially incurable hematologic malignancy originating from clonal plasma cells. This study evaluated the usefulness of the radiotracers 11C-methionine (MET) and 18F-2`-deoxy-2`-fluorodeoxyglucose (FDG) for staging and re-staging in MM. 43 patients with MM underwent both MET- and FDG-PET/CT for staging or re-staging within 3±2 days. Scans were compared on a patient and on a lesion basis. Tracer uptake was correlated with the degree of bone marrow (BM) involvement and standard clinical parameters of disease activity. Additionally, BM samples were stained for L-type amino acid transporter 1 (LAT1) expression in 15 patients. MET-PET detected focal lesions (FL) in 39/43 subjects (90.7%), whereas 10 patients were missed in FDG-PET/CT (detection rate, 33/43; 76.7%; p<0.05). MET depicted more FL in 28/43 patients (65.1%; p<0.001), whereas in the remainder (34.9%, n=15) both tracers yielded comparable results. LAT1 was highly expressed on the cell surface of myeloma cells. Both FDG and MET uptake correlated significantly with biopsy-proven BM involvement (p<0.001), with MET demonstrating a stronger correlation (SUVmean, r=0.9 vs r=0.6; SUVmax, r=0.88 vs r=0.58). Abnormal beta-2-microglobulin and free light chain levels correlated with the presence of focal intramedullary lesions detected in MET- or FDG-PET/CT (MET, p=0.006 and p=0.01, respectively; FDG, p=0.02 and p=0.01). MET appears to be superior to FDG for staging and re-staging of both intra- and extramedullary MM lesions. Tracer uptake correlates with BM involvement, β2m and FLC levels and appears to be a more accurate marker of tumor burden and disease activity. PMID:26877783

  10. A novel eukaryotic Na+ Methionine selective symporter is essential for mosquito development

    PubMed Central

    Meleshkevitch, Ella A.; Voronov, Dmitri A.; Miller, Melissa M.; Penneda, Maria; Fox, Jeffrey M.; Metzler, Ryan; Boudko, Dmitri Y.

    2013-01-01

    AeNAT5 (NCBI, ABZ81822), an orphan member of the insect-specific Nutrient Amino acid Transporter subfamily of SoLute Carrier family 6 (NAT-SLC6) and the first representative of a novel eukaryotic methionine-selective transport system (M), was cloned from cDNA of the vector mosquito, Aedes aegypti. It has orphan orthologs throughout several mosquito genomes, but not in Drosophila or outside Diptera. It shows the highest apparent affinity to L-Met (K0.5 = 0.021 mM) and its metabolites Homocysteine and Cysteine (K0.5 = 0.89 and 2.16 mM), but weakly interact with other substrates. It has a Na+ - coupled mechanism (K0.5 Na+ ~ 46 mM) with 1AA:1Na+ stoichiometry that maintains ~ 60% activity in Cl− - free media. In situ hybridization showed accumulation of AeNAT5 transcript in the absorptive and secretory epithelia, as well as in specific peripheral neurons and the central ganglia of mosquito larvae. The labeling pattern is distinct from that of the previously characterized AeNAT1. RNAi of AeNAT5 increases larval mortality during ecdysis and dramatically suppresses adult emergence. Our results showed that in addition to previously characterized broad spectra and aromatic amino acid selective transport systems, the mosquito NAT-SLC6 subfamily evolved a unique mechanism for selective absorption of sulfur-containing substrates. We demonstrated specific patterns of alimentary and neuronal transcription of AeNAT5 in mosquito larvae that is collateral with the indispensable function of this transporter in mosquito development. PMID:23748165

  11. Identification of methionine synthase (Sal k 3), as a novel allergen of Salsola kali pollen.

    PubMed

    Assarehzadegan, Mohammad Ali; Sankian, Mojtaba; Jabbari, Farahzad; Tehrani, Mohsen; Falak, Reza; Varasteh, Abdolreza

    2011-01-01

    Salsola kali pollen is a common cause of pollinosis during summer and early fall in desert and semi-desert regions. The aim of this study was the identification and characterization of Sal k 3, a new allergen from S. kali pollen. S. kali pollen extract was fractionated by SDS-PAGE and the allergenic profile was determined by IgE-immunoblotting using twelve S. kali allergic patients. Protein identification was carried out by the means of mass spectrometry. Using degenerated primers, two DNA fragments encoding N- and C-terminal domain of Sal k 3 were amplified by PCR, then cloned into the PTZ57R/T vector and sequenced. The open reading frame of Sal k 3 fragments were subcloned in the pET-32b(+) vector, expressed in E. coli, and purified by Ni2+ affinity chromatography. The IgE-binding capacity of rSal k 3 fragments was then studied by IgE-immunoblotting, inhibition assays, and skin prick tests. A 45-kDa allergen was identified as a fragment of the cobalamin-independent methionine synthase (MetE) by mass spectrometry and was detected in the sera of 8/12 (66.6%) of S. kali allergic patients. Moreover, inhibition assays demonstrated that the purified rSal k 3 fragments were similar to their counterparts in the crude extract. Sal k 3 represents a new allergen of S. kali pollen and seems to be an important allergenic compound in S. kali pollen.

  12. Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.

    PubMed

    Munkhjargal, Tserendorj; Ishizaki, Takahiro; Guswanto, Azirwan; Takemae, Hitoshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-05-15

    Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this.

  13. Effects of zinc-methionine on growth performance, intestinal flora and immune function in pigeon squabs.

    PubMed

    Wang, Y; Yi, L; Zhao, M L; Wu, J Q; Wang, M Y; Cheng, X C

    2014-01-01

    1. Different concentrations of zinc-methionine (Zn-Met) were given to pigeon squabs, and the resulting effects on growth, immune functions and intestinal microflora were investigated from hatching to 28 d of age. A total of 180 artificially hatched pigeon squabs were randomly allotted to each of three treatments with three replicates of 20 squabs. The three treatments given were either one ml (2 mg/ml) Zn-Met, one ml (10 mg/ml) Zn-Met or one ml 0.9% NaCl solution. 2. The results showed that Zn-Met improved the growth performance of squabs. The average daily and average weekly weight gain was significantly greater in squabs treated with Zn-Met than in the control group. 3. The group given 2 and 10 mg supplemental Zn-Met had heavier thymus, spleen and bursa of Fabricius than the control group at d 28. 4. Maternal antibody titres against Newcastle disease haemagglutination inhibition and alpha-naphthyl acetate esterase were significantly higher in squabs treated with supplemental 2 and 10 mg Zn-Met compared to the control group at d 14 and d 28. 5. Additionally, the squabs given supplemental 2 mg Zn-Met exhibited significantly higher Bacillaceae, Lactobacillus, Enterococcus and Bifidobacterium populations at d 14 and d 28, but lower Escherichia coli populations at d 28 compared to the control group. On the contrary, Lactobacillus, Enterococcus and Bifidobacterium populations were significantly decreased with 10 mg Zn-Met at d 28. 6. This study indicates that supplementation with Zn-Met has a positive effect on growth performance, immune function and regulation of intestinal flora in pigeons. An inclusion level of 2 mg seems to be better than 10 mg Zn-Met per day per bird.

  14. In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response

    PubMed Central

    Stephen, Abish S.; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J.; Burnett, Gary R.; Allaker, Robert P.

    2016-01-01

    Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation

  15. Histamine chloramine reactivity with thiol compounds, ascorbate, and methionine and with intracellular glutathione.

    PubMed

    Peskin, Alexander V; Winterbourn, Christine C

    2003-11-15

    Histamine is stored in granules of mast cells and basophils and released by inflammatory mediators. It has the potential to intercept some of the HOCl generated by the neutrophil enzyme, myeloperoxidase, to produce histamine chloramine. We have measured rate constants for reactions of histamine chloramine with methionine, ascorbate, and GSH at pH 7.4, of 91 M(-1)s(-1), 195 M(-1)s(-1), and 721 M(-1)s(-1), respectively. With low molecular weight thiols, the reaction was with the thiolate and rates increased exponentially with decreasing thiol group pK(a). Comparing rate constants for different chloramines reacting with ascorbate or a particular thiol anion, these were higher when there was less negative charge in the vicinity of the chloramine group. Histamine chloramine was the most reactive among biologically relevant chloramines. Consumption of histamine chloramine and oxidation of intracellular GSH were examined for human fibroblasts. At nontoxic doses, GSH loss over 10 min was slightly greater than that with HOCl, but the cellular uptake of histamine chloramine was 5-10-fold less. With histamine chloramine, GSSG was a minor product and most of the GSH was converted to mixed disulfides with proteins. HOCl gave a different profile of GSH oxidation products, with significantly less GSSG and mixed disulfide formation. There was irreversible oxidation and losses to the medium, as observed with HOCl and other cell types. Thus, histamine chloramine shows high preference for thiols both in isolation and in cells, and in this respect is more selective than HOCl.

  16. Stress Signaling in the Methionine-Choline-Deficient Model of Murine Fatty Liver Disease

    PubMed Central

    Soon, Russell K.; Yan, Jim S.; Grenert, James P.; Maher, Jacquelyn J.

    2010-01-01

    Background & Aims Stress signaling, both within and outside the endoplasmic reticulum, has been linked to metabolic dysregulation and hepatic steatosis. Methionine-choline-deficient (MCD) diets cause severe fatty liver disease and have the potential to cause many types of cellular stress. The purpose of this study was to characterize hepatic stress in MCD-fed mice and explore the relationship between MCD-mediated stress and liver injury. Methods Stress signaling was examined in mice fed MCD formulas for 4–21 days. Signaling was also evaluated in mice fed MCD formulas supplemented with clofibrate, which inhibits hepatic triglyceride accumulation. The role of the pro-apoptotic stress protein C/EBP homologous protein (CHOP) in MCD-mediated liver injury was assessed by comparing the responses of wild-type and CHOP-deficient mice to an MCD diet. Results MCD feeding caused steatohepatitis coincident with the activation of cJun N-terminal kinase and caspase-12. In contrast, MCD feeding did not activate inositol-requiring protein-1 and actually suppressed the expression of X-box protein-1s. MCD feeding caused weak stimulation of PKR-like ER-resident kinase, but robust activation of general control non-derepressible-2, followed by the phosphorylation of eukaryotic initiating factor-2α and induction of CHOP. Clofibrate eliminated MCD-mediated hepatic steatosis but did not inhibit diet-induced stress. CHOP deficiency did not alleviate, and in fact worsened, MCD-mediated liver disease. Conclusions MCD feeding causes an integrated stress response in the liver rather than a classical unfolded protein response. This stress response does not by itself lead to liver injury. CHOP, despite its identity as a mediator of stress-related cell death, does not play a central role in the pathogenesis of MCD-mediated liver disease. PMID:20682321

  17. Increasing dietary crude protein does not increase the methionine requirement in kittens.

    PubMed

    Strieker, M J; Morris, J G; Kass, P H; Rogers, Q R

    2007-12-01

    The objective of this study was to determine if the methionine (met) requirement of kittens is correlated with the concentration of dietary crude protein (CP). The study used 48 male kittens in two replications of six 4 x 4 Latin squares, each representing one concentration of met (1.5, 2.5, 3.5, 4.5, 6.0 or 9.0 g/kg diet) with four CP concentrations (150, 200, 300 and 500 g/kg diet) in 2-week periods. Cystine was present in the lowest CP diet at 5.3 g/kg diet and increased as dietary CP increased. Body weight gain, food intake, nitrogen balance and plasma amino acids, glucose, insulin, cortisol, somatomedin C, T(3) and T(4) concentrations on day 12 were measured. From breakpoint analysis of the nitrogen retention curves, the met requirement of kittens was found to be 3.1, 3.8, 3.1 and 2.4 g met/kg for the 150, 200, 300 and 500 g CP/kg diets, respectively. When met was limiting (1.5 or 2.5 g/kg diet), increasing dietary CP did not decrease, but rather increased food intake, body weight gain and nitrogen retention. Plasma met concentrations increased as dietary met increased and at 2.5-3.5 g met/kg diet were not different among kittens fed the various CP diets. Total plasma T(3) and T(4) increased significantly as dietary CP increased in kittens given the 2.5 and 4.5 g met/kg diets. Results indicate that food intake and possibly altered hormonal secretion play a role in this growth response. In conclusion, the met requirement of growing kittens, unlike omnivores and herbivores studied, was not positively correlated with the concentration of dietary CP.

  18. The Pathogenesis of Ethanol versus Methionine and Choline Deficient Diet-Induced Liver Injury

    PubMed Central

    Gyamfi, Maxwell Afari; Damjanov, Ivan; French, Samuel; Wan, Yu-Jui Yvonne

    2008-01-01

    The differences and similarities of the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) were examined. Mice (6/group) received 1 of 4 Lieber-Decarli liquid diets for 6 weeks: (1) paired-fed control diet; (2) control diet with ethanol (ethanol); (3) paired-fed methionine/choline deficient (MCD) diet; and (4) MCD plus ethanol (combination). Hepatotoxicity, histology, and gene expression changes were examined. Both MCD and ethanol induced macrovesicular steatosis. However, the combination diet produced massive steatosis with minor necrosis and inflammation. MCD and combination diets, but not ethanol, induced serum ALT levels by 1.6- and 10-fold, respectively. MCD diet, but not ethanol, also induced serum alkaline phosphatase levels suggesting bile duct injury. Ethanol increased liver fatty acid binding protein (L-FABP) mRNA and protein levels. In contrast, the combination diet decreased L-FABP mRNA and protein levels and increased hepatic free fatty acid and lipid peroxide levels. Ethanol, but not MCD, reduced hepatic S-adenosylmethionine (SAM) and GSH levels. Hepatic TNFα protein levels were increased in all treatment groups, however, IL-6, a hepatoprotective cytokine which promotes liver regeneration was increased in ethanol-fed mice (2-fold), but decreased in the combination diet-treated mice. In addition, the combination diet reduced phosphorylated STAT3 and Bcl-2 levels. While MCD diet might cause bile duct injury and cholestasis, ethanol preferentially interferes with the SAM-GSH oxidative stress pathway. The exacerbated liver injury induced by the combination diet might be explained by reduced L-FABP, increased free fatty acids, oxidative stress, and decreased IL-6 protein levels. The combination diet is an efficient model of steatohepatitis. PMID:18036573

  19. A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies.

    PubMed

    Stracke, Jan; Emrich, Thomas; Rueger, Petra; Schlothauer, Tilman; Kling, Lothar; Knaupp, Alexander; Hertenberger, Hubert; Wolfert, Andreas; Spick, Christian; Lau, Wilma; Drabner, Georg; Reiff, Ulrike; Koll, Hans; Papadimitriou, Apollon

    2014-01-01

    Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

  20. The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

    PubMed

    Anufrieva, Natalya V; Faleev, Nicolai G; Morozova, Elena A; Bazhulina, Natalia P; Revtovich, Svetlana V; Timofeev, Vladimir P; Tkachev, Yaroslav V; Nikulin, Alexei D; Demidkina, Tatyana V

    2015-09-01

    In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  1. Effect of Dietary Combination of Methionine and Fish Oil on Cellular Immunity and Plasma Fatty Acids in Infectious Bursal Disease Challenged Chickens

    PubMed Central

    Kasim, Azhar; Yong Meng, Goh; Teck Chwen, Loh; Kamalidehghan, Behnam; Soleimani Farjam, Abdoreza

    2013-01-01

    This study was carried out to investigate the modulatory effects of dietary methionine and fish oil on immune response, plasma fatty acid profile, and blood parameters of infectious bursal disease (IBD) challenged broiler chickens. A total of 300 one-day-old male broiler chicks were assigned to one of six dietary treatment groups in a 3 × 2 factorial arrangement. There were three levels of fish oil (0, 2.5 and 5.5%), and two levels of methionine (NRC recommendation and twice NRC recommendation). The results showed that the birds fed with 5.5% fish oil had higher total protein, white blood cell count, and IL-2 concentration than those of other groups at 7 days after IBD challenge. Inclusion of fish oil in diet had no effect on IFN-γ concentration. However, supplementation of methionine twice the recommendation enhanced the serum IFN-γ and globulin concentration. Neither of fish oil nor methionine supplementation affected the liver enzymes concentration. It can be suggested that a balance of moderate level of fish oil (2.5%) and methionine level (twice NRC recommendation) might enhance immune response in IBD challenged broiler chickens. PMID:24198724

  2. Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amonio acid and in crystallne sperm whale (Physeter catodon) myoglobin

    SciTech Connect

    Keniry, M.A.; Rothgeb, T.M.; Smith, R.L.; Gutowsky, H.S.; Oldfield, E.

    1983-04-12

    Deuterium (/sup 2/H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T/sub 1/) were obtained of L-(epsilon-/sup 2/H/sub 3/)methionine, L-(epsilon-/sup 2/H/sub 3/)methionine in a D,L lattice, and (S-methyl-/sup 2/H/sub 3/)methionine in the crystalline solid state, as a function of temperature, in addition to obtaining /sup 2/H T/sub 1/ and line-width results as a function of temperature on (epsilon-/sup 2/H/sub 3/)methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering. Also recorded were /sup 13/C cross-polarization ''magic-angle'' sample-spinning NMR spectra of (epsilon-/sup 13/C)methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. (JMT)

  3. High-resolution sup 13 C NMR study of the topography and dynamics of methionine residues in detergent-solubilized bacteriorhodopsin

    SciTech Connect

    Seigneuret, M.; Neumann, J.M.; Levy, D.; Rigaud, J.L. )

    1991-04-23

    The proton transport membrane protein bacteriorhodopsin has been biosynthetically labeled with (methyl-{sup 13}C)methionine and studied by high-resolution {sup 13}C NMR after solubilization in the detergent Triton X-100. The nine methionine residues of bacteriorhodopsin give rise to four well-resolved {sup 13}C resonances, two of which are shifted upfield or downfield due to nearby aromatic residues. Methionine residues located on the hydrophilic surfaces, on the hydrophobic surface, and in the interior of the protein could be discriminated by studying the effects of papain proteolysis, glycerol-induced viscosity increase, and paramagnetic broadening by spin-labels on NMR spectra. Such data were used to evaluate current models of the bacteriorhodopsin transmembrane folding and tertiary structure. T{sub 2} and NOE measurements were performed to study the local dynamics of methionine residues in bacteriorhodopsin. For the detergent-solubilized protein, hydrophilic and hydrophobic external residues undergo a relatively large extent of side chain wobbling motion while most internal residues are less mobile. In the native purple membrane and in reconstituted bacteriorhodopsin liposomes, almost all methionine residues have their wobbling motion severely restricted, indicating a large effect of the membrane environment on the protein internal dynamics.

  4. Effect of dietary combination of methionine and fish oil on cellular immunity and plasma fatty acids in infectious bursal disease challenged chickens.

    PubMed

    Maroufyan, Elham; Kasim, Azhar; Yong Meng, Goh; Ebrahimi, Mahdi; Teck Chwen, Loh; Mehrbod, Parvaneh; Kamalidehghan, Behnam; Soleimani Farjam, Abdoreza

    2013-01-01

    This study was carried out to investigate the modulatory effects of dietary methionine and fish oil on immune response, plasma fatty acid profile, and blood parameters of infectious bursal disease (IBD) challenged broiler chickens. A total of 300 one-day-old male broiler chicks were assigned to one of six dietary treatment groups in a 3 × 2 factorial arrangement. There were three levels of fish oil (0, 2.5 and 5.5%), and two levels of methionine (NRC recommendation and twice NRC recommendation). The results showed that the birds fed with 5.5% fish oil had higher total protein, white blood cell count, and IL-2 concentration than those of other groups at 7 days after IBD challenge. Inclusion of fish oil in diet had no effect on IFN- γ concentration. However, supplementation of methionine twice the recommendation enhanced the serum IFN- γ and globulin concentration. Neither of fish oil nor methionine supplementation affected the liver enzymes concentration. It can be suggested that a balance of moderate level of fish oil (2.5%) and methionine level (twice NRC recommendation) might enhance immune response in IBD challenged broiler chickens.

  5. Effects of Dietary Copper-Methionine on Matrix Metalloproteinase-2 in the Lungs of Cold-Stressed Broilers as an Animal Model for Pulmonary Hypertension.

    PubMed

    Bagheri Varzaneh, Mina; Rahmani, Hamidreza; Jahanian, Rahman; Mahdavi, Amir Hossein; Perreau, Corinne; Perrot, Gwenn; Brézillon, Stéphane; Maquart, François-Xavier

    2016-08-01

    The objective of the present study was to investigate the effects of different levels of copper (as supplemental copper-methionine) on ascites incidence and matrix metalloproteinase-2 (MMP-2) changes in the lungs of cold-stressed broilers. For this purpose, 480 1-day-old Ross 308 broiler chickens were randomly assigned to six treatments. Treatments consisted of two ambient temperatures (thermoneutral and cold stress) each combined with 0, 100, and 200 mg supplemental copper/kg as copper-methionine in a 2 × 3 factorial arrangement in a completely randomized design with four replicates. Ascites was diagnosed based on abdominal and pericardial fluid accumulation at 45 days of age. Fourty-eight broilers were killed at 38 and 45 days of age, and their lungs were collected for biological analysis. Results showed that MMP-2 increased in the lungs of ascitic broilers and that copper-methionine supplementation significantly reduced MMP-2 in cold-stressed broiler chickens. Treatments did not affect tissue inhibitor of metalloproteinase-2 (TIMP-2) at 38 and 45 days of age, and no difference was observed between 100 and 200 mg/kg copper-methionine treatments. In conclusion, copper-methionine at higher than conventional levels of supplementation decreased ascites incidence in low temperature through reduced MMP-2 concentration. Further research is warranted to investigate the effect of copper on MMP-2 concentrations in other tissues with high oxygen demand.

  6. Dimethyl sulfoxide elevates hydrogen peroxide-mediated cell death in Saccharomyces cerevisiae by inhibiting the antioxidant function of methionine sulfoxide reductase A.

    PubMed

    Kwak, Geun-Hee; Choi, Seung Hee; Kim, Hwa-Young

    2010-09-01

    Dimethyl sulfoxide (DMSO) can be reduced to dimethyl sulfide by MsrA, which stereospecifically catalyzes the reduction of methionine-S-sulfoxide to methionine. Our previous study showed that DMSO can competitively inhibit methionine sulfoxide reduction ability of yeast and mammalian MsrA in both in vitro and in vivo, and also act as a non-competitive inhibitor for mammalian MsrB2, specific for the reduction of methionine-R-sulfoxide, with lower inhibition effects. The present study investigated the effects of DMSO on the physiological antioxidant functions of methionine sulfoxide reductases. DMSO elevated hydrogen peroxide-mediated Saccharomyces cerevisiae cell death, whereas it protected human SK-Hep1 cells against oxidative stress. DMSO reduced the protein-carbonyl content in yeast cells in normal conditions, but markedly increased protein-carbonyl accumulation under oxidative stress. Using Msr deletion mutant yeast cells, we demonstrated the DMSO's selective inhibition of the antioxidant function of MsrA in S. cerevisiae, resulting in an increase in oxidative stress-induced cytotoxicity.

  7. 1H NMR studies of substrate hydrogen exchange reactions catalyzed by L-methionine gamma-lyase.

    PubMed

    Esaki, N; Nakayama, T; Sawada, S; Tanaka, H; Soda, K

    1985-07-16

    Hydrogen exchange reactions of various L-amino acids catalyzed by L-methionine gamma-lyase (EC 4.4.1.11) have been studied. The enzyme catalyzes the rapid exchange of the alpha- and beta-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of alpha-hydrogen exchange was about 40 times faster than that of the enzymatic elimination reaction of the sulfur-containing amino acids. The enzyme also catalyzes the exchange reaction of alpha- and beta-hydrogens of the following straight-chain L-amino acids which are not susceptible to elimination: norleucine, norvaline, alpha-aminobutyrate, and alanine. The exchange rates of the alpha-hydrogen and the total beta-hydrogens of L-alanine and L-alpha-aminobutyrate with deuterium followed first-order kinetics. For L-norvaline, L-norleucine, S-methyl-L-cysteine, and L-methionine, the rate of alpha-hydrogen exchange followed first-order kinetics, but the rate of total beta-hydrogen exchange decreased due to a primary isotope effect at the alpha-position. One beta-hydrogen of S-methyl-L-cysteine was exchanged faster than the other, although both the beta-hydrogens were exchanged completely with deuterium ultimately. L-Phenylalanine and L-tryptophan slowly underwent alpha-hydrogen exchange. The pro-R hydrogen of glycine was deuterated stereospecifically. None of the following amino acids were susceptible to the enzymatic hydrogen exchange: D isomers of the above amino acids, branched chain L-amino acids, acidic L-amino acids, and basic L-amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Role of structural and functional elements of mouse methionine-S-sulfoxide reductase in its subcellular distribution.

    PubMed

    Kim, Hwa-Young; Gladyshev, Vadim N

    2005-06-07

    Oxidized forms of methionine residues in proteins can be repaired by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). In mammals, three MsrBs are present, which are targeted to various subcellular compartments. In contrast, only a single mammalian MsrA gene is known whose products have been detected in both cytosol and mitochondria. Factors that determine the location of the protein in these compartments are not known. Here, we found that MsrA was present in cytosol, nucleus, and mitochondria in mouse cells and tissues and that the major enzyme forms detected in various compartments were generated from a single-translation product rather than by alternative translation initiation. Both cytosolic and mitochondrial forms were processed with respect to the N-terminal signal peptide, and the distribution of the protein occurred post-translationally. Deletion of amino acids 69-108, 69-83, 84-108, or 217-233, which contained elements important for MsrA structure and function, led to exclusive mitochondrial location of MsrA, whereas a region that affected substrate binding but was not part of the overall fold had no influence on the subcellular distribution. The data suggested that proper structure-function organization of MsrA played a role in subcellular distribution of this protein in mouse cells. These findings were recapitulated by expressing various forms of mouse MsrA in Saccharomyces cerevisiae, suggesting conservation of the mechanisms responsible for distribution of the mammalian enzyme among different cellular compartments.

  9. Alcohol, folate, methionine, and risk of incident breast cancer in the American Cancer Society Cancer Prevention Study II Nutrition Cohort.

    PubMed

    Feigelson, Heather Spencer; Jonas, Carolyn R; Robertson, Andreas S; McCullough, Marjorie L; Thun, Michael J; Calle, Eugenia E

    2003-02-01

    Recent studies suggest that the increased risk of breast cancer associated with alcohol consumption may be reduced by adequate folate intake. We examined this question among 66,561 postmenopausal women in the American Cancer Society Cancer Prevention Study II Nutrition Cohort. A total of 1,303 incident cases had accrued during the first 5 years of follow-up. Cox proportional hazards models and stratified analysis were used to examine the relationship between alcohol, dietary and total folate intake, multivitamin use, dietary methionine, and breast cancer. We observed an increasing risk of breast cancer with increasing alcohol consumption (P for trend = 0.01). In the highest category of consumption (15 or more grams of ethanol/day), the risk of breast cancer was 1.26 (95% confidence interval, 1.04-1.53) compared with nonusers. We observed this association with higher alcohol consumption for in situ, localized, and regional disease. We found no association between risk of breast cancer and dietary folate, total folate, multivitamin use, or methionine intake. Furthermore, we found no evidence of an interaction between levels of dietary folate (P for interaction = 0.10) or total folate (P for interaction = 0.61) and alcohol. Nor did we find evidence of an interaction between alcohol consumption and recent or long-term multivitamin use (P for interaction = 0.27). Our results are consistent with a positive association with alcohol but do not support an association with folate or methionine intake or an interaction between folate and alcohol intake on risk of breast cancer.

  10. Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

    PubMed Central

    Veskovic, Milena; Mladenovic, Dusan; Jorgacevic, Bojan; Stevanovic, Ivana; de Luka, Silvio

    2015-01-01

    Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency. PMID:25193852

  11. Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates.

    PubMed

    St John, G; Brot, N; Ruan, J; Erdjument-Bromage, H; Tempst, P; Weissbach, H; Nathan, C

    2001-08-14

    Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met