The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Ruma Banerjee - One of the best experts on this subject based on the ideXlab platform.
-
Kinetic and thermodynamic characterization of the common polymorphic variants of human Methionine Synthase reductase.
Biochemistry, 2004Co-Authors: Horatiu Olteanu, Kirsten R. Wolthers, Nigel S. Scrutton, Andrew W. Munro, Ruma BanerjeeAbstract:Human Methionine Synthase reductase (MSR) is a protein containing both FAD and FMN, and it reactivates Methionine Synthase that has lost activity due to oxidation of cob(I)alamin to cob(II)alamin. In this study, anaerobic redox titrations were employed to determine the midpoint reduction potentials for the flavin cofactors in two highly prevalent polymorphic variants of MSR, I22/L175 and M22/S175. The latter is a genetic determinant of plasma homocysteine levels and has been linked to premature coronary artery disease, Down's syndrome, and neural tube defects. The I22/L175 polymorphism has been described in a homocystinuric patient. Interestingly, this polymorphism is in the extended linker region between the two flavin domains, which may mediate or facilitate interaction with Methionine Synthase. In MSR I22/L175, the FMN potentials are −103 mV (oxidized/semiquinone) and −175 mV (semiquinone/hydroquinone) at pH 7.0 and 25 °C, and the corresponding FAD potentials are −252 and −285 mV, respectively. For the...
-
Redundancy in the pathway for redox regulation of mammalian Methionine Synthase: reductive activation by the dual flavoprotein, novel reductase 1.
The Journal of biological chemistry, 2003Co-Authors: Horatiu Olteanu, Ruma BanerjeeAbstract:Abstract Methionine Synthase is an essential cobalamin-dependent enzyme in mammals that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to give tetrahydrofolate and Methionine. It is oxidatively labile and requires for its sustained activity an auxiliary repair system that catalyzes a reductive methylation reaction. Genetic and biochemical studies have demonstrated that the soluble dual flavoprotein oxidoreductase, Methionine Synthase reductase, serves as a redox partner for Methionine Synthase in an NADPH-dependent reaction. However, three reports suggest the possibility of redundancy in this redox pathway. First, a hyperhomocysteinemic patient has been reported who has an isolated functional deficiency of Methionine Synthase but appears to be distinct from the cblE and cblG classes of patients with defects in Methionine Synthase reductase and Methionine Synthase, respectively. Second, another dual flavoprotein oxidoreductase with significant homology to Methionine Synthase reductase, NR1, has been described recently, but its function is unknown. Third, Methionine Synthase can be activated in vitro by a two-component redox system comprised of soluble cytochrome b5 and P450 reductase. In this study, we demonstrate a function for human NR1 in vitro. It is able to fully activate Methionine Synthase in the presence of soluble cytochrome b5 with a Vmax of 2.8 ± 0.1 μmol min–1 mg–1 protein, which is comparable with that seen with Methionine Synthase reductase. The KactNR1 is 1.27 ± 0.16 μm, and a 20-fold higher stoichiometry of reductase to Methionine Synthase is required for NR1 versus Methionine Synthase reductase, suggesting that it may represent a minor pathway in the cell, assuming that the two proteins are present at similar levels.
-
Differences in the efficiency of reductive activation of Methionine Synthase and exogenous electron acceptors between the common polymorphic variants of human Methionine Synthase reductase.
Biochemistry, 2002Co-Authors: Horatiu Olteanu, Troy Munson, Ruma BanerjeeAbstract:Methionine Synthase reductase (MSR) catalyzes the conversion of the inactive form of human Methionine Synthase to the active state of the enzyme. This reaction is of paramount physiological importance since Methionine Synthase is an essential enzyme that plays a key role in the Methionine and folate cycles. A common polymorphism in human MSR has been identified (66A → G) that leads to replacement of isoleucine with Methionine at residue 22 and has an allele frequency of 0.5. Another polymorphism is 524C → T, which leads to the substitution of serine 175 with leucine, but its allele frequency is not known. The I22M polymorphism is a genetic determinant for mild hyperhomocysteinemia, a risk factor for cardiovascular disease. In this study, we have examined the kinetic properties of the M22/S175 and I22/S175 and the I22/L175 and I22/S175 pairs of variants. EPR spectra of the semiquinone forms of variants I22/S175 and M22/S175 are indistinguishable and exhibit an isotropic signal at g = 2.00. In addition, the...
-
Human Methionine Synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent Methionine Synthase activation.
The Journal of biological chemistry, 2001Co-Authors: Horatiu Olteanu, Ruma BanerjeeAbstract:Abstract Methionine Synthase is a key enzyme in the Methionine cycle that catalyzes the transmethylation of homocysteine to Methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent two-protein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of Methionine Synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative Methionine Synthase reductase in the cblE class of patients with an isolated functional deficiency of Methionine Synthase suggested a role for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H. Q., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S., and Gravel, R. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3059–3064). In this study, we have cloned and expressed the cDNA encoding human Methionine Synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of Methionine Synthase at a level that is comparable with that seen in thein vitro assay that utilizes artificial reductants. Methionine Synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine Synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 μmol min−1 mg−1 at 25 °C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is ∼100-fold slower than that of P-450 reductase. TheK m for NADPH is 2.6 ± 0.5 μm, and the K act for Methionine Synthase reductase is 80.7 ± 13.7 nm for NADPH-dependent activity of Methionine Synthase.
-
Targeted Disruption of the Methionine Synthase Gene in Mice
Molecular and cellular biology, 2001Co-Authors: Deborah A. Swanson, Ruma Banerjee, Mei-lan Liu, Priscilla J. Baker, Lisa Garrett, Michael L. Stitzel, Michelle A. Harris, Barry Shane, Lawrence C. BrodyAbstract:Alterations in homocysteine, Methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine Synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and Methionine. Human patients with Methionine Synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypoMethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of Methionine Synthase deficiency, we utilized gene-targeting technology to inactivate the Methionine Synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and Methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in Methionine Synthase. Whether any human patients with Methionine Synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that Methionine Synthase-deficient patients have residual Methionine Synthase activity or that humans have a compensatory mechanism that is absent in mice.
William A. Gibbons - One of the best experts on this subject based on the ideXlab platform.
-
The Effect of Ethanol and Its Metabolites Upon Methionine Synthase Activity In Vitro
Alcohol (Fayetteville N.Y.), 1998Co-Authors: Susan H. Kenyon, Anna Nicolaou, William A. GibbonsAbstract:The association of alcoholism with macrocytic anaemia has lead to investigation of the role of cobalamin-dependent Methionine Synthase in mediating alcohol toxicity. Several studies have found that long-term ingestion of large quantities of ethanol causes inhibition of liver Methionine Synthase activity in vivo: however, ethanol has not been found to inhibit the enzyme directly. The effect of ethanol and its breakdown products, acetate and acetaldehyde, on highly purified rat liver Methionine Synthase was tested in vitro. Enzyme activity was not inhibited by ethanol or acetate. Acetaldehyde was found to inhibit Methionine Synthase activity, with an apparent IC50 of 2 mM. The reported inhibition by acetaldehyde was found to become irreversible over time. Acetaldehyde-induced inhibition of liver Methionine Synthase activity is thus proposed as the most likely explanation of the reported in vivo effect of ethanol upon Methionine Synthase.
-
The Inactivation of Methionine Synthase in Isolated Rat Hepatocytes by Sodium Nitroprusside
European journal of biochemistry, 1997Co-Authors: Anna Nicolaou, Susan H. Kenyon, Catherine J. Waterfield, William A. GibbonsAbstract:Methionine Synthase, the enzyme that catalyses the transfer of a methyl group from 5-methyl tetrahydrofolate to homocysteine via the cofactor methylcobalamin, is one of the two established mammalian enzymes that utilise a biologically active vitamin B-12 derivative. Through its substrates, products and downstream metabolites, Methionine Synthase is directly involved in the sulphur amino acid pathways, polyamine biosynthesis, biological methylations and one-carbon-unit transfers. Rat liver Methionine Synthase was shown to be inactivated by the nitric oxide donor sodium nitroprusside. The inactivation occurred during the treatment of isolated rat hepatocytes in a time-dependent and dose-dependent manner with an apparent IC50 value of 170 μM. Highly purified rat liver Methionine Synthase was inactivated in a partially irreversible manner with an apparent IC50 value of 10 μM. The inactivation has been attributed to nitric oxide released by sodium nitroprusside. Since biomolecules possessing transition state metals are targets for nitric oxide, the possibility of a nitric oxide-cobalamin interaction could explain the observed inactivation. Nitric oxide is directly involved in different aspects of liver metabolic functions both under physiological and pathological conditions like sepsis and inflammation. The nitric-oxide-induced inactivation of Methionine Synthase could offer a rational explanation for the cellular and cyto-toxic effects of this highly reactive molecule.
-
Stimulation in vitro of vitamin B12-dependent Methionine Synthase by polyamines.
Biochemical Journal, 1996Co-Authors: Susan H. Kenyon, Anna Nicolaou, Tamara Ast, William A. GibbonsAbstract:Vitamin B12-dependent Methionine Synthase is an important enzyme for sulphur amino acid, folate polyamine metabolism, S-adenosylMethionine metabolism and also in the methylation pathway of DNA, RNA, proteins and lipids. Consequently, studies aiming at exploring the control and regulation of Methionine Synthase are of particular interest. Here we report the modulation of enzyme activity in vitro by polyamines. Although putrescine, cadaverine, spermine and spermidine all stimulated enzyme activity, the last two were the most potent, causing increases in enzyme activity up to 400%. The EC50 for spermine was determined as 8 microM and for spermidine 40 microM. The physiological concentration for spermine has been reported to be 15-19 microM. Spermine was found to increase both the Km and the V(max) with respect to methyltetrahydrofolate for the enzyme. These data support the hypothesis that spermine and spermidine are feedback regulators of Methionine Synthase both in vivo and in vitro and are consistent with the polyamines' regulating cell signalling pathways.
-
In vitro inactivation of mammalian Methionine Synthase by nitric oxide
European journal of clinical investigation, 1996Co-Authors: Anna Nicolaou, Susan H. Kenyon, Tamara Ast, John M. Gibbons, William A. GibbonsAbstract:The research described here provides one mechanism of uniting current effects of nitric oxide (NO) with the elevated levels of homocysteine detected in patients with cardiovascular and other disease. Time- and dose-dependent studies of the inhibition of purified mammalian Methionine Synthase by NO were performed. The in vitro study gave an effective IC 50 value of 3 μmol L -1 . Methionine Synthase converts cellular homocysteine to Methionine and is a major enzyme in the biosynthetic pathways for folates, S-adenosylMethionine and biological methylations, sulphur amino acids and polyamines. Nitric oxide-induced inactivation of Methionine Synthase alters the levels of these metabolites and could therefore provide a connection between the cardiovascular effects of NO, the plasma homocysteine levels and cardiovascular diseases that is complementary to the more traditional NO-induced stimulation of guanylate cyclase and the convertion of homocysteine to oxidized sulphur amino acids.
-
In vitro phosphorylation of vitamin B12 dependent Methionine Synthase by protein kinase A.
Biochemical Society transactions, 1995Co-Authors: Susan H. Kenyon, Tamara Ast, William A. GibbonsAbstract:MethionineSynthase( L-homocysteine-S-methyltransferase) is a cytosolic enzyme catalysing the formation of Methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate. It is one of two vitamin B,, (cobalamin) dependent enzymes, in mammals, the other being methylmalonyl CoA mutase. In bacteria a cobalamin independent form of Methionine Synthase also exists’. In vitamin B,, deficiency Methionine Synthase activity is affected. Symptoms of vitamin B,, deficiency include: pernicious anaemia, neurological and psychiatric abnormalities*. Inhibition of Methionine Synthase is considered to be important in producing, at least some, of these symptoms as it stands at the convergence point of the folate metabolic pathway as well as the sulphur amino acid pathway. Inactivation of Methionine Synthase will thus affect folate metabolism, Methionine, polyamine & S-adenosylMethionine production (and hence methylation reactions) and homocysteine metabolism’. I t can be seen that Methionine Synthase is biologically important. So it was of interest to us to study the regulation of this enzyme. There are a couple of reports about the regulation of gene transcription in bacteria4. I t has also been suggested that enzyme activity could be regulated by controlling the avaliability of cobalamin. This seems unlikely as most of the body’s vitamin B,, is stored in the enzyme bound form’. Nitric oxide has been shown to inhibit Methionine Synthase, although it is uncertain whether this could be a physiological or toxic effect6. A logical first step to initiate studies on possible mechanisms for regulating Methionine Synthase was to ascertain whether protein kinases changed the activity of the enzyme, as kinases are well known regulators of many enzymatic processes. Here we report preliminary investigations into the possible regulation of Methionine Synthase by protein kinase A. Methionine Synthase activity was assayed for, using a modified version of the method of Weisbech et al’ and incubating for various times up to 30 mins. Protein kinase A, 5 picounits (from Sigma 1.3 pmolar units is equivalent to Ipg) was introduced into the standard assay mixture, along with ImM ATP, 2.2mM MgClz and 12.6pM CAMP. Control assays contained all elements except the kinase, to account for the magnesium induced stimulation of the Methionine Synthase. A time dependent study was performed and can be seen in figure I . The experiment was performed on two Methionine Synthase samples at different stages of purification6. The purer sample showed a four fold increase in stimulation (table I ) . I 1 I I I I I Sample
Roy A. Gravel - One of the best experts on this subject based on the ideXlab platform.
-
Restricted role for Methionine Synthase reductase defined by subcellular localization
Molecular genetics and metabolism, 2008Co-Authors: D.s. Froese, Jun Zhang, R. Dumas, W.m. Schoel, Matthias Amrein, Roy A. GravelAbstract:Methionine Synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of Methionine Synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target the cytosol where Methionine Synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target mitochondria was also suggested by the work of Leal et al. [N.A. Leal, H. Olteanu, R. Banerjee, T.A. Bobik, Human ATP:Cob(I)alamin adenosyltransferase and its interaction with Methionine Synthase reductase, J. Biol. Chem. 279 (2004) 47536-47542.] who showed that it can act as the reducing enzyme in combination with MMAB (ATP:Cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol, but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin.
-
Metabolic derangement of Methionine and folate metabolism in mice deficient in Methionine Synthase reductase
Molecular genetics and metabolism, 2007Co-Authors: C. Lee Elmore, Daniel Leclerc, Rima Rozen, Erica D. Watson, Teodoro Bottiglieri, Natalia I. Krupenko, Sergey A. Krupenko, James C. Cross, Roy A. GravelAbstract:Abstract Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form Methionine. In humans, Methionine Synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of Methionine Synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying Methionine Synthase reductase, an enzyme essential for the activity of Methionine Synthase. This model is a hypomorph, with reduced Methionine Synthase reductase activity, thus avoiding the lethality associated with the absence of Methionine Synthase activity. Mtrr gt/gt mice have increased plasma homocyst(e)ine, decreased plasma Methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrr gt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocyst(e)inemia may be useful in understanding biological effects of elevated homocyst(e)ine.
-
Effects of polymorphisms of Methionine Synthase and Methionine Synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study.
Atherosclerosis, 2003Co-Authors: Paul F. Jacques, Roy A. Gravel, Andrew G. Bostom, Jacob Selhub, Sharron Rich, R. Curtis Ellison, John H. Eckfeldt, Rima RozenAbstract:Abstract The metabolism of homocysteine requires contributions of several enzymes and vitamin cofactors. Earlier studies identified a common polymorphism of methylenetetrahydrofolate reductase that was associated with mild hyperhomocysteinemia. Common variants of two other enzymes involved in homocysteine metabolism, Methionine Synthase and Methionine Synthase reductase, have also been identified. Methionine Synthase catalyzes the remethylation of homocysteine to form Methionine and Methionine Synthase reductase is required for the reductive activation of the cobalamin-dependent Methionine Synthase. The Methionine Synthase gene (MTR) mutation is an A to G substitution, 2756A→G, which converts an aspartate to a glycine codon. The Methionine Synthase reductase gene (MTRR) mutation is an A to G substitution, 66A→G, that converts an isoleucine to a Methionine residue. To determine if these polymorphisms were associated with mild hyperhomocysteinemia, we investigated subjects from two of the NHLBI Family Heart Study field centers, Framingham and Utah. Total plasma homocysteine concentrations were determined after an overnight fast and after a 4-h Methionine load test. MTR and MTRR genotype data were available for 677 and 562 subjects, respectively. The geometric mean fasting homocysteine was unrelated to the MTR or MTRR genotype categories (AA, AG, GG). After a Methionine load, a weak positive association was observed between change in homocysteine after a Methionine load and the number of mutant MTR alleles ( P -trend=0.04), but this association was not statistically significant according to the overall F-statistic ( P =0.12). There was no significant interaction between MTR and MTRR genotype or between these genotypes and any of the vitamins with respect to homocysteine concentrations. This study provides no evidence that these common MTR and MTRR mutations are associated with alterations in plasma homocysteine.
-
Infant Methionine Synthase variants and risk for spina bifida
Journal of medical genetics, 1999Co-Authors: Gary M. Shaw, Roy A. Gravel, Daniel Leclerc, Karen Todoroff, Richard H. Finnell, Edward J. Lammer, Rima RozenAbstract:Editor—The mechanisms underlying the reduction in risk for neural tube defect (NTD) affected pregnancies by maternal folic acid supplementation are unknown. Current research efforts are focusing on candidate genes that encode enzymes involved in folate metabolism. Since homocysteine levels have been raised in some women who delivered infants with NTDs,1 2 the enzymes involved in both folate and homocysteine metabolism have received particular attention. Some, but not all, epidemiological data suggest an association between the C677T mutation in methylenetetrahydrofolate reductase (MTHFR) and increased risk for NTDs.3-6 No association between mutations in cystathionine β Synthase and NTD risk has been observed.7 The epidemiological data pertaining to genetic variants of Methionine Synthase are quite limited.8 9 Methionine Synthase catalyses the methylation of homocysteine to Methionine using 5-methyltetrahydrofolate, the product of the MTHFR reaction, as the carbon donor. Thus, the investigation of genetic variants of Methionine Synthase is a prudent area of enquiry for exploring …
-
human Methionine Synthase cdna cloning and identification of mutations in patients of the cblg complementation group of folate cobalamin disorders
Human Molecular Genetics, 1996Co-Authors: Daniel Leclerc, Rima Rozen, David S. Rosenblatt, Benedicte Christensen, E Campeau, Philippe Goyette, C E Adjalla, M Ross, P Eydoux, Roy A. GravelAbstract:Methionine Synthase catalyzes the remethylation of homocysteine to Methionine in a methylcobalamin-dependent reaction. We used specific regions of homology within the Methionine Synthase sequences of several lower organisms to clone a human Methionine Synthase cDNA by a combination of RT-PCR and inverse PCR. The enzyme is 1265 amino acids in length and contains the seven residue structure-based sequence fingerprint identified for cobalamin-containing enzymes. The gene was localized to chromosome 1q43 by the FISH technique. We have identified one missense mutation and a 3 bp deletion in patients of the cblG complementation group of inherited homocysteine/folate disorders by SSCP and sequence analysis, as well as an amino acid substitution present in high frequency in the general population. We discuss the possibility that a mild deficiency of Methionine Synthase activity could be associated with mild hyperhomocysteinemia, a risk factor for cardiovascular disease and possibly neural tube defects.
Rowena G. Matthews - One of the best experts on this subject based on the ideXlab platform.
-
Vitamin‐B12‐Independent Methionine Synthase from a Higher Plant (Catharanthus Roseus)
European Journal of Biochemistry, 2008Co-Authors: Johannes Eichel, Rowena G. Matthews, Julio C. González, Michael Hotze, Joachim SchröderAbstract:Methionine Synthases catalyze the formation of Methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in l-Methionine biosynthesis, and it also serves to regenerate the methyl group of 5-adenosylMethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a Methionine Synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50 % identity with the cobalamin-independent Methionine Synthase from Escherichia coli (MetE) and 41 % identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require 5-adenosylMethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent Methionine Synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plant extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of Methionine biosynthesis in plants.
-
mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin dependent Methionine Synthase
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Diane A Hall, Craig Vander W Kooi, Chad N Stasik, Shawn Y Stevens, Erik R P Zuiderweg, Rowena G. MatthewsAbstract:Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin Synthase, pyruvate formate lyase, and cobalamin-dependent Methionine Synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide Synthase, and Methionine Synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and Methionine Synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.
-
Interaction of flavodoxin with cobalamin-dependent Methionine Synthase.
Biochemistry, 2000Co-Authors: Diane A Hall, Martha L. Ludwig, Tuajuanda C. Jordan-starck, Rachel O. Loo, Rowena G. MatthewsAbstract:Cobalamin-dependent Methionine Synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and Methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylMethionine serves as the methyl donor. In humans, the electron is thought to be provided by Methionine Synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and Methionine Synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and Methionine Synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of Methionine Synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of Methionine Synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and Methionine Synthase.
-
methylenetetrahydrofolate reductase and Methionine Synthase biochemistry and molecular biology
European Journal of Pediatrics, 1998Co-Authors: Rowena G. Matthews, Christal A Sheppard, Celia W GouldingAbstract:Methylenetetrahydrofolate reductase and cobalamin-dependent Methionine Synthase catalyze the penultimate and ultimate steps in the biosynthesis of Methionine in prokaryotes, and are required for the regeneration of the methyl group of Methionine in mammals. Defects in either of these enzymes can lead to hyperhomocysteinemia. The sequences of the human methylenetetrahydrofolate reductase and Methionine Synthase are now known, and show clear homology with their bacterial analogues. Mutations in both enzymes that are known to occur in humans and to be associated with hyperhomocysteinemia affect residues that are conserved in the bacterial enzymes. Structure/function studies on the bacterial proteins, summarized in this review, are therefore relevant to the function of the human enzymes; in particular studies on the effects of bacterial mutations analogous to those causing hyperhomocysteinemia in human may shed light on the defects associated with these mutations.
-
Cobalamin-dependent Methionine Synthase and serine hydroxymethyltransferase: targets for chemotherapeutic intervention?
Advances in enzyme regulation, 1998Co-Authors: Rowena G. Matthews, James T. Drummond, Heather K. WebbAbstract:Chemotherapeutic drugs targeted at folate-dependent reactions have typically been directed at a limited number of target enzymes: dihydrofolate reductase, thymidylate Synthase, and GAR and AICAR transformylase. This review discusses two other potential targets for chemotherapeutic inhibition: cobalamin-dependent Methionine Synthase and serine hydroxymethyltransferase. Brief reviews of the catalytic properties of these two enzymes are presented, and possible strategies for chemotherapeutic intervention are discussed.
Anna Nicolaou - One of the best experts on this subject based on the ideXlab platform.
-
Inhibition of cobalamin-dependent Methionine Synthase by substituted benzo-fused heterocycles.
The FEBS journal, 2006Co-Authors: Elizabeth C. Banks, Stephen W. Doughty, Steven M. Toms, Richard T. Wheelhouse, Anna NicolaouAbstract:The cobalamin-dependent cytosolic enzyme, Methionine Synthase (EC.2.1.1.13), catalyzes the remethylation of homocysteine to Methionine using 5-methyltetrahydrofolate as the methyl donor. The products of this remethylation--Methionine and tetrahydrofolate--participate in the active Methionine and folate pathways. Impaired Methionine Synthase activity has been implicated in the pathogenesis of anaemias, cancer and neurological disorders. Although the need for potent and specific inhibitors of Methionine Synthase has been recognized, there is a lack of such agents. In this study, we designed, synthesized and evaluated the inhibitory activity of a series of substituted benzimidazoles and small benzothiadiazoles. Kinetic analysis revealed that the benzimidazoles act as competitive inhibitors of the rat liver Methionine Synthase, whilst the most active benzothiadiazole (IC(50) = 80 microm) exhibited characteristics of uncompetitive inhibition. A model of the methyltetrahydrofolate-binding site of the rat liver Methionine Synthase was constructed; docking experiments were designed to elucidate, in greater detail, the binding mode and reveal structural requirements for the design of inhibitors of Methionine Synthase. Our results indicate that the potency of the tested compounds is related to a planar region of the inhibitor that can be positioned in the centre of the active site, the presence of a nitro functional group and two or three probable hydrogen-bonding interactions.
-
The Effect of Ethanol and Its Metabolites Upon Methionine Synthase Activity In Vitro
Alcohol (Fayetteville N.Y.), 1998Co-Authors: Susan H. Kenyon, Anna Nicolaou, William A. GibbonsAbstract:The association of alcoholism with macrocytic anaemia has lead to investigation of the role of cobalamin-dependent Methionine Synthase in mediating alcohol toxicity. Several studies have found that long-term ingestion of large quantities of ethanol causes inhibition of liver Methionine Synthase activity in vivo: however, ethanol has not been found to inhibit the enzyme directly. The effect of ethanol and its breakdown products, acetate and acetaldehyde, on highly purified rat liver Methionine Synthase was tested in vitro. Enzyme activity was not inhibited by ethanol or acetate. Acetaldehyde was found to inhibit Methionine Synthase activity, with an apparent IC50 of 2 mM. The reported inhibition by acetaldehyde was found to become irreversible over time. Acetaldehyde-induced inhibition of liver Methionine Synthase activity is thus proposed as the most likely explanation of the reported in vivo effect of ethanol upon Methionine Synthase.
-
The Inactivation of Methionine Synthase in Isolated Rat Hepatocytes by Sodium Nitroprusside
European journal of biochemistry, 1997Co-Authors: Anna Nicolaou, Susan H. Kenyon, Catherine J. Waterfield, William A. GibbonsAbstract:Methionine Synthase, the enzyme that catalyses the transfer of a methyl group from 5-methyl tetrahydrofolate to homocysteine via the cofactor methylcobalamin, is one of the two established mammalian enzymes that utilise a biologically active vitamin B-12 derivative. Through its substrates, products and downstream metabolites, Methionine Synthase is directly involved in the sulphur amino acid pathways, polyamine biosynthesis, biological methylations and one-carbon-unit transfers. Rat liver Methionine Synthase was shown to be inactivated by the nitric oxide donor sodium nitroprusside. The inactivation occurred during the treatment of isolated rat hepatocytes in a time-dependent and dose-dependent manner with an apparent IC50 value of 170 μM. Highly purified rat liver Methionine Synthase was inactivated in a partially irreversible manner with an apparent IC50 value of 10 μM. The inactivation has been attributed to nitric oxide released by sodium nitroprusside. Since biomolecules possessing transition state metals are targets for nitric oxide, the possibility of a nitric oxide-cobalamin interaction could explain the observed inactivation. Nitric oxide is directly involved in different aspects of liver metabolic functions both under physiological and pathological conditions like sepsis and inflammation. The nitric-oxide-induced inactivation of Methionine Synthase could offer a rational explanation for the cellular and cyto-toxic effects of this highly reactive molecule.
-
Stimulation in vitro of vitamin B12-dependent Methionine Synthase by polyamines.
Biochemical Journal, 1996Co-Authors: Susan H. Kenyon, Anna Nicolaou, Tamara Ast, William A. GibbonsAbstract:Vitamin B12-dependent Methionine Synthase is an important enzyme for sulphur amino acid, folate polyamine metabolism, S-adenosylMethionine metabolism and also in the methylation pathway of DNA, RNA, proteins and lipids. Consequently, studies aiming at exploring the control and regulation of Methionine Synthase are of particular interest. Here we report the modulation of enzyme activity in vitro by polyamines. Although putrescine, cadaverine, spermine and spermidine all stimulated enzyme activity, the last two were the most potent, causing increases in enzyme activity up to 400%. The EC50 for spermine was determined as 8 microM and for spermidine 40 microM. The physiological concentration for spermine has been reported to be 15-19 microM. Spermine was found to increase both the Km and the V(max) with respect to methyltetrahydrofolate for the enzyme. These data support the hypothesis that spermine and spermidine are feedback regulators of Methionine Synthase both in vivo and in vitro and are consistent with the polyamines' regulating cell signalling pathways.
-
In vitro inactivation of mammalian Methionine Synthase by nitric oxide
European journal of clinical investigation, 1996Co-Authors: Anna Nicolaou, Susan H. Kenyon, Tamara Ast, John M. Gibbons, William A. GibbonsAbstract:The research described here provides one mechanism of uniting current effects of nitric oxide (NO) with the elevated levels of homocysteine detected in patients with cardiovascular and other disease. Time- and dose-dependent studies of the inhibition of purified mammalian Methionine Synthase by NO were performed. The in vitro study gave an effective IC 50 value of 3 μmol L -1 . Methionine Synthase converts cellular homocysteine to Methionine and is a major enzyme in the biosynthetic pathways for folates, S-adenosylMethionine and biological methylations, sulphur amino acids and polyamines. Nitric oxide-induced inactivation of Methionine Synthase alters the levels of these metabolites and could therefore provide a connection between the cardiovascular effects of NO, the plasma homocysteine levels and cardiovascular diseases that is complementary to the more traditional NO-induced stimulation of guanylate cyclase and the convertion of homocysteine to oxidized sulphur amino acids.