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Query: UNIPROT:P06889 (Mol)
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Crystals of a cobalamin-binding domain (M(r) = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5, starting from solutions of intact (M(r) = 133,000) cobalamin-dependent methionine synthase. The crystals are orthorhombic in space group P2(1)2(1)2(1), with cell dimensions a = 96.9 A, b = 55.4 A, c = 103.8 A. For two molecules per asymmetric unit, the calculated VM value is 2.45 A3/Da. A native data set has been collected to 3 A resolution.
J Mol Biol 1992 May 20
PMID:Crystallization and preliminary X-ray diffraction studies of the cobalamin-binding domain of methionine synthase from Escherichia coli. 159 36

The biosynthesis of methionine in Escherichia coli is under complex regulation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S-adenosyl-methionine which functions as a corepressor for the MetJ protein. Recently, a new regulatory locus, metR, has been identified. The MetR protein is required for both metE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA-binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metabolic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metE gene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group.
Mol Microbiol 1991 Jul
PMID:Regulation of methionine synthesis in Escherichia coli. 194 95

We have cloned the genes for the two homocysteine transmethylases of Escherichia coli K12. The vitamin B12-independent enzyme is encoded by the metE gene while the metH gene codes for the vitamin B12-requiring enzyme. Overexpression of the gene products and Tn1000 mutagenesis have enabled the metE and metH gene products to be identified as 99 kDa and 130 kDa polypeptides, respectively. The truncated polypeptides generated by Tn1000 insertion were used to determine the direction of transcription of the metE and metH genes. Negative complementation suggests that the MetH enzyme exists as an oligomer. Investigation of the expression of the chromosomal- and plasmid-encoded gene products confirms that metE is subject to negative control by vitamin B12 and methionine, and that metH is under positive control by the cofactor and negative control by methionine. For vitamin B12 and methionine to act as regulatory effectors in metE control, functional metH and metJ genes are required, respectively. The use of stable Tn1000-generated fragments of the metE product as electrophoretic markers for the plasmid-encoded metE gene product demonstrated that the two regulatory proteins involved in negative control of metE are present in excess. Under conditions whereby both forms of negative metE control are non-functional, the metE gene product represented about 90% of the total protein, and cell growth was severely impaired.
Mol Gen Genet 1988 Jan
PMID:Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli. 283 Apr 70

The effects of phenytoin (DPH) on folate metabolism have been studied in female Swiss Webster mice. Doses of DPH which produce steady-state plasma levels of DPH in the therapeutic range of 10-20 micrograms/ml were found to decrease plasma folate levels. In addition, the in vivo oxidation rate of [14C] formate and [2-14C] histidine to 14CO2 was increased. The increased metabolic rates were accompanied by a decrease in the hepatic activity of N5, N10-methylenetetrahydrofolate (5, 10-CH2-H4folate) reductase. N5-methyltetrahydrofolate-homocysteine transmethylase (methionine synthase); EC 2.1.1.13) activity in the liver was unchanged. The distribution of folates in the liver was determined by high-pressure liquid chromatography (HPLC) and it was found that the concentration of tetrahydrofolate (H4folate) was increased in DPH-treated mice whereas the concentration of N5-methyltetrahydrofolate was decreased. These effects were observed in mice treated with DPH for 4 days, but not in mice given a single DPH injection 24 hr previously. Decreased activity of hepatic 5, 10-CH2-H4folate reductase is postulated to account for the other effects which were observed. Decreased activity presumably results in increased tissue concentrations of 5, 10-CH2-H4folate, which is in equilibrium with its dissociation products, H4folate and formaldehyde. Increased concentrations of H4folate lead to increases in the oxidation rate of formate and histidine. These effects on folate metabolism may have important implications in the pharmacological and toxicological effects of DPH.
Mol Pharmacol 1984 May
PMID:Decreased hepatic 5, 10-methylenetetrahydrofolate reductase activity in mice after chronic phenytoin treatment. 637 25

Nitrous oxide administration to experimental animals leads to significant alterations in the hepatic folate pathway. This pathway is closely linked to the metabolism of methionine and S-adenosylmethionine (AdoMet), two compounds that play a central role in biologically important methylation reactions. This study was carried out to assess whether nitrous oxide administration to animals can affect the metabolism of AdoMet and the AdoMet-dependent methylation reactions. Exposure of rats to a mixture of nitrous oxide and oxygen (50:50) for 2 hr reduced hepatic AdoMet levels. However, when methionine was administered to these rats, hepatic AdoMet rapidly increased to levels that were significantly higher than those observed in air-exposed animals. Concomitant with this increase, there was a significant and marked increase in the rate of methylation of phospholipids and carboxymethylation of proteins. Thus, nitrous oxide, in addition to its inhibitory effect on 5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthase, EC 2.1.1.13) activity, possesses another effect. It increases the rate of conversion of exogenously administered methionine into AdoMet with a subsequent increase in the rate of methylation of key cellular constituents.
Mol Pharmacol 1983 Jul
PMID:Effect of nitrous oxide and methionine treatments on hepatic S-adenosylmethionine and methylation reactions in the rat. 686 21

Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.
Plant Mol Biol 1995 Dec
PMID:Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes. 861 21

Two cDNA clones coding for S-adenosyl-L-methionine synthase (SAMs, EC 2.5.1.6) have been isolated from a cDNA library of gibberellic acid-treated unpollinated pea ovaries. Both cDNAs were sequenced showing a high degree of identity but coding for different SAMs polypeptides. The presence of two SAMs genes in pea was further confirmed by Southern analysis. Expression of the SAMs genes in the pea plant was found at different levels in vegetative and reproductive tissues. We characterized the expression levels of SAMs genes during the development or senescence of pea ovaries. Northern analysis showed that transcription of SAMs genes in parthenocarpic fruits was upregulated by auxins in the same manner as in fruits from pollinated ovaries. In both pollinated and 2,4-dichlorophenoxyacetic acid-treated ovaries, and benzyladenine, although able to induce parthenocarpic development, did not affect SAMs mRNA levels. These data are consistent with an active participation of auxins in the upregulation of SAMs during fruit setting in pea and suggest that, at the molecular level, parthenocarpic development of pea ovaries is different for gibberellin- and cytokinin-treated ovaries than for auxin-induced parthenocarpic biosynthesis since treatment of the ovaries with aminoethoxyvinylglycine resulted in a delay of senescence and prevention of SAMs mRNA accumulation. A possible mechanism for hormonal regulation of SAMs during ovary development is discussed.
Plant Mol Biol 1996 Feb
PMID:Hormonal regulation of S-adenosylmethionine synthase transcripts in pea ovaries. 862 12

Inborn errors resulting in isolated functional methionine synthase deficiency fall into two complementation groups, cblG and cblE. Using biochemical approaches we demonstrate that one cblG patient has greatly reduced levels of methionine synthase while in another, the enzyme is specifically impaired in the reductive activation cycle. The biochemical data suggested that low levels of methionine synthase activity in the first patient may result from mutations in the catalytic domains of the enzyme, reduced transcription, or generation of unstable message or protein. Using Northern analysis, we demonstrate that the molecular basis for the biochemical phenotype in this patient is associated with greatly diminished steady-state levels of methionine synthase mRNA. The biochemical data on the second patient cell line implicated mutations specific to reductive activation, a function that is housed in the C-terminal AdoMet-binding domain and the intermediate B12-binding domain, in the highly homologous bacterial enzyme. We have detected two mutations in a compound heterozygous state, one that results in conversion of a conserved proline (1173) to a leucine residue and the other a deletion of an isoleucine residue (881). The crystal structure of the C-terminal domain of the Escherichia coli MS predicts that the Pro to Leu mutation could disrupt activation since it is embedded in a sequence that makes direct contacts with the bound AdoMet. Deletion of isoleucine in the B12-binding domain would result in shortening of a beta-sheet. Our data provide the first evidence for mutations in the methionine synthase gene being culpable for the cblG phenotype. In addition, they suggest directly that mutations in methionine synthase can lead to elevated homocysteine, implicated both in neural tube defects and in cardiovascular diseases.
Hum Mol Genet 1996 Dec
PMID:Defects in human methionine synthase in cblG patients. 896 36

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.
Hum Mol Genet 1996 Dec
PMID:Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. 896 37

Flavodoxin reductase from Escherichia coli is an FAD-containing oxidoreductase that transports electrons between flavodoxin or ferredoxin and NADPH. Together with flavodoxin, the enzyme is involved in the reductive activation of three E. coli enzymes: cobalamin-dependent methionine synthase, pyruvate formate lyase and anaerobic ribonucleotide reductase. An additional function for the oxidoreductase appears to be to protect the bacteria against oxygen radicals. The three-dimensional structure of flavodoxin reductase has been solved by multiple isomorphous replacement, and has been refined at 1.7 A to an R-value of 18.4% and Rfree 24.8%. The monomeric molecule contains one beta-sandwich FAD domain and an alpha/beta NADP domain. The overall structure is similar to other reductases of the NADP-ferredoxin reductase family in spite of the low sequence similarities within the family. Flavodoxin reductase lacks the loop which is involved in the binding of the adenosine moiety of FAD in other FAD binding enzymes of the superfamily but is missing in the FMN binding phthalate dioxygenase reductase. Instead of this loop, the adenine interacts with an extra tryptophan at the C terminus. The FAD in flavodoxin reductase has an unusual bent conformation with a hydrogen bond between the adenine and the isoalloxazine. This is probably the cause of the unusual spectrum of the enzyme. There is a pronounced cleft close to the isoalloxazine that appears to be well suited for binding of flavodoxin/ferredoxin. Two extra short strands of the NADP-binding domain probably act as an anchor point for the binding of flavodoxin.
J Mol Biol 1997 Apr 25
PMID:The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 A resolution. 914 48


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