Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5'-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.
Plant Mol Biol 1996 Feb
PMID:A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana. 862 5

In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation.
J Mol Biol 1996 Apr 19
PMID:The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties. 863 80

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue.
J Mol Biol 1996 Jun 07
PMID:Functional analysis of conserved motifs in EcoP15I DNA methyltransferase. 865 25

The major symptoms of Parkinson disease (PD) are tremors, hypokinesia, rigidity, and abnormal posture, caused by the degeneration of dopamine (DA) neurons in the substantia nigra (SN) and deficiency of DA in the neostriatal DA terminals. Norepinephrine (NE) and serotonin (5-HT) levels in the neostriatum and tyrosine hydroxylase and melanin pigments in the substantia nigra are also decreased, and brain cholinergic activity is increased. The cause of PD is unknown, but PD is an age-related disorder, suggesting that changes that occur during the aging process may help to precipitate PD. Methylation increases in aging animals. Increased methylation can deplete DA, NE, and 5-HT; increase acetylcholine; and cause hypokinesia and tremors. These effects are similar to changes seen in PD, and interestingly also, they are similar to some of the changes that are associated with the aging process. It is suggested, therefore, that increased methylation may be an inducing factor in parkinsonism. Accordingly, the effects of an increase in methylation in the brain of rats were studied. S-adenosylmethionine (AdoMet), the limiting factor in the methylation process, was injected into the lateral ventricle of rats. Specific behavioral changes that resemble changes seen in PD were investigated. The results showed that AdoMet caused tremors, rigidity, hypokinesia, and depleted DA. The hypokinetic effects of a single dose of AdoMet lasted for about 90 min. AdoMet has a dose-dependent hypokinetic effect. A dose of 9.4 nmol reduced movement time (MT) by 68.9% and increased rest time (RT) by 20.7%, and a dose of 400 nmol reduced MT by 92.4% and increased RT by 27.6%. The normethyl analog of AdoMet, S-adenosylhomocysteine, did not cause hypokinesia or tremors, but it blocked the AdoMet-induced motor effects. L-dopa, the precursor of DA, also blocked the AdoMet-induced motor effects. These data suggest that the methyl group of AdoMet as well as DA depletion are involved in the AdoMet-induced motor effects. A dose of 0.65 mumol of AdoMet depleted DA in the ipsilateral caudate nucleus (CN) or neostriatum by 50.1%, and DA in the contralateral CN was reduced by 9.3%. Double the dose of AdoMet did not increase the depletion of DA on the ipsilateral CN, but DA in the contralateral CN was decreased by 26.3%. Taken together, the results suggest that increased methylation may contribute to the symptoms of PD.
Mol Chem Neuropathol 1995 Dec
PMID:Striatal dopamine depletion, tremors, and hypokinesia following the intracranial injection of S-adenosylmethionine: a possible role of hypermethylation in parkinsonism. 874 29

Multiple sequence alignment of distantly related viral proteins remains a challenge to all currently available alignment methods. The hidden Markov model approach offers a new, flexible method for the generation of multiple sequence alignments. The results of studies attempting to infer appropriate parameter constraints for the generation of de novo HMMs for globin, kinase, aspartic acid protease, and ribonuclease H sequences by both the SAM and HMMER methods are described.
Proc Int Conf Intell Syst Mol Biol 1996
PMID:Parameterization studies for the SAM and HMMER methods of hidden Markov model generation. 887 15

Mammalian S-adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver-type and non-hepatic-type enzymes. To investigate the possible role of AdoMet synthetase in proliferating cells, we have examined the expression of these two isozyme genes in regenerating rat liver after partial hepatectomy using Northern blot analysis. In normal adult rat liver the non-hepatic-type isozyme mRNA was not detectable, however, when partial hepatectomy was performed, there was an obvious appearance of the non-hepatic-type enzyme mRNA after operation. The levels of non-hepatic-type isozyme mRNA was peaked at 4h and maintained the level at least till 8 h after operation, then decreased. In addition, the liver-type AdoMet synthetase gene expression was also induced by partial hepatectomy with similar time course. These results indicate that these two AdoMet synthetase isozymes may play an important role during the prereplicative phase which precedes DNA synthesis.
Biochem Mol Biol Int 1996 Nov
PMID:Expression of S-adenosylmethionine synthetase isozyme genes in regenerating rat liver after partial hepatectomy. 895 39

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

We have investigated the disposition of potentially endotoxic homocysteine (Hcy) and its transsulfuration metabolite cysteine (Cys) in 98 individuals (age range 20-66 years). Our study reports on the relationship between Hcy and two important dietary factors likely to influence plasma levels of this thiol: dietary folate and dietary methionine. chi2 analysis shows a low frequency of elevated plasma Hcy at high folate intake. This frequency for Hcy >10 micromol/liter with a folate intake >350 microg/day is significant (P < 0.02). The data reflect a tendency for elevated Hcy values to be associated with low dietary folate, although many subjects with a low dietary folate also had a low plasma Hcy. Intake of dietary methionine was found to be significantly higher in males than in females (P < .0001). This may account for the looser relationship between Hcy and its transsulfuration product, Cys, in females (R2 = 0.30) compared to males (R2 = 0.73), since conversion of methionine to SAM in males would activate cystathionine beta synthase and commit excess Hcy to transsulfuration. The generally lower methionine intake of females means that more Hcy is utilized in the remethylation cycle in which methionine is produced from the de novo methyl group of 5-methyltetrahydrofolate or from the preformed methyl group of betaine. Clearly a Hcy moiety locked up in remethylation would be further removed from Cys, the end product of transsulfuration. An increasing number of studies are clarifying the relationship between Hcy, folate, and other B vitamins. However, less attention seems to be given to the influence of dietary methionine on the disposition of Hcy. The present study supports biochemical theory and indicates that more focus should be given to the effect of dietary methionine on Hcy. These findings have particular significance since even moderate increases in plasma Hcy are associated with a toxic vascular effect. Consequently the relationship between dietary folate and Hcy levels should be a factor in evaluating recommended dietary allowances for this vitamin. The simplicity of our dietary folate questionnaire also raises the possibility of a screening test in which individuals can ascertain whether their folate intake is adequate to reduce Hcy levels to a benign value.
Biochem Mol Med 1996 Dec
PMID:The influence of dietary folate and methionine on the metabolic disposition of endotoxic homocysteine. 898 31

The reconstruction of phylogenetic history is predicated on being able to accurately establish hypotheses of character homology, which involves sequence alignment for studies based on molecular sequence data. In an empirical study investigating nucleotide sequence alignment, we inferred phylogenetic trees for 43 species of the Apicomplexa and 3 of Dinozoa based on complete small-subunit rDNA sequences, using six different multiple-alignment procedures: manual alignment based on the secondary structure of the 18S rRNA molecule, and automated similarity-based alignment algorithms using the PileUp, ClustalW, TreeAlign, MALIGN, and SAM computer programs. Trees were constructed using neighboring-joining, weighted-parsimony, and maximum-likelihood methods. All of the multiple sequence alignment procedures yielded the same basic structure for the estimate of the phylogenetic relationship among the taxa, which presumably represents the underlying phylogenetic signal. However, the placement of many of the taxa was sensitive to the alignment procedure used; and the different alignments produced trees that were on average more dissimilar from each other than did the different tree-building methods used. The multiple alignments from the different procedures varied greatly in length, but aligned sequence length was not a good predictor of the similarity of the resulting phylogenetic trees. We also systematically varied the gap weights (the relative cost of inserting a new gap into a sequence or extending an already-existing gap) for the ClustalW program, and this produced alignments that were at least as different from each other as those produced by the different alignment algorithms. Furthermore, there was no combination of gap weights that produced the same tree as that from the structure alignment, in spite of the fact that many of the alignments were similar in length to the structure alignment. We also investigated the phylogenetic information content of the helical and nonhelical regions of the rDNA, and conclude that the helical regions are the most informative. We therefore conclude that many of the literature disagreements concerning the phylogeny of the Apicomplexa are probably based on differences in sequence alignment strategies rather than differences in data or tree-building methods.
Mol Biol Evol 1997 Apr
PMID:Effects of nucleotide sequence alignment on phylogeny estimation: a case study of 18S rDNAs of apicomplexa. 910 Mar 73

"beta CATECHIN", a preparation of natural vitamins and phytosubstances, has been designed as an "universal antioxidant' drink. Having previously demonstrated the scavenging action of a variety of free radicals by "beta CATECHIN", we now report the effect of its long term administration on the life span of senesescence accelerated mice (SAM-P8), a murine model system that offers many characteristics of mammalian aging with a shortened life span. Both male and female SAM (age: 10 weeks) maintained on "beta CATECHIN" containing drinking water (1ml/kg b.w) showed significant extension of the mean life span as compared to their respective controls that received normal drinking water. Results show that administration of "beta CATECHIN" increased the 50% mean survival rate by 8 weeks in case of female, and 7.5 weeks for male SAM. In addition to offering fresh evidence to the "free radical theory of aging", the results emphasize on the importance of a daily supplement of natural antioxidants, especially as a combination, to achieve the goal of a long and disease-free life.
Biochem Mol Biol Int 1997 Apr
PMID:Effect of "beta CATECHIN" on the life span of senescence accelerated mice (SAM-P8 strain). 913 32


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