Dependent Methyltransferase Research Articles

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases

The gene cluster (ery) responsible for production of the macrolide antibiotic erythromycin by Saccharopolyspora erythraea is also known to contain ermE, the gene conferring resistance to the antibiotic. The nucleotide sequence has been determined of a 4.5 kb portion of the biosynthetic gene cluster, from a region lying between 3.7 kb and 8.2 kb 3' of ermE. This has revealed the presence of four complete open reading frames, including the previously known ery gene eryG, which catalyses the last step in the biosynthetic pathway. Comparison of the amino acid sequence of EryG with the sequence of other S-adenosylmethionine (SAM)-dependent methyltransferases has revealed that one of the sequence motifs previously suggested to be part of the SAM-binding site is present not only in EryG but also in many other recently sequenced SAM-dependent methyltransferases. Previous genetic studies have shown that this region also contains gene(s) involved in hydroxylation of the intermediate 6-deoxyerythronolide B. One of the three other open reading frames (eryF) in fact shows very high sequence similarity to known cytochrome P450 hydroxylases. An adjacent gene (ORF5) shows a strikingly high degree of similarity to prokaryotic and eukaryotic acyltransferases and thioesterases.

Studies on naturally occurring proteinous inhibitor for transmethylation reactions.

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.

The effect of methyltransferase inhibitors on histamine release from human dispersed lung mast cells activated with anti-human IgE and calcium ionophore A23187

Inhibitors of adenosylmethionine (AdoMet)-dependent methyltransferases reduce histamine release from enzymatically dispersed human lung mast cells activated with either anti-human IgE or calcium ionophore A23187. The ic 25 values for adenosine and 3-deazaadenosine (DZA) inhibiting anti-IgE-induced histamine release were 395 μM and 301 μM respectively. The addition of homocysteine thiolactone (Hcy) potentiated the effects of adenosine and DZA, reducing their ic 25 values to 32 μM and 10.5 μM respectively. The adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) inhibited anti-IgE-induced histamine release with an ic 50 of 162 μM. This inhibition was not potentiated by Hcy. The combination of DZA and Hcy effectively inhibited histamine release induced by concentrations of A23187 which released a similar amount of histamine to anti-IgE. However the combination was 17 times less potent against A23187- compared with anti-IgE-induced release. These observations suggest that AdoMet-dependent methyltransferases play an important role in IgE-dependent histamine release from human lung mast cells but their role in A23187-induced release is less clear.

Studies directed toward the total synthesis of sinefungin. I. Synthesis of 4-(5'-deoxyuridin-5'-yl)-4-nitrobutyronitrile, 4-(5'-deoxyadenosin-5'-yl)-4-nitrobutyramide and closely related nucleosides.

S-Adenosylhomocysteine analogues, 1-(5, 6-dideoxy-6-nitro-β-D-ribo-hexofuranosyl) uracil (7a), 9-(5, 6-dideoxy-6-nitro-β-D-ribo-hexofuranosyl) adenine (7b), 4-(5'-deoxyuridin-5'-yl)-4-nitrobutyronitrile (15) and 4-(5'-deoxyadenosin-5'-yl)-4-nitrobutyramide (20) were synthesized as potential inhibitors of S-adenosylmethionine (SAM)-dependent methyltransferases and S-adenosylhomocysteine hydrolases. The chemistry developed for the preparation of these compounds should be useful in the total synthesis of the nucleoside antibiotics sinefungin and A9145C, which are potent inhibitors of certain SAM-dependent methyltransferases.

Regulation of cobalamin and folate metabolism by methionine in human bone marrow cultures.

In cobalamin deficiency folate metabolism is disturbed. In the liver this deranged metabolism can be overcome by methionine, however, methionine failed to overcome this abnormality in bone marrow cultures from cobalamin deficient patients. In cobalamin deficient E. coli mutant bacteria, methionine under different conditions could either inhibit or potentiate the growth of the organism. This study was therefore initiated to test the effect of methionine, under different conditions, on bone marrow cultures. The defective DNA synthesis in megaloblastic bone marrow due to cobalamin deficiency could be corrected by the in vitro addition of low 0.27 mumol (40 micrograms) but not high 6.7 mumol (1 mg) amounts of methionine. This was measured by the ability of deoxyuridine to suppress the 3H-thymidine incorporation into DNA. The effect of methionine in facilitating de novo DNA synthesis is probably due to the catalytic action of SAM which activates cobalamin dependent methyltransferase enzyme thus potentiating the effect of cobalamin. In contrast high concentrations of methionine may inhibit this enzyme.

Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions.

Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage. The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions. Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+). Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5. Tn5 insertions were obtained at the known regulatory loci metJ and metK. Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion. Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12. Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained. The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12. The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12.

Potentiation of the growth inhibitory effects of adenosine 3',5'-monophosphate analogues by homocysteine

The growth inhibitory effects of N 6-monobutyryl adenosine 3',5' monophosphate (mbcAMP) and N 6, O 2'-dibutyryl adenosine 3',5' monophosphate (dbcAMP) towards Walker carcinoma in vitro are significantly potentiated by the addition of l-homocysteine to the culture medium. This effect is not seen with l-cysteine or when exogenous cAMP or prostaglandin E 2(PGE 2) replace the butyrylated cyclic nucleotide. Combinations of mbcAMP or dbcAMP and l-homocysteine significantly inhibit nucleic acid methylations. Both the butyrylated cyclic nucleotides cause an elevation of the intracellular level of S-adenosyl- l-homocysteine (SAH), a potent inhibitor of S-adenosyl- l-methionine (SAM) dependent methyltransferases, and this is significantly enhanced in combination with l-homocysteine. The increase in SAH level produced by such combinations is proportional to the inhibition of methyl group incorporation into 5-methyl cytosine and 7-methyl guanine. These results suggest that l-homocysteine potentiates accumulation of SAH in the presence of mbcAMP and dbcAMP and that the resultant inhibition of methylation accounts for the enhanced growth inhibition.

Affinity chromatography of an S-adenosylmethionine-dependent methyltransferase using immobilized S-adenosylhomocysteine : Purification of the indolethylamine N-methyltransferases of Phalaris Tuberosa

In the cases that have been studied so far, S-adenosylhomocysteine (SAH) is a powerful inhibitor of S-adenosylmethionine (SAM) binding to SAM-dependent methyltransferases. We deduced, from the available data on the binding of SAM and SAH analogues to SAM dependent methyltransferases, that linkage of SAH through the carboxyl group to an immobilized support would lead to a more general affinity adsorbent for SAM-dependent methyltransferases than linkage through other functional groups. This paper describes the synthesis of this affinity adsorbent and its use to purify the two indolethylamine N-methyltransferases of Phalaris tuberosa.

The effect of vitamin B 12 inhibition [formula omitted]: Impaired folate polyglutamate biosynthesis indicating that 5-methyltetrahydropteroylglutamate is not its usual substrate

Using 5-[ 14C] methyltetrahydropteroylglutamate and [ 3H] pteroylglutamate it was found that vitamin B 12 dependent methyltransferase can be inhibited in vivo in mice by nitrous oxide. Examination of the liver [ 3H] folates showed as expected that both controls and nitrous oxide treated mice had synthesised the reduced 5-methyl form. However, the controls had converted [ 3H] almost exclusively into a polyglutamate with virtually no monoglutamate present, while nitrous oxide treated mice had failed to add glutamates to over half of the incorporated folate. Thus in vivo demethylation may be necessary prior to polyglutamate biosynthesis and act as a method of controlling folate accumulation, or be used to create a concentration gradient with the circulating folate.

N5, 10-methylenetetrahydrofolate reductase deficiency and schizophrenia: A working hypothesis

Publisher Summary This chapter presents a working theory of N 5.10 -methylenetetrahydrofolate reductase deficiency and schizophrenia. Patients with genetically determined deficient activity of N 5.10 -methylenetetrahydrofolate reductase had a progressive history of aberrant behavior and disturbed thought processes diagnosed independently by several psychiatrists as schizophrenia. Treatment with folic acid increased the availability of N5-methyltetrahydrofolate, as judged by the alleviation of homocystine excretion. Concomitantly, there was a striking improvement in behavior and mental processes. As a working hypothesis, we suggest that decreased methylation of biogenic amines by the N 5 -methylenetetrahydrofolate -dependent methyltransferase can be a significant factor in the psychotic manifestations of the syndrome of schizophrenia.

Biosynthesis of choline in vitro

Phospholipids play an essential role as a barrier between cell content and the extracellular environment and regulate various cell signaling processes. Phosphatidylcholine (PtdCho) is one of the most abundant phospholipids in plant, animal, and some prokaryote cell membranes. In plants and some parasites, the biosynthesis of PtdCho begins with the amino acid serine, followed mainly through a phosphoethanolamine N-methyltransferase (PMT)-mediated biosynthetic pathway to phosphocholine (pCho). Because the PMT-mediated pathway, referred to as the phosphobase methylation pathway, produces a series of important primary and specialized metabolites for plant development and stress response, understanding the PMT enzyme is a key aspect of engineering plants with improved stress tolerance and fortified nutrients. Importantly, given the very limited phylogenetic distribution of PMTs, functional analysis and the identification of inhibitors targeting PMTs have potential and positive impacts in humans and in veterinary and agricultural fields. Here, we describe detailed basic knowledge and practical research methods to enable the systematic study of the biochemical and biophysical functions of PMT. The research methods described in this chapter are also applicable to the studies of other ubiquitous S-adenosyl-l-methionine (SAM)-dependent methyltransferases in all kingdoms.