Reduction of methionine sulfoxide to methionine by Escherichia coli.

L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml.     

The quantitative oxidation of methionine to methionine sulfoxide by peroxynitrite

Both peroxynitrous acid and peroxynitrite react with methionine, k(acid) = (1.7 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 8.6 +/- 0.2 M(-1) s(-1), respectively, and with N-acetylmethionine k(acid) = (2.8 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 10.0 +/- 0.1 M(-1) s(-1), respectively, to form sulfoxides. In contrast to the results of Pryor et al. (1994, Proc. Natl. Acad. Sci. USA 91, 11173-11177), a linear correlation between k(obs) and [met] was obtained. Surprisingly, for every two sulfoxides and nitrites formed, one peroxynitrite is converted to nitrate. Thus, methionine also catalyzes the isomerization of peroxynitrite to nitrate. Neither the pH nor the concentration of methionine affected the distribution of the yields of nitrite, nitrate, and methionine sulfoxide, which were the only products detected. No products other than nitrite, nitrate, and methioninesulfoxide could be detected. The reactions of methionine and N-acetylmethionine with peroxynitrous acid and peroxynitrite are simple bimolecular reactions that do not involve an activated form of peroxynitrous acid or of peroxynitrite. Nitrite, produced together with methionine sulfoxide, or present as a contamination in the peroxynitrite preparation, is not innocuous, but oxidizes methionine by one electron, which leads to the formation of methional and ethylene.     

Chromatographic separation of methionine,methionine sulphoxide,methionine sulphone,and their products of oral microbial metabolism

The metabolic pathways of methionine sulphoxide and methionine sulphone were investigated employing a combination of gas chromatography, thin-layer chromatography, paper chromatography, and radioactive methods of analyses. Gas chromatographic analysis demonstrated that methionine, methionine sulphoxide, and methionine sulphone all yielded qualitatively similar volatile sulphur compounds, namely, methyl mercaptan, dimethyl disulphide, and small amounts of dimethyl sulphide. The study indicated that the principal pathway of methionine sulphoxide and methionine sulphone metabolism is mediated via methionine which gives rise to methyl mercaptan, part of which is oxidized to dimethyl disulphide. Whereas methionine sulphoxide was readily reduced to methionine, the reduction of methionine sulphone proceeded at a considerably slower rate.     

N-terminal methionine removal and methionine metabolism in Saccharomyces cerevisiae

Methionine aminopeptidase (MetAP) catalyzes removal of the initiator methionine from nascent polypeptides. In eukaryotes, there are two forms of MetAP, type 1 and type 2, whose combined activities are essential, but whose relative intracellular roles are unclear. Methionine metabolism is an important aspect of cellular physiology, involved in oxidative stress, methylation, and cell cycle. Due to the potential of MetAP activity to provide a methionine salvage pathway, we evaluated the relationship between methionine metabolism and MetAP activity in Saccharomyces cerevisiae. We provide the first demonstration that yeast MetAP1 plays a significant role in methionine metabolism, namely, preventing premature activation of MET genes through MetAP function in methionine salvage. Interestingly, in cells lacking MetAP1, excess methionine dramatically inhibits cell growth. Growth inhibition is independent of the ability of methionine to repress MET genes and does not result from inhibition of synthesis of another metabolite, rather it results from product inhibition of MetAP2. Inhibition by methionine is selective for MetAP2 over MetAP1. These results provide an explanation for the previously observed dominance of MetAP1 in terms of N-terminal processing and cell growth in yeast. Additionally, differential regulation of the two isoforms may be indicative of different intracellular roles for the two enzymes.     

Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase.

Methionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR.     

Myeloperoxidase-mediated oxidation of methionine

The myeloperoxidase-mediated oxidation of methionine was studied using a purified canine myeloperoxidase preparation. The system required the simultaneous presence of myeloperoxidase, H₂O₂, and a halide anion. When 0.1 mM H₂O₂ was used, the system has a pH optimum of approximately pH 5–5.5. Bromide and iodide were much more effective than chloride in the myeloperoxidase-mediated oxidation of methionine. Horseradish peroxidase was unable to oxidize methionine whether chloride or iodide was used. In contrast, lactoperoxidase oxidized methionine in the presence of iodide but not chloride. Based on studies of (1) the effect of various inhibitors and singlet oxygen quenchers, as well as (2) the effect of D₂O on the oxidation of methionine, by the myeloperoxidase system, OCl^?, or methylene blue, it was shown that the oxidation of methionine by the myeloperoxidase system was not mediated by OCl^? or ¹O₂. The mechanism of the myeloperoxidase-mediated oxidation of methionine remains unclear. However, it may be one mechanism by which the myeloperoxidase system damage microorganisms.     

Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli

Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in alpha-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.     

The biosynthesis of methionine

Summary The methylation of the thiol group of homocysteine leading to methionine is a biochemical reaction of particular interest since it represents a crossroad of the action of two vitamins, folic acid and cobalamin, both in bacteria and in animals. This enzymic reaction, its mechanism and its regulation which has been studied in detail in several laboratories is discussed. Another route which does not require cobalamin occurs in bacteria and plants. Bacteria possessing both pathways of methionine synthesis show regulatory interconnections between them. Plants which generally are devoid of cobalamin synthesize methionine solely by the cobalaminindependent pathway the mechanism of which is as yet not fully understood.an invited article.     

Cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from N5-methyltetrahydrofolate to homocysteine, producing tetrahydrofolate and methionine. Insufficient availability of cobalamin, or inhibition of methionine synthase by exposure to nitrous oxide, leads to diminished activity of this enzyme. In humans, severe inhibition of methionine synthase results in the development of megaloblastic anemia, and eventually in subacute combined degeneration of the spinal cord. It also results in diminished intracellular folate levels and a redistribution of folate derivatives. In this review, we summarize recent progress in understanding the catalysis and regulation of this important enzyme from both bacterial and mammalian sources. Because inhibition of mammalian methionine synthase can restrict the incorporation of methyltetrahydrofolate from the blood into cellular folate pools that can be used for nucleotide biosynthesis, it is a potential chemotherapeutic target. The review emphasizes the mechanistic information that will be needed in order to design rational inhibitors of the enzyme.     

Analyses of methionine sulfoxide reductase activities towards free and peptidyl methionine sulfoxides

There have been insufficient kinetic data that enable a direct comparison between free and peptide methionine sulfoxide reductase activities of either MsrB or MsrA. In this study, we determined the kinetic parameters of mammalian and yeast MsrBs and MsrAs for the reduction of both free methionine sulfoxide (Met-O) and peptidyl Met-O under the same assay conditions. Catalytic efficiency of mammalian and yeast MsrBs towards free Met-O was >2000-fold lower than that of yeast fRMsr, which is specific for free Met-R-O. The ratio of free to peptide Msr activity in MsrBs was 1:20-40. In contrast, mammalian and yeast MsrAs reduced free Met-O much more efficiently than MsrBs. Their k(cat) values were 40-500-fold greater than those of the corresponding MsrBs. The ratio of free to peptide Msr activity was 1:0.8 in yeast MsrA, indicating that this enzyme can reduce free Met-O as efficiently as peptidyl Met-O. In addition, we analyzed the in vivo free Msr activities of MsrBs and MsrAs in yeast cells using a growth complementation assay. Mammalian and yeast MsrBs, as well as the corresponding MsrAs, had apparent in vivo free Msr activities. The in vivo free Msr activities of MsrBs and MsrAs agreed with their in vitro activities.     

Metabolism of glycosaminoglycans in rats during methionine deficiency and administration of excess methionine

Methionine deficiency in rats caused significant decrease in the concentration of many sulphated glycosaminoglycans in the aorta and other tissues, while administration of excess methionine caused an increase in these constituents. The activity of some important biosynthetic enzymes decreased in methionine deficiency and increased on administration of excess methionine. No uniform pattern was observed in the changes in the activity of enzymes concerned with degradation of glycosaminoglycans. The concentration of 3′-phosphoade-nosine 5′-phosphosulphate and the activities of the sulphate activating system and sulpho-transferase were decreased in methionine deficiency, while feeding excess methionine did not affect these parameters as compared to controls.     

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

In order to improve the methionine yield of the isolate B. heali, attempts were made to isolate mutants resistant to the methionine analogue DL-ethionine after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The minimum inhibitory concentration (MIC) of ethionine for B. heali was found to be 2 mM. After mutagenesis and screening, five mutants resistant to 50 mM of ethionine were isolated. The yield of the best ethionine resistant mutant, B. heali Br EthR, was 13 mg/l of methionine medium under optimum cultivation conditions.     

Pharmacokinetic studies of methionine in rats using deuterium-labeled methionine quantitative assessment of remethylation

The pharmacokinetics of methionine has been studied in rats by means of stable isotope methodology. After the i.v. bolus injection of [2H7]methionine (5 mg/kg body wt.), the plasma concentrations of [2H7]methionine, demethylated [2H4]homocysteine and remethylated [2H4]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry. The half-life for [2H7]methionine were 35.0 +/- 6.9 min. The appearance of the metabolites, [2H4]homocysteine and [2H4]methionine, in the plasma was very rapid. The fraction of [2H7]methionine that remethylated to [2H4]methionine through [2H4]homocysteine were 0.185 +/- 0.028. The administered [2H7]methionine did not influence the plasma levels of endogenous methionine and homocysteine. The present stable isotope methodology has made it possible to evaluate the pharmacokinetics of methionine, including the estimation of remethylation.