The physiological role of reversible methionine oxidation

Sulfur-containing amino acids such as cysteine and methionine are particularly vulnerable to oxidation. Oxidation of cysteine and methionine in their free amino acid form renders them unavailable for metabolic processes while their oxidation in the protein-bound state is a common post-translational modification in all organisms and usually alters the function of the protein. In the majority of cases, oxidation causes inactivation of proteins. Yet, an increasing number of examples have been described where reversible cysteine oxidation is part of a sophisticated mechanism to control protein function based on the redox state of the protein. While for methionine the dogma is still that its oxidation inhibits protein function, reversible methionine oxidation is now being recognized as a powerful means of triggering protein activity. This mode of regulation involves oxidation of methionine to methionine sulfoxide leading to activated protein function, and inactivation is accomplished by reduction of methionine sulfoxide back to methionine catalyzed by methionine sulfoxide reductases. Given the similarity to thiol-based redox-regulation of protein function, methionine oxidation is now established as a novel mode of redox-regulation of protein function. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Effects of excess methionine on collagen metabolism: a study on newborn rat skin

We studied the biochemical effects of excess methionine intake on the skin of newborn rats. Group 1 pups were intubated with methionine dissolved in 0.1 ml physiological saline solution in the amount of 0.1 g/100 body wt as a control using a gastric needle. Group 2 pups were given 0.2 g/100 g in the same manner as group 1 as an experimental group. They were intubated every other day from day 3 to 13. On Day 15, [14C]proline was injected intraperitoneally into pups and their skin was removed. 14C total and hydroxyproline uptake was examined in the tissue as well as in the sequential extracts. Although excess methionine intake by the pups did not alter the collagen content of their skin, it caused an increase in the content of type III collagen and a decrease in crosslinked collagen. In addition, newly synthesized collagen in the neutral salt extract increased in the excess methionine group, indicating that crosslinked collagen decreases as excess methionine was intubated. The present study demonstrated that excess methionine in the early lactational period altered the nature of the skin collagen of suckling newborn rat pups.     

Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [Me-13C, Me-2H(3)]methionine sulfoxide and [Me-13C, Me-2H(3)]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0 +/- 1.0 microM (means +/- SD, n = 8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.     

Betaine-Homocysteine Transmethylase in Pseudomonas denitrificans, a Vitamin B12 Overproducer

A pantothenate-methionine auxotroph (J741) of Pseudomonas denitrificans was isolated whose growth requirement for methionine could not be satisfied by known precursors of the amino acid, including homocysteine. However, some "methyl rich" compounds such as betaine and dimethylacetothetin (DMT) could satisfy the requirement. S-Methyl-methionine and S-adenosylmethionine were ineffective. Extracts were found to contain an enzyme, betaine-homocysteine transmethylase (BHTase), that uses betaine or DMT as a methyl donor and homocysteine as an acceptor to produce methionine. Growth of J741 in methionine leads to a total repression of the BHTase, whereas the use of DMT leads to a three- to sixfold stimulation of enzyme synthesis compared to betaine-grown cells. The pantothenate requirement is unrelated to the methionine auxotrophy, since the growth of other single auxotrophic mutants as well as revertants of J741 still have their methionine requirement satisfied by betaine or DMT. Another methionine auxotroph that could not use betaine for growth was devoid of BHTase activity.     

Thioredoxin-dependent redox regulation of cellular signaling and stress response through reversible oxidation of methionines

The sensitive oxidations of sulfur containing amino acids (i.e., cysteines and methionines) commonly control protein function, and act as important signaling mechanisms to modify metabolic responses to environmental stressors. Mechanisms associated with cysteine oxidation to form sulfenic acid and disulfides (i.e., cystine and glutathione adducts), and their reversibility through thioredoxin-dependent mechanisms, are broadly appreciated as important regulatory mechanisms that control the function of a range of different proteins. Less commonly understood are the cellular consequences of methionine oxidation to form methionine sulfoxide, as the structural requirements for their thioredoxin-dependent reduction by methionine sulfoxide reductases limit the reversibility of methionine oxidation to sequences within surface exposed and conformationally disordered regions of proteins. Surface exposed methionines are commonly involved in molecular recognition between transient protein signaling complexes, where their oxidation disrupts productive protein-protein interactions linked to a range of cellular responses. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress responses in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the physical basis for differences in the sensitivity of individual methionines within plant and animal calmodulin to reactive oxygen species (ROS), the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes. It is suggested that, in combination with high-throughput proteomic methods and current generation informatics tools, these mechanistic insights permit useful predictions of oxidatively sensitive signaling proteins that act as redox and stress sensors in response to methionine oxidation.     

Bioengineering approaches to improve the nutritional values of seeds by increasing their methionine content

Methionine is a nutritionally essential, sulfur-containing amino acid found at low levels in plants and in their seeds. Methionine levels often limit the plant’s value as a source of dietary protein for humans and animals. Despite recent accumulated knowledge of methionine metabolism in vegetative tissues, there is still little knowledge of methionine metabolism in seeds. In this review, we summarize the efforts made to increase the levels of methionine in seeds using genetic engineering methods. Two main approaches were tested: the first was the expression of methionine-rich storage proteins in a seed-specific manner, with the goal of trapping the soluble methionine into protein form and competing with the catabolism of methionine to its essential metabolites. However, in many cases this approach does not lead to a significant increase in total methionine content. The second approach aimed to increase the soluble content of methionine in seeds. Despite the nutritional significance of methionine, the factors regulating soluble methionine content in seeds are not fully known. Evidence shows that two biosynthetic pathways, the aspartate family pathway and the S-methylmethionine pathway, contribute to soluble methionine content in seeds. However, their roles in soluble methionine synthesis and accumulation are not fully understood. In recent years, combinations of these two approaches have been tested; however, they have not yet succeeded in elevating total methionine content in seeds. More emphasis should be applied to gaining knowledge of the biosynthesis pathways that could contribute to an increase in methionine content in seeds.     

Mitochondrially encoded methionine is inversely related to longevity in mammals

Methionine residues in proteins react readily with reactive oxygen species making them particularly sensitive to oxidation. However, because oxidized methionine can be reduced back in a catalyzed reaction, it has been suggested that methionine residues act as oxidant scavengers, protecting not only the proteins where they are located but also the surrounding macromolecules. To investigate whether methionine residues may be selected for or against animal longevity, we carried out a meta-examination of mitochondrial genomes from mammalian species. Our analyses unveiled a hitherto unnoticed observation: mitochondrially encoded polypeptides from short-lived species are enriched in methionine when compared with their long-lived counterparts. We show evidence suggesting that methionine addition to proteins in short-lived species, rather than methionine loss from proteins in long-lived species, is behind the reported difference in methionine usage. The inverse association between longevity and methionine, which persisted after correction for body mass and phylogenetic interdependence, was paralleled by the methionine codon AUA, but not by the codon AUG. Although nuclear encoded mitochondrial polypeptides exhibited higher methionine usage than nonmitochondrial proteins, correlation with longevity was only found within the group of those polypeptides located in the inner mitochondrial membrane. Based on these results, we propose that short-lived animals subjected to higher oxidative stress selectively accumulate methionine in their mitochondrially encoded proteins, which supports the role of oxidative damage in aging.     

Inhibition of Ethylene Production in Penicillium digitatum

Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and α-ketoglutarate as ethylene precursors, was inhibited by methionine, methionine sulfoxide, methionine sulfone, and methionine sulfoximine. Rhizobitoxine did not affect ethylene production but its ethoxy and methoxy analogues were effective inhibitors of ethylene production; its saturated methoxy analogue and kainic acid stimulated ethylene production. Tracer studies showed that the inhibitors blocked the conversion of [³H]glutamate into [³H]ethylene.

In shake cultures of this fungus, which utilize methionine as the ethylene precursor, rhizobitoxine and its unsaturated analogues all inhibited, while the saturated methoxy analogue stimulated ethylene production. In both types of cultures inhibition was irreversible and was diminished by increasing concentrations of the ethylene precursor. The inhibitory activity or lack of it by rhizobitoxine and its analogues appears to be a function of their structural resemblance to glutamate and methionine as well as of their size and stereoconfiguration. These data suggest similarities between the ethylene-forming system in the fungus and in higher plants despite differences in precursors under some cultural conditions.

     

Biochemical and regulatory effects of methionine analogues in Saccharomyces cerevisiae.

The effect of three methionine analogues, ethionine, selenomethionine, and trifluoromethionine, on the biosynthesis of methionine in Saccharomyces cerevisiae has been investigated. We have found the following to be true. (i) A sharp decrease in the endogenous methionine concentration occurs after the addition of any one of these analogues to growing cells. (ii) All of them can be transferred to methionine transfer ribonucleic acid in vitro as well as in vivo with, as a consequence, their incorporation into proteins. In the absence of radioactive trifluoromethionine, this conclusion results from experiments of an indirect nature and must be taken as an indication rather than a direct demonstration. (iii) Ethionine and selenomethionine can be activated as homologues of S-adenosylmethionine, whereas trifluoromethionine cannot. (iv) All of them can act as repressors of the methionine biosynthetic pathway. This has been shown by measuring the de novo rate of synthesis of methionine in a culture grown in the presence of any one of the three analogues.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Comparison of Rice (Oryza sativa L.) Seed Proteins from Several Varieties

Enzymatically modified proteins (EMP) with different methionine levels were produced from soy protein isolate using an improved plastein reaction. The products having methionine at approximate levels of 4%, 7%, and 14%, designated as EMP₄, EMP₇, and EMP₁₄, respectively, were investigated to characterize their chemical properties particularly in terms of the state and location of the methionine residues. Leucine aminopeptidase treatment of the EMP products did not find any significant amount of methionine residues at the N-terminals, but carboxypeptidase A treatment liberated methionine efficiently in accordance with the methionine levels in the EMP products. Treatment with LiBH₄ reduced the methionine content of EMP₁₄ by approximately 64%. A significant amount of homoserine was produced when EMP₁₄ was treated with BrCN. All these data indicate that the covalently attached methionine molecules are localized at or near the C-terminals of the EMP molecules, probably as oligomers.     

Temperature-sensitive mutants of Saccharomyces cerevisiae variable in the methionine content of their protein.

Temperature-sensitive mutants were derived from Saccharomyces cerevisiae Y5alpha by ethyl methane sulfonate mutagenesis, in a search for mutants that would produce methionine-rich protein at the nonpermissive temperature. A total of 132 mutant strains were selected which showed adequate growth on minimal medium at 25 degrees C but little or no growth on the same medium supplemented with a high concentration (2 mg/ml) of l-methionine at 37 degrees C. Several of these mutants were found to increase the proportion of methionine in their protein to much higher levels than that of the wild-type parent after a temperature shift from 25 to 37 degrees C. Two strains, 476 and 438, which were temperature sensitive only in the presence of methionine, produced cellular protein with methionine contents as high as 3.6 and 4.3%, respectively, when incubated in the presence of methionine. The former strain contained 2.5% methionine even when incubated at 37 degrees C in the absence of methionine. Wild strain Y5alpha, on the other hand, had 1.75% methionine under all conditions tested. Most temperature-sensitive mutants isolated had the same methionine content as the wild strain. It is concluded that the proportion of a specific amino acid, such as methionine, in S. cerevisiae protein can be altered by culturing certain temperature-sensitive mutants at an elevated temperature.     

Differential inhibition of glutamine and gamma-glutamylcysteine synthetases by alpha-alkyl analogs of methionine sulfoximine that induce convulsions.

The alpha-methyl and alpha-ethyl analogs of methionine sulfoximine, like methionine sulfoximine, induce convulsions in mice and inhibit glutamine synthetase irreversibly; alpha-ethylmethionine sulfoximine is approximately 50% as inhibitory as methionine sulfoximine and alpha-methylmethionine sulfoximine. However, whereas alpha-methylmethionine sulfoximine and methionine sulfoximine inhibit gamma-glutamylcysteine synthetase markedly, alpha-ethylmethionine sulfoximine does not, nor does administration of the alpha-ethyl analog produce the decrease in tissue glutathione levels found after giving methionine sulfoximine or its alpha-methyl analog. The findings strongly indicate that methionine sulfoximine-induced convulsions are closely associated with inhibition of glutamine synthetase rather than with inhibition of gamma-glutamylcysteine synthetase. The alpha-alkyl methionine sulfoximine analogs cannot be catabolized via the corresponding alpha-keto or alpha-imino acids, and, like other alpha-substituted amino acids, are probably not metabolized to a significant extent in vivo; this suggests that the amino acid sulfoximine molecules themselves, rather than their metabolites, are directly involved in the induction of convulsions. Possible explanations for the reported lack of correlation between the occurrence of convulsions and the levels of glutamine synthetase activity (and its substrates and product) are considered. The findings suggest that studies on the mechanism of induction of convulsions may be extended significantly and refined in biochemical terms by the use of other structurally modified convulsant molecules.     

Incorporation of methionine from met-tRNA-Met-F into internal positions of polypeptides by mouse liver polysomes

Two methionine accepting tRNA species corresponding to tRNA_F^Met and tRNA_M^Met from mouse ascites tumor cells were tested for their ability to donate methionine into internal positions of growing polypeptide chains on mouse liver polysomes. Both tRNA species can function in the elongation of polypeptide chains as judged by their ability to incorporate methionine into protein in the absence of chain initiation. The insertion of methionine into internal positions of polypeptide chains from Met-tRNA_F^Met was confirmed by Edman degradation and CNBr cleavage. When both tRNA^Met species were present in saturating concentrations in the cell-free system a strong preference for the incorporation of methionine from Met-tRNA_M^Met became apparent.     

New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag"

Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.     

Quantitative analysis of pathways of methionine metabolism and their regulation in lemna

Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 growing under standard conditions, and used to quantitate pathways of methionine metabolism. Synthesis of S-adenosylmethionine (AdoMet) is the major pathway for methionine metabolism, with over 4 times as much methionine metabolized by this route as accumulates in protein. More than 90% of AdoMet is used for transmethylation. Methyl groups of choline, phosphatidylcholine, and phosphorylcholine are major end products of this pathway. Flux through methylthio recycling is about one-third the amount of methionine accumulating in protein. Spermidine synthesis accounts for at least 60% of the flux through methylthio recycling. The results obtained here, together with those reported for methionine-supplemented plants (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839), indicate that methionine supplementation reduced methylneogenesis by no more than the small amount expected from the reduced entry of sulfate sulfur into methionine (Giovanelli, Mudd, Datko, 1985 Plant Physiol 77: 450-455). Methionine supplementation had no significant effect on transmethylation or methylthio recycling. The combined data provide the first comprehensive estimates of the quantitative relationships of major pathways for methionine metabolism and their control in plants.     

Stimulation of Methionine Sulfoxide Reduction and Ethylene Production by Isoprothiolane in Relation to Its Activity against Non-parasitic Damping-off (MURENAE Disease) of Rice Seedlings

A rice blast controlling agent, isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate), stimulated the reduction of methionine sulfoxide to methionine by the rice plant. In the presence of isoprothiolane, the methionine/(methionine + its sulfoxide) ratio was increased to 129~208% of the control. The ethylene production by the plant was also enhanced by isoprothiolane, probably because methionine is an important precursor of ethylene. The non-parasitic damping-off caused by chilling stress on rice seedlings was effectively prevented with the application of isoprothiolane as well as ethephon, which easily decomposes to ethylene and acids. Therefore, the ethylene level modified by isoprothiolane and ethephon can contribute to their protective activity against the non-parasitic damping-off of rice seedlings. Indeed, a close relationship between the ethylene level and the protective activity against damping-off was obtained with isoprothiolane, but not with ethephon. Endogenous ethylene seems to be more effective in controlling the damping-off than exogenous ethylene from ethephon.     

不同形式蛋氨酸对建鲤生长性能及血清游离氨基酸含量的影响

为考察不同形式蛋氨酸对建鲤生长的作用效果, 实验以豆粕、鱼粉、棉粕为蛋白源, 配制缺乏蛋氨酸的基础饲料(对照组, 蛋氨酸含量为0.48%), 在基础饲料中分别添加晶体蛋氨酸、微囊蛋氨酸、蛋氨酸羟基类似物(MHA)及蛋氨酸羟基类似物钙盐(MHA-Ca), 使蛋氨酸含量达到0.58%, 获得5个饲料处理组, 饲养平均体重为(8.61.0) g的建鲤(Cyprinus carpio var Jian)8周。结果显示: 各组鱼体增重率分别为343.51%、350.77%、382.80%、384.02%和385.59%; 饲料系数分别为1.58、1.55、1.42、1.42和1.41; 晶体蛋氨酸组鱼体增重率、饲料系数与对照组无显著差异(P0.05), 微囊蛋氨酸组、MHA组、MHA-Ca组增重率较对照组提高11.4%、11.8%、12.2% (P0.05), 饲料系数降低10.1%、10.1%、10.8% (P0.05)。各处理组在肌肉水分、脂肪含量间无显著差异(P0.05), MHA组肌肉粗蛋白含量较晶体蛋氨酸组显著下降, 其他各组间无显著差异(P0.05)。对摄食后不同时间的血清游离氨基酸浓度变化的分析表明, 对照组在摄食后2h或3h达到峰值, 晶体蛋氨酸组、MHA组在摄食后1h达到吸收峰值, 微囊蛋氨酸组在摄食后1h或2h达到峰值, 而MHA-Ca组则在摄食后3h达到峰值。上述结果表明, 在蛋氨酸缺乏的颗粒饲料中补充晶体蛋氨酸, 对建鲤生长性能无改善作用, 而添加微囊蛋氨酸、蛋氨酸羟基类似物、蛋氨酸羟基类似物钙盐则显著提高了鱼体生长性能, 降低饲料系数。       

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Adaptive Biosynthesis of Thiazole and Bypassing of the Thiazole Requirement in Exobasidium by Acetyl Phosphate, Tween and Ethanol

The tested strain of Exobasidium vaccinii (Fuck.) Woron. required thiamine or its two moieties, thiazole and pyrimidine, for good growth. When grown on pyrimidine medium, rapid growth or thiazole synthesis appears after 600–1000 hours. As methionine, a supposed precursor in the biosynthesis of thiazole, stimulates this inductive growth, the influence of methionine precursors or metabolites stimulating methionine synthesis were tested for their ability to shorten the lag phase of E. vaccinii cultured on a thiamine-deficient synthetic medium. None of the tested methionine precursors replaced methionine or significantly stimulated the inductive growth. This result does not exclude acetylhomoserine as a central metabolite in methionine synthesis. Acetyl phosphate, alcohols and the fatty acids of Tween, possible deliverers of the active acetyl groups, induced growth in thiamine-free media. This growth seems to be possible by utilization of the compounds through a metabolic pathway not requiring thiamine. No evidence for participation of the glyoxylate cycle was found.