Methionine methyl group metabolism in lemna

To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl-methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed.     

1H, 13C and 15N resonance assignment of a zinc-binding methionine sulfoxide reductase type-B from the thermophilic archeabacterium Methanothermobacter thermoautotrophicus

The ¹H, ¹³C and ¹⁵N resonance assignment of a type-B zinc-binding methionine sulfoxide reductase lacking a ‘recycling’ cysteine from the thermophilic archeabacterium Methanothermobacter thermoautotrophicus is reported.     

Biosynthesis of methylphosphomannosyl residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. Evidence that the methyl group is derived from methionine

The phosphorylated oligosaccharides of Dictyostelium discoideum contain methylphosphomannosyl residues which are stable to mild-acid and base hydrolysis (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we present evidence that these methyl groups are derived from [methyl-3H]methionine, in vivo and [methyl-3H]S-adenosylmethionine in vitro. About 18% of the macromolecules secreted from vegetative cells labeled with [methyl-3H]methionine are released by digestion with preparations of endoglycosidase/peptide N-glycosidase F. The majority of the released molecules are sulfated, anionic high mannose-type oligosaccharides. Strong acid hydrolysis of the [3H]methyl-labeled molecules yields [3H]methanol with kinetics of release similar to those found for the generation of Man-6-P from chemically synthesized methylphosphomannose methylglycoside. Treatment of the [3H]methyl-labeled molecules with a phosphodiesterase from Aspergillus niger which is known to cleave this phosphodiester also releases [3H]methanol from a portion of the oligosaccharides. In vitro incorporation of [methyl-3H]S-adenosylmethionine into endogenous acceptors found in membrane preparations shows that the [3H]methyl group of the methylphosphomannose residues can be derived from this molecule.     

A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.     

RELATION OF DL-HOMOCYSTEINE,DL-METHIONINE,AND METHYL DONORS TO THE APHANOMYCES ROOT ROT OF PEAS

Davey , C. B., and G. C. Papavizas . (Crops Res. Div., ARS, USDA, Beltsville, Md.) Relation of dl -homocysteine, dl .-methionine, and methyl donors to the Aphanomyces root rot of peas. Amer. Jour. Bot. 50(1): 67–72. 1963.—dl -Homocysteine, a compound lacking a methyl (CH₃) group, completely prevented Aphanomyces root rot of peas, whereas several CH₃ donors (betaine, choline, dimethyl-β-propiothetin chloride, S-methylmethionine) did not. dl -Homocysteine was taken up by pea plants from nutrient solution and converted to methionine. The methylation of dl -homocysteine in pea tissue occurred without an exogenous source of labile CH₃ groups. More d - than l -methionine was taken up by the plants and more methionine of both forms was taken up when methionine served as the sole sulfur source than when it was supplemented with sulfate. dl -Methionine sulfoxide but not dl -methionine sulfone was converted to methionine in pea tissues. Methionine derived from dl -methionine sulfoxide was proportionately more abundant in the aerial portions of the plants.     

Insights into Function,Catalytic Mechanism,and Fold Evolution of Selenoprotein Methionine Sulfoxide Reductase B1 through Structural Analysis

Methionine sulfoxide reductases protect cells by repairing oxidatively damaged methionine residues in proteins. Here, we report the first three-dimensional structure of the mammalian selenoprotein methionine sulfoxide reductase B1 (MsrB1), determined by high resolution NMR spectroscopy. Heteronuclear multidimensional spectra yielded NMR spectral assignments for the reduced form of MsrB1 in which catalytic selenocysteine (Sec) was replaced with cysteine (Cys). MsrB1 consists of a central structured core of two β-sheets and a highly flexible, disordered N-terminal region. Analysis of pH dependence of NMR signals of catalytically relevant residues, comparison with the data for bacterial MsrBs, and NMR-based structural analysis of methionine sulfoxide (substrate) and methionine sulfone (inhibitor) binding to MsrB1 at the atomic level reveal a mechanism involving catalytic Sec⁹⁵ and resolving Cys⁴ residues in catalysis. The MsrB1 structure differs from the structures of Cys-containing MsrBs in the use of distal selenenylsulfide, residues needed for catalysis, and the mode in which the active form of the enzyme is regenerated. In addition, this is the first structure of a eukaryotic zinc-containing MsrB, which highlights the structural role of this metal ion bound to four conserved Cys. We integrated this information into a structural model of evolution of MsrB superfamily.     

In vivo methylation of elongation factor Tu of Escherichia coli.

A protein existing mainly in the supernatant fraction of Escherichia coli was found to be methylated by accepting the methyl moiety originating from methionine. The protein was identified as peptide synthesis elongation factor Tu (EF-Tu) by the following criteria. 1) The methylatable protein separated at the same position as purified EF-Tu on two-dimensional gel electrophoresis. 2) The methylatable protein interacted with antiserum specific for EF-Tu. Amino acid analysis of the methyl-labeled protein suggested that the site of methylation was an epsilon-amino group of lysine.     

Metabolism of S-methylcysteine and its sulfoxide in Chinese cabbage, Brassica pekinensis Rupr.

The metabolism of sulfur of S-methyl-L-cysteine and ($)S-methyI-L-cysteinesulfoxide in Chinese cabbage was studied. Results showed thatthese sulfur-bearing amino acids are metabolized to cysteineby demethylation. This conclusion is based on; 1) the relativelyhigh recovery of radioactivity in the cysteine of the insolublefraction a short time after the administration of 35S-labeledS-methyl-L-cysteine or ($)S-methyl- L-cysteine sulfoxide; 2)the similarity in the ratios of ³H to ³⁵S in the cysteine ofthe insoluble fraction and in the double-labeled cysteine partof S-methyl-L-cysteine and its sulfoxide fed to detached leavesand 3) the obvious structural relationship between cysteineand its methyl derivative, S-methyl-L-cysteine. Moreover, we ascertained that the sulfur of S-methyl-L-cysteinewas also utilized when supplied to plants; i.e. tomato, tobaccoand cucumber, in which this sulfur amino acid has not been found. (Received June 30, 1971; )     

Characterization of N-methyl-L-methionine sulfoxide and isethionic acid from the red alga Grateloupia doryphora

Isethionic acid (2‐hydroxyethane sulfonic acid) and N‐methyl‐L‐methionine sulfoxide (4‐methane sulfinyl‐2‐methylamino butyric acid) were isolated from the red alga Grateloupia doryphora (Cryptonemiales) collected from Brittany (France); they were identified as major organic solutes together with floridoside (α‐D‐galacto‐pyranosyl‐(1–2)‐glycerol). The presence of isethionic acid has recently been reported in certain red algae, however, the occurrence of N‐methyl‐L‐methionine sulfoxide is still very rare. This report deals with the first isolation of isethionic acid and N‐methyl‐L‐methionine sulfoxide from G. doryphora and their subsequent NMR characterization.     

In Vitro and In Situ Absorption of Methionine Sulfoxide in Rat Small Intestine

Absorption of methionine and its sulfoxide was investigated in vitro with everted sacs and in situ with circulated loops of rat small intestine. Transmural transport and tissue accumulation of methionine sulfoxide in the everted sacs were in fair agreement with those of methionine. Apparent kinetic parameters for the difference of transmural transport in the absence and presence of 10^?5 m carbonylcyanide m-chlorophenylhydrazone, i.e. for the energy-dependent active transport, were similar for both methionine and its sulfoxide. Methionine was found at a low level in the serosal fluid of the everted sac on incubation with methionine sulfoxide. It was attributed to the methionine leaked out from the tissue but not to that formed by reduction of methionine sulfoxide during the course of intestinal transport. Similar transport was also observed in situ in circulated intestinal loops for methionine and its sulfoxide. The absorption efficiency of methionine sulfoxide in the small intestine is not the reason for the decreased nutritional availability of the most likely oxidation product of methionine.     

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.     

Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol.

S-Adenosylmethionine serves as the methyl donor for many biological methylation reactions and provides the propylamine group for the synthesis of polyamines. S-Adenosylmethionine is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase. The cellular factors regulating S-adenosylmethionine synthesis have not been well defined. Here we show that in rat hepatocytes S-nitrosoglutathione monoethyl ester, a cell-permeable nitric oxide donor, markedly reduces cellular S-adenosylmethionine content via inactivation of methionine adenosyltransferase by S-nitrosylation. Removal of the nitric oxide donor from the incubation medium leads to the denitrosylation and reactivation of methionine adenosyltransferase and to the rapid recovery of cellular S-adenosylmethionine levels. Nitric oxide inactivates methionine adenosyltransferase via S-nitrosylation of cysteine 121. Replacement of the acidic (aspartate 355) or basic (arginine 357 and arginine 363) amino acids located in the vicinity of cysteine 121 by serine leads to a marked reduction in the ability of nitric oxide to S-nitrosylate and inactivate hepatic methionine adenosyltransferase. These results indicate that protein S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target cysteine.     

DNA methylation by dimethyl sulfoxide and methionine sulfoxide triggered by hydroxyl radical and implications for epigenetic modifications

In this Letter, we demonstrate the formation of m⁵dC from dC or in DNA by dimethylsulfoxide (DMSO) and methionine sulfoxide (MetO), under physiological conditions in the presence of the Fenton reagent in vitro. DMSO reportedly affects the cellular epigenetic profile, and enhances the metastatic potential of cultured epithelial cells. The methionine sulfoxide reductase (Msr) gene was suggested to be a metastatis suppressor gene, and the accumulation of MetO in proteins may induce metastatic cancer. Our findings are compatible with these biological data and support the hypothesis that chemical cytosine methylation via methyl radicals is one of the mechanisms of DNA hypermethylation during carcinogenesis. In addition to m⁵dC, the formation of 8-methyldeoxyguanosine (m⁸dG) was also detected in DNA under the same reaction conditions. The m⁸dG level in human DNA may be a useful indicator of DNA methylation by radical mechanisms.     

Effect of mineral deficiency on amine formation in higher plants

Potassium deficiency caused putrescine accumulation in the leaves of barley, radish, pea, bean and spinach plants. Magnesium deficiency caused putrescine accumulation in barley, pea and bean leaves, and also in the leaves of older radish plants. In young radish plants less putrescine was found in magnesium deficiency, and in spinach magnesium deficiency was without effect on putrescine levels. Putrescine content may be a useful guide to the mineral status of legumes, since accumulation of this amine may be detected before deficiency symptoms appear. Radioactivity from l-arginine-[U-¹⁴C] fed to barley seedlings was detected in agmatine within 2 hr, and probably also in the hordatines after 24 hr, feeding. After 2 hr the label in the agmatine was greatest in the potassium-deficient plants, but after 24 hr the level declined to that found in the agmatine of the leaves of the magnesium-deficient and control seedlings. The rate of putrescine formation was high in both potassium and magnesium deficiency. Incorporation of radioactivity in spermidine and spermine on feeding putrescine-[1,4-¹⁴C] to barley seedlings was estimated in the dansylated amines after separation by TLC. Activity was higher in spermidine and lower in spermine in the potassium-deficient plants than in the controls. The spermidine/spermine ratio declined on excision of barley leaves.     

ISOLATION OF NITROSOGUANIDINE-INDUCED CHLORELLA VULGARIS MUTANTS WITH HIGH METHIONINE CONTENT1

Mutants of Chlorella vulgaris induced by N-methyl-N′-nitro-N-nitrosoguanidine (NG), and, selected for the resistance to either ethionine or 6-methylpurine, were tested for the relative rate of incorporation into protein of ³H-methionine and ¹⁴C-leucine. A highly significant, correlation between the ³H-to-¹⁴C ratio in the protein and its methionine content was found. 6-Methylpurine proved to be more effective than ethionine as a screening agent for high methionine strains. Screening for 6-methylpurine resistance, followed by a second screening for the highest methionine-to-leucine incorporation ratio, led to isolation of the mutants with a content up to 45% higher in methionine and up to 3 times higher in cysteine with respect to the wild strain.     

The impact of red and blue light-emitting diode illumination on radish physiological indices

The objective was to evaluate the effect of different combinations of red (638 nm) and blue (455 nm) light produced by solid-state light-emitting diodes (LEDs) on physiological indices (net assimilation rate, hypocotyl-to-leaf ratio, leaf area, leaf dry weight, hypocotyl length and diameter, plant length, developed leaves), variation of photosynthetic pigments and non-structural carbohydrates in radish (Raphanus sativus L., var. ‘Faraon’). Lighting experiments were performed under controlled conditions (total PPFD - 200 μmol m⁻² s⁻¹; 16 h photoperiod; 14/18°C night/day temperature). The LED conditions: 638 nm; 638 + 5% 455 nm; 638 + 10% 455 nm; 638 + 10% 455 + 731 nm; 638 + 10% 455 + 731 + 669 nm. Our results showed that radishes grown under red (638 nm) alone were elongated, and the formation of hypocotyl was weak. The net assimilation rate, hypocotyl-to-leaf ratio, and leaf dry weight also were low due to the low accumulation of photosynthetic pigments and non-structural carbohydrates in leaves. The supplemented blue (455 nm) light was necessary for the non-structural carbohydrates distribution between radish storage organs and leaves which resulted in hypocotyl thickening. Red alone (638 nm) or in combination with far-red (731 nm), or red669 for radish generative development was required.     

The influence of pH on arsenazo III.

None of the methods already reported for elimination of pectins from rRNA extracts allowed the complete removal of methylated polysaccharides from methyl-labeled cytoplasmic 17 and 26 S rRNA preparations of sycamore (Acer pseudoplatanus L.) cells. An improved procedure for purifying large amounts of higher plant cytoplasmic rRNA labeled on the methyl groups was investigated. Bulk cellular RNA from sycamore cells incubated for 24 to 36 h with methyl-labeled methionine was extracted at 4°C by the phenol-extraction procedure. Most of the pectic compounds (that accounted for about 30% of the total label of RNA extracts) was selectively precipitated, before the 66% ethanol precipitation of nucleic acid, by bringing the deproteinized aqueous layer to 10% ethanol ?0.15 m sodium acetate. Cytoplasmic rRNA, 17 and 26 S, were isolated by repeated sucrose gradient sedimentations and further chromatographed on a methylated albumin kieselgurh (MAK) column. The old-fashioned MAK chromatography proved to be very useful for elimination of residual pectins, since these compounds eluted in the void volume of the column. This purification procedure gave in a reproducible way cytoplasmic 17 and 26 rRNA virtually free of any labeled DNA, mRNA, plastid rRNA, and pectic compounds.     

Corynebacterium diphtheriae Methionine Sulfoxide Reductase A Exploits a Unique Mycothiol Redox Relay Mechanism

Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.     

Mechanisms of methyl-carbon transfer from S-methylcysteine to pectin in Chinese cabbage (Brassica pekinensis Rupr.)

In order to learn how the methyl group of S-methylcysteine wasmetabolized and its carbon was incorporated into the methylester of pectin in Chinese cabbage, leaves were fed S-methylcysteinewhich was labeled in the methyl group with both ³H and ¹⁴C.Incorporation of the radioactive isotopes into S-adenosylmethioninewas detected with little reduction in the ³H/¹⁴C ratio betweenthe methyl groups. The changes in the ³H/¹⁴C ratio between thepectin methyl ester recovered from the leaves and the S-methylcysteinefed to them indicate that there are at least two pathways inthe transfer of the methyl-carbon from S-methylcysteine to themethyl ester of pectin: one is the intact methyl group transfer,probably through S-adenosylmethionine, and the other is carbontransfer after the degradation of the methyl group. Cysteinesulfoxide lyase (EC 4.4.1.4 [EC] ) was found in the leaves and rootsin Chinese cabbage and its involvement in the methyl-carbontransfer is discussed. (Received December 8, 1975; )