Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

In vitro incorporation of selenomethionine into protein by astragalus polysomes

Selenium-accumulator plants synthesize selenium compounds that differ from those produced by nonaccumulators. To determine if there are any subcellular differences between accumulators and nonaccumulators in the use of selenomethionine in vitro, polysomes from Astragalus crotalariae (accumulator) and Astragalus lentiginosis (nonaccumulator) were translated in the presence of selenomethionine. Polysomes from both species efficiently used selenomethionine in vitro during the translation process. Inasmuch as no differences in the incorporation of selenomethionine into protein were observed between polysomes from the two types of Astragalus, it can be inferred that in accumulators there exists a mechanism that either prevents synthesis of selenomethionine or modifies this selenocompound to a derivative that cannot be incorporated into protein.     

Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae*

Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity.     

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Effect of Selenomethionine on Growth of Escherichia coli and Bacillus megaterium

Studies with methionineless strains of Escherichia coli WWU and Bacillus megaterium KM have shown that selenomethionine only partially satisfies their methionine requirement.     

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900 cm⁻¹ due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.     

Selenoprotein inAspergillus terreus

Aspergillus terreus, a moderately selenium-tolerant fungus, metabolized⁷⁵ Se-selenite into several protein seleno-amino acids: selenomethionine and selenocysteine, as well as, nonprotein seleno-amino acids, selenocystathionine, and y-glutamyl selenomethyl selenocysteine. The results indicate the failure of the fungus to discriminate between sulphur and selenium. Selenium was also incorporated into several proteins of different molecular weights, mostly of low molecular weight proteins. Labeled studies showed the presence of high levels of selenomethionine and selenocysteine in the protein hydrolysate. The actual incorporation of protein selenoamino acids into the fungal protein was proven. The results demonstrated a finding that detracts from previous held views.     

Microbial Transformations of Selenium

Resting cell suspensions of a strain of Corynebacterium isolated from soil formed dimethyl selenide from selenate, selenite, elemental selenium, selenomethionine, selenocystine, and methaneseleninate. Extracts of the bacterium catalyzed the production of dimethyl selenide from selenite, elemental selenium, and methaneseleninate, and methylation of the inorganic Se compounds was enhanced by S-adenosylmethionine. Neither trimethylselenonium nor methaneselenonate was metabolized by the Corynebacterium. Resting cell suspensions of a methionine-utilizing pseudomonad converted selenomethionine to dimethyl diselenide. Six of 10 microorganisms able to grow on cystine used selenocystine as a sole source of carbon and formed elemental selenium, and one of the isolates, a pseudomonad, was found also to produce selenide. Soil enrichments converted trimethylselenonium to dimethyl selenide. Bacteria capable of utilizing trimethylselenonium, dimethyl selenide, and dimethyl diselenide as carbon sources were isolated from soil.     

Chemical form of selenium greatly affects metal uptake and responses by cultured human lymphocytes

In this work, possible interference with functional activities of human lymphocytes after in vitro treatment with selenium was examined. Sodium selenite and selenomethionine compounds were tested in parallel, and their capability to inhibit or to increase the antibody production by lymphocytes was investigated. Furthermore, after incubation for 7 d, total cell-associated Se was measured by a fluorimetric method. The in vitro doses of Se employed in this study mainly reflect those measured in blood of individuals with different Se intake. Low doses of Se (0.5–2.0μM) added either as sodium selenite or selenomethionine did not alter the secretion of antibodies. When Se was added at higher levels, instead, an inhibitory effect was found using selenite, whereas a progressive increase in immunoglobulin production was observed after exposure to selenomethionine. In both cases, modifications were detected at 5 μM (395 μg Se/L), and were significant at 10 μM (789 μg Se/L). A different trend between the two chemical forms was also observed with regard to Se uptake by cells. Interestingly, both Se uptake and cell sensitivity were influenced by the density of the cells in culture. Our data suggest that the biological effects of Se in mammalian systems are strongly influenced by its chemical form, and caution should be exerted to avoid toxic effects of selenium.     

Enhancement of ethylene formation by selenoamino acids

Selenomethionine and selenoethionine enhanced ethylene production in senescing flower tissue of Ipomoea tricolor Cav. and in auxin-treated pea (Pisum sativum L.) stem sections. This enhancement was fully inhibited by the aminoethoxy analog of rhizobitoxine. Methionine did not have a comparable promotive effect, and ethionine partly inhibited ethylene production. When [¹⁴C]methionine was applied to flower or pea stem tissue followed by treatment with unlabeled selenomethionine or selenoethionine, the specific radioactivity of the ethylene evolved was considerably reduced. The dilution of the specific radioactivity of ethylene by selenomethionine, and in pea stem sections also by selenoethionine, was greater than the dilution by nonradioactive methionine at the same concentration. These results indicate that both selenoamino acids serve as precursors of ethylene and that they are converted to ethylene more efficiently than is methionine.     

Characteristics of transport of selenoamino acids by epithelial amino acid transporters

Selenoamino acids are the main form of organic selenium derived from the diet. They are efficiently absorbed in the intestine and reabsorbed in kidney, but the transporter proteins that mediate their cellular uptake have not yet been identified. We here describe the transport pathways of selenoamino acids and derivatives, including selenomethionine, methylselenocysteine, selenocystine, selenobetaine and selenocystamine. Transport studies employed the Xenopus laevis oocyte system expressing the amino acid transporters SIT1, b^0,+rBAT, B⁰ or PAT1 and intestinal Caco-2 and renal OK cell lines that possess a multitude of amino acid transporters. Our results suggest that the major route for the uptake of selenomethionine is the system b^0,+ rBAT in Caco-2 cells and B⁰ in OK cells. Affinity of selenomethionine or methionine for these transporters did not differ, but for SIT1 selenomethionine shows a higher affinity than methionine. Methylselenocysteine displayed a higher affinity than cysteine for all transporters tested and in both OK and Caco-2 cells, system B⁰ seems to be the primary uptake route. Selenocystine is taken up well by the b^0,+ rBAT system, while selenobetaine is a low-affinity substrate only for SIT1 and PAT1. Selenocystamine was not transported by any of the transport systems investigated. When cells were exposed to selenoamino acids, intracellular selenium levels in OK cells considerably exceeded those in Caco-2 cells, indicating effective renal reabsorption capacity. In conclusion, selenoamino acids but not the seleno-derivatives selenobetaine and selenocystamine, are effectively transported by various intestinal and renal amino acid transporters and are thus available for selenium metabolism and therapeutic approaches.     

Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.     

The bioavailability of various selenium compounds to a marine wading bird

The uptake of dietary selenium (about 3.5 mg/kg AF dry wt) as selenomethionine, selenocystine, selenite, selenate, and fish selenium in the plasma and red blood cells (RBC) of the oystercatcher has been investigated. The birds received the various selenium compounds subsequently, for at least 9 wk. After dietary supplementation of selenocystine, selenite, and selenate, plasma selenium was about 350 μg/L and RBC selenium 2.1 mg/kg dry wt. After supplementation of selenomethionine, the plasma concentration increased to 630 μg/L, and the RBC concentration to 4.1 mg/kg dry wt. When the fodder contained 3.1 mg/kg fish Se, an average plasma and RBC concentration of 415 μg/L and 14.4 mg/kg dry wt, respectively, was measured. The maximal increase of the selenium concentration in the plasma was attained at first sampling, 14 d after a change in dietary selenium (selenomethione or fish Se); the uptake seemed to be a concentration-regulated process. RBC concentrations (γ in mg/kg dry wt) increased with time (X in d) according toY=a?be^?cX . Fifty percent of the total increase was attained within 17d, suggesting that diffusion into the RBC played a role. The selenium concentration in the plasma was positively correlated with the (fish) Se concentration in the fodder; the RBC concentration (60 d after the change in diet) was positively correlated with the plasma concentration. When the diet contained fish Se, the blood selenium concentrations of the captive birds were similar to the concentrations measured in field birds. Fish Se is a yet undetermined selenium compound. The present experiment showed that fish Se differed from selenomethionine, selenocystine, selenite, or selenate in uptake from the food and uptake in the RBC.     

Selenoprotein P Is the Major Selenium Transport Protein in Mouse Milk

Selenium is transferred from the mouse dam to its neonate via milk. Milk contains selenium in selenoprotein form as selenoprotein P (Sepp1) and glutathione peroxidase-3 (Gpx3) as well as in non-specific protein form as selenomethionine. Selenium is also present in milk in uncharacterized small-molecule form. We eliminated selenomethionine from the mice in these experiments by feeding a diet that contained sodium selenite as the source of selenium. Selenium-replete dams with deletion of Sepp1 or Gpx3 were studied to assess the effects of these genes on selenium transfer to the neonate. Sepp1 knockout caused a drop in milk selenium to 27% of the value in wild-type milk and a drop in selenium acquisition by the neonates to 35%. In addition to decreasing milk selenium by eliminating Sepp1, deletion of Sepp1 causes a decline in whole-body selenium, which likely also contributes to the decreased transfer of selenium to the neonate. Deletion of Gpx3 did not decrease milk selenium content or neonate selenium acquisition by measurable amounts. Thus, when the dam is fed selenium-adequate diet (0.25 mg selenium/kg diet), milk Sepp1 transfers a large amount of selenium to neonates but the transfer of selenium by Gpx3 is below detection by our methods.     

Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide

Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent (N,N',N'-trimethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N'-(p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.     

Selenium Alleviates Porcine Nephrotoxicity of Ochratoxin A by Improving Selenoenzyme Expression In Vitro

Ochratoxin A (OTA), a mycotoxin, is a potent nephrotoxin in humans and animals. Selenium (Se) is an essential micronutrient for humans and animals, and plays a key role in antioxidant defense. To date, little is known about the effect of Se on OTA-induced nephrotoxicity. In this study, the protective effects of selenomethionine against OTA-induced nephrotoxicity were investigated using the porcine kidney 15 (PK15) cells as a model. The results showed that OTA induced nephrotoxicity in a dose-dependent manner. Se at 0.5, 1, 2 and 4 μM had significant protective effects against OTA-induced nephrotoxicity. Furthermore, selenomethionine enhanced the activity and mRNA and protein expression of glutathione peroxidase 1 (GPx1), mRNA expression of GPx4, and mRNA expression of thioredoxin reductase 1 in the presence and absence of OTA. Among them, promoting effect of selenomethionine on GPx1 was maximal. Knock-down of GPx1 by using a GPx1-specific siRNA eliminated the protective effects of selenomethionine against OTA-induced nephrotoxicity. The results suggest that selenomethionine alleviates OTA-induced nephrotoxicity by improving selenoenzyme expression in PK15 cells. Therefore, selenomethionine supplementation may be an attractive strategy for protecting humans and animals from the risk of kidney damage induced by OTA.     

One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

X-ray structure of the metal-sensor CnrX in both the apo- and copper-bound forms

Both the X-ray structures of the apo- and the copper-bound forms of the metal-sensor domain (residues 31-148) of CnrX from Cupriavidus metallidurans CH34 were obtained at 1.74 Å resolution from a selenomethionine derivative. This four-helix hooked-hairpin is the first structure of a metal-sensor in an ECF-type signaling pathway. The copper ion is bound in a type 2-like center with a 3N1O coordination in the equatorial plane and shows an unprecedented remote fifth axial ligand with Met93 contributing a weak S-Cu bond. The signal onset cannot be explained by conformational changes associated with CnrX metallation.     

Structure of an atypical periplasmic adaptor from a multidrug efflux pump of the spirochete Borrelia burgdorferi

Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35 Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.     

Induction of retrovirus gene expression by selenium compounds

Sodium selenite, sodium selenate, selenium oxide, selenophypoxanthine, selenopurine, selenocysteine, selenoethionine and selenomethionine were tested for their ability to induce endogenous retrovirus expression in cultured AKR mouse embryo fibroblasts. All except selenoethionine were highly toxic to the cells. Only selenomethionine however, had the ability to induce virus expression under the conditions used. The level of virus induction (plaque-forming-units/10(5) cells) was roughly proportional to dose over the range of concentrations from 0.25 mM to 5.0 mM. Induction was best observed when a treatment duration of 48 h was used and required the treatment of actively dividing cells. The induction and the cytotoxic effects of selenomethionine could be abrogated by simultaneous treatment with methionine. A ratio of methionine to selenomethionine of 1:10 inhibited induction by approx. 60% while equivalent amounts of methionine inhibited selenomethionine-mediated induction by greater than 96%, indicating that methionine was more efficiently recognized by the cells than was selenomethionine. A possible mechanism for selenomethionine induction involving the production of undermethylated DNA is presented.