Propargylic Se-adenosyl-l-selenomethionine: A Chemical Tool for Methylome Analysis.

Devising synthetic strategies to construct a covalent bond is a common research topic among synthetic chemists. A key driver of success is the high tunability of the conditions, including catalysts, reagents, solvents, and reaction temperature. Such flexibility of synthetic operations has allowed for the rapid exploration of a myriad of artificial synthetic transformations in recent decades. However, if we turn our attention to chemical reactions controlled in living cells, the situation is quite different; the number of hit substrates for the reaction-type is relatively small, while the crowded environment is chemically complex and inflexible to control.A specific objective of this Account is to introduce our chemical methylome analysis as an example of bridging the gap between chemistry and biology. Protein methylation, catalyzed by protein methyltransferases (MTases) using S-adenosyl-l-methionine (SAM or AdoMet) as a methyl donor, is a simple but important post-translational covalent modification. We aim to efficiently identify MTase substrates and methylation sites using activity-based protein profiling (ABPP) with propargylic Se-adenosyl-l-selenomethionine (ProSeAM, also called SeAdoYn). Specifically, we draw heavily from quantitative proteomics that yields information about the differences between two samples utilizing LC-MS/MS analysis. By exploiting the use of ProSeAM, we have prepared the requisite two samples for quantitative methylome analysis. The structural difference between ProSeAM and the parent SAM is so small that the quantity of modification of the protein substrate with this artificial cofactor reflects, to a large extent, levels of activity of the MTase of interest with SAM. First, we identified that the addition of exogenous recombinant MTase (methylation accel), a natural catalyst, enhances the generation of the corresponding propargylated product even in the cell lysate. Then, we applied the principle to isotope label-free quantification with HEK293T cell lysates. By comparing the intensity of LC-MS/MS signals in the absence and presence of the MTase, we have successfully correlated the MTase substrates. We have currently applied the concept to the stable isotope label-based quantification, SILAC (stable isotope labeling by amino acids in cell culture). The strategy merging ProSeAM/MTase/SILAC (PMS) is uniquely versatile and programmable. We can choose suitable cell lines, subcellular fractions (i.e.; whole lysate or mitochondria), and genotypes as required. In particular, we would like to emphasize that the use of cell lysates derived from disease-associated MTase knockouts (KOs) holds vast potential to discover functionally unknown but biologically important methylation events. By adding ProSeAM and a recombinant MTase to the lysates derived from KO cells, we successfully characterized unprecedented nonhistone substrates of several MTases. Furthermore, this chemoproteomic procedure can be applied to explore MTase inhibitors (methylation brake). The combined strategy with ProSeAM/inhibitor/SILAC (PIS) offers intriguing opportunities to explore nonhistone methylation inhibitors.Considering that SAM is the second most widely used enzyme-substrate following ATP, the interdisciplinary research between chemistry and biology using SAM analogs has a potentially huge impact on a wide range of research fields associated with biological methylation. We hope that this Account will help to further delineate the biological function of this important class of enzymatic reaction.

Direct Imaging of Protein-Specific Methylation in Mammalian Cells.

Abundant post-translational modification through methylation alters the function, stability, and/or localization of a protein. Malfunctions in post-translational modification are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now, their usage was limited to cell lysates. Herein, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and FRET detected by means of fluorescence lifetime imaging microscopy. Through this approach, methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines has been successfully imaged. To further demonstrate its potential, the localization-dependent methylation state of Foxo1 in the cellular context has been visualized.

N-ϒ-(l-Glutamyl)-l-Selenomethionine Inhibits Fat Storage via the Stearoyl-CoA Desaturases FAT-6 and FAT-7 and the Selenoprotein TRXR-1 in Caenorhabditis elegans.

SCOPE: Selenium is an important nutrient for human health. The influence of dietary selenium on lipid metabolism remains largely unknown. N-γ-(l-glutamyl)-l-selenomethionine (Glu-SeMet) on inhibition of fat accumulation and its underlying mechanisms in the nematode Caenorhabditis elegans are investigated. METHODS AND RESULTS: Triacylglyceride quantification and post-fixed Nile red staining methods are conducted to evaluate fat accumulation in wild-type N2 worms in normal or high-glucose diet. Glu-SeMet (0.01 µm) treatment effectively reduces fat storage in wild-type N2 C. elegans in both a normal and high-glucose diet. Further evidence shows that Glu-SeMet (0.01 µm) decreases the ratio of oleic acid/stearic acid (C18:1Δ9/C18:0) using gas chromatography-mass spectrometry analysis. The mRNA levels of fatty acid stearoyl-CoA desaturases, FAT-6 and FAT-7, and the mediator-15 (MDT-15) are downregulated while the wild-type N2 worms are co-treated with high glucose and Glu-SeMet (0.01 µm). The effect of reduced fat accumulation is absent in fat-6, fat-7, and trxr-1 mutant worms under high glucose and Glu-SeMet (0.01 µm) co-treatment. CONCLUSIONS: This study demonstrates that Glu-SeMet inhibiting fat accumulation may be associated with FAT-6 and FAT-7 and the selenoprotein TRXR-1 in C. elegans. This study implies a potential for Glu-SeMet as a new treatment for obesity or its complications.

Selenium species-dependent toxicity, bioavailability and metabolic transformations in Caenorhabditis elegans.

The essential micronutrient selenium (Se) is required for various systemic functions, but its beneficial range is narrow and overexposure may result in adverse health effects. Additionally, the chemical form of the ingested selenium contributes crucially to its health effects. While small Se species play a major role in Se metabolism, their toxicological effects, bioavailability and metabolic transformations following elevated uptake are poorly understood. Utilizing the tractable invertebrate Caenorhabditis elegans allowed for an alternative approach to study species-specific characteristics of organic and inorganic Se forms in vivo, revealing remarkable species-dependent differences in the toxicity and bioavailability of selenite, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys). An inverse relationship was found between toxicity and bioavailability of the Se species, with the organic species displaying a higher bioavailability than the inorganic form, yet being less toxic. Quantitative Se speciation analysis with HPLC/mass spectrometry revealed a partial metabolism of SeMet and MeSeCys. In SeMet exposed worms, identified metabolites were Se-adenosylselenomethionine (AdoSeMet) and Se-adenosylselenohomocysteine (AdoSeHcy), while worms exposed to MeSeCys produced Se-methylselenoglutathione (MeSeGSH) and γ-glutamyl-MeSeCys (γ-Glu-MeSeCys). Moreover, the possible role of the sole selenoprotein in the nematode, thioredoxin reductase-1 (TrxR-1), was studied comparing wildtype and trxr-1 deletion mutants. Although a lower basal Se level was detected in trxr-1 mutants, Se toxicity and bioavailability following acute exposure was indistinguishable from wildtype worms. Altogether, the current study demonstrates the suitability of C. elegans as a model for Se species dependent toxicity and metabolism, while further research is needed to elucidate TrxR-1 function in the nematode.

Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy.

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. In vivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

N-γ-(L-Glutamyl)-L-selenomethionine enhances stress resistance and ameliorates aging indicators via the selenoprotein TRXR-1 in Caenorhabditis elegans.

SCOPE: Selenium is an essential trace nutrient for human health. This study investigates the organic form of selenium, N-γ-(L-Glutamyl)-L-selenomethionine (Glu-SeMet), for its effects on aging indicators and stress resistance. The role of the selenoprotein TRXR-1 was also evaluated in Caenorhabditis elegans. METHODS AND RESULTS: Glu-SeMet-treated wild-type N2 worms showed increased survival upon oxidative and thermal stress challenges. However, Glu-SeMet treatment did not extend the lifespan of wild-type N2 C. elegans under normal conditions (p = 0.128 for 0.01 µM and p = 0.799 for 10 µM Glu-SeMet). Under stress conditions, Glu-SeMet significantly increased the survival of wild-type N2 C. elegans, but the phenomenon was absent from trxr-1 null mutant worms. Furthermore, Glu-SeMet treatments significantly ameliorated aging indicators, including body bends, pumping rate, defecation duration, and lipofuscin accumulation in wild-type N2 nematodes. Nevertheless, the ameliorative effects by Glu-SeMet were absent in the trxr-1 null mutant worms. CONCLUSION: The findings indicate that enhanced stress resistance and improved aging indicators by Glu-SeMet in C. elegans are mediated by the selenoprotein TRXR-1. Glu-SeMet has potential for improving health and also provides new insights into selenium's regulatory mechanisms in intact organisms.

Trifluoroselenomethionine: A New Unnatural Amino Acid.

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N-(tert-butoxycarbonyl)-l-aspartic acid tert-butyl ester. TFSeM shows enhanced methioninase-induced cytotoxicity, relative to selenomethionine (SeM), toward HCT-116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase.

Selenoglycoproteins attenuate adhesion of tumor cells to the brain microvascular endothelium via a process involving NF-κB activation.

Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.

Large-scale, protection-free synthesis of Se-adenosyl-L-selenomethionine analogues and their application as cofactor surrogates of methyltransferases.

S-adenosyl-L-methionine (SAM) analogues have previously demonstrated their utility as chemical reporters of methyltransferases. Here we describe the facile, large-scale synthesis of Se-alkyl Se-adenosyl-L-selenomethionine (SeAM) analogues and their precursor, Se-adenosyl-L-selenohomocysteine (SeAH). Comparison of SeAM analogues with their equivalent SAM analogues suggests that sulfonium-to-selenonium substitution can enhance their compatibility with certain protein methyltransferases, favoring otherwise less reactive SAM analogues. Ready access to SeAH therefore enables further application of SeAM analogues as chemical reporters of diverse methyltransferases.

Profiling protein methylation with cofactor analog containing terminal alkyne functionality.

Enzymatic transmethylation from the cofactor S-adenosyl-L-methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne-containing SAM mimic, Se-adenosyl-L-selenomethionine (ProSeAM), and a cleavable azido-azo-biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM-labeled proteins with the azido-azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal-alkyne-containing SAM analog cofactors.

O antigen is the receptor of Vibrio cholerae serogroup O1 El Tor typing phage VP4.

Bacteriophage VP4 is a lytic phage of the Vibrio cholerae serogroup O1, and it is used in phage subtyping of V. cholerae biotype El Tor. Studies of phage infection mechanisms will promote the understanding of the basis of phage subtyping as well as the genetic differences between sensitive and resistant strains. In this study, we investigated the receptor that phage VP4 uses to bind to El Tor strains of V. cholerae and found that it infects strains through adsorbing the O antigen of V. cholerae O1. In some natural isolates that are resistant to VP4 infection, mutations were identified in the wb* cluster (O-antigen gene cluster), which is responsible for the biosynthesis of O antigen. Mutations in the manB, wbeE, and wbeU genes caused failure of adsorption of VP4 to these strains, whereas the observed amino acid residue mutations within wbeW and manC have no effect on VP4 infection. Additionally, although mutations in two resistant strains were found only in manB and wbeW, complementing both genes did not restore sensitivity to VP4 infection, suggesting that other resistance mechanisms may exist. Therefore, the mechanism of VP4 infection may provide a basis for subtyping the phage. Elaborate mutations of the O antigen may imbue V. cholerae strains with resistance to phage infection.

Se-adenosyl-L-selenomethionine cofactor analogue as a reporter of protein methylation.

Posttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM's utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.

Bis-pentafluorobenzyl derivatives of N-acetyl-L-methionine and N-acetyl-L-selenomethionine for the quantitative determination in human plasma by gas chromatography-negative ion chemical ionisation mass spectrometry.

Targeted anti-cancer combination therapy with infusion of N-acetyl-L-methionine (NALM) and N-acetyl-L-selenomethionine (NASeLM) shows promising results in cancer treatment. Selenium has been recognised as a valuable additive in cancer therapeutics due to its ability to minimise side effects of chemotherapy and its role in cancer prevention and therapy. Due to the promising results of this new therapeutic approach evaluation of pharmacokinetic data for NALM and NASeLM is of ultimate importance. We have therefore elaborated a method for the quantitative measurement of these compounds in human plasma based on GC-negative ion chemical ionisation-MS. The derivatisation sequence elaborated can be regarded as a novel strategy for the chemical modification of delicate sulphur- and selenium-containing compounds, and underlines the enhanced reactivity of selenium-analogues of sulphur-containing amino acids. The target compounds were extracted from plasma with ethyl acetate and converted to the S/Se-pentafluorobenzyl-homocysteine pentafluorobenzyl ester derivative. Reaction conditions were optimised for derivative yield. Calibration graphs were established in the range of 2.938-481.105 ng/0.5 mL plasma (NALM) and 0.233-59.543 ng/0.5 mL plasma (NASeLM). Accuracy, precision and stability data were elaborated. The method was applied to pharmacokinetic profiling of the compounds after infusion into human volunteers.

AglP is a S-adenosyl-L-methionine-dependent methyltransferase that participates in the N-glycosylation pathway of Haloferax volcanii.

While pathways for N-glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post-translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S-layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N-glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S-adenosyl-L-methionine-dependent methyltransferase.

One-pot synthesis of highly functionalized seleno amino acid derivatives.

We herein provide a new and rapid protocol to generate derivatives of seleno amino acid, including methyl selenocysteine, selenomethionine, and selenocystine. Applying the isocyanide-based multicomponent reaction Ugi-4C-5C reaction, we show that each of the commercially available seleno amino acids are good substrate for these reactions and can be used together with complementary oxocomponents and isocyanides to generate highly diverse functionalized selenium-containing compounds. These compounds might become useful tools for applications in chemical biology to elucidate the role of selenium in biochemistry.

Experimental and theoretical evidence for cyclic selenurane formation during selenomethionine oxidation.

The oxidation products of selenomethionine (SeMet) have been studied via experimental (77)Se NMR and theoretical (77)Se chemical shifts. Four signals are observed: a diastereomeric pair of selenoxides at 840 ppm and two unidentified resonances at 703 and 716 ppm. Theoretical DeltaG and chemical shifts suggest the 703 and 716 ppm resonances correspond to hypervalent selenium heterocycles, called selenuranes, formed by reaction with the amine or acid group of the amino acid and the selenoxide. To identify which of these selenuranes is formed, the amine and acid groups were individually protected. The N-formyl SeMet formed only the selenoxide pair at 840 ppm. The oxidized SeMet methyl ester produced signals at 703 and 716 ppm which are assigned as the Se-N selenurane.

Insight into the polar reactivity of the onium chalcogen analogues of S-adenosyl-L-methionine.

S-Adenosyl-L-methionine (AdoMet) is one of Nature's most diverse metabolites, used not only in a large number of biological reactions but amenable to several different modes of reactivity. The types of transformations in which it is involved include decarboxylation, electrophilic addition to any of the three carbons bonded to the central sulfur atom, proton removal at carbons adjacent to the sulfonium, and reductive cleavage to generate 5'-deoxyadenosyl 5'-radical intermediates. At physiological pH and temperature, AdoMet is subject to three spontaneous degradation pathways, the first of which is racemization of the chiral sulfonium group, which takes place in a pH-independent manner. The two remaining pathways are pH-dependent and include (1) intramolecular attack of the alpha-carboxylate group onto the gamma-carbon, affording L-homoserine lactone (HSL) and 5'-methylthioadenosine (MTA), and (2) deprotonation at C-5', initiating a cascade that results in formation of adenine and S-ribosylmethionine. Herein, we describe pH-dependent stability studies of AdoMet and its selenium and tellurium analogues, Se-adenosyl-L-selenomethionine and Te-adenosyl-L-telluromethionine (SeAdoMet and TeAdoMet, respectively), at 37 degrees C and constant ionic strength, which we use as a probe of their relative intrinsic reactivities. We find that with AdoMet intramolecular nucleophilic attack to afford HSL and MTA exhibits a pH-rate profile having two titratable groups with apparent pK(a) values of 1.2 +/- 0.4 and 8.2 +/- 0.05 and displaying first-order rate constants of <0.7 x 10(-6) s(-1) at pH values less than 0.5, approximately 3 x 10(-6) s(-1) at pH values between 2 and 7, and approximately 15 x 10(-6) s(-1) at pH values greater than 9. Degradation via deprotonation at C-5' follows a pH-rate profile having one titratable group with an apparent pK(a) value of approximately 11.5. The selenium analogue decays significantly faster via intramolecular nucleophilic attack, also exhibiting a pH-rate profile with two titratable groups with pK(a) values of approximately 0.86 and 8.0 +/- 0.1 with first-order rate constants of <7 x 10(-6) s(-1) at pH values less than 0.9, approximately 32 x 10(-6) s(-1) at pH values between 2 and 7, and approximately 170 x 10(-6) s(-1) at pH values greater than 9. Degradation via deprotonation at C-5' proceeds with one titratable group displaying an apparent pK(a) value of approximately 14.1. Unexpectedly, TeAdoMet did not decay at an observable rate via either of these two pathways. Last, enzymatically synthesized AdoMet was found to racemize at rates that were consistent with earlier studies (Hoffman, J. L. (1986) Biochemistry 25, 4444-4449); however, SeAdoMet and TeAdoMet did not racemize at detectable rates. In the accompanying paper, we use the information obtained in these model studies to probe the mechanism of cyclopropane fatty acid synthase via use of the onium chalcogens of AdoMet as methyl donors.

Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase.

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.

Identification of selenium species in urine by ion-pairing HPLC-ICP-MS using laboratory-synthesized standards.

This study focused on the detection/identification of possible selenium metabolites in human urine. Organoselenium compounds not commercially unavailable were synthesized and characterized by electrospray mass spectrometry. Separation of selenomethionine, methylselenomethionine, trimethylselonium, selenoethionine, and selenoadenosylmethionine was achieved by ion-pairing HPLC with a mobile phase of 2 mmol L(-1) hexanesulfonic acid, 0.4% acetic acid, 0.2% triethanolamine (pH 2.5), and 5% methanol. The column effluent was introduced on-line to inductively coupled plasma-mass spectrometry for selenium-specific detection ((77)Se and (78)Se). For selenium speciation in urine, solid-phase extraction was carried out using C(18) cartridges modified with hexanesulfonic acid. Selective retention of cationic species was observed from acidified urine (perchloric acid, pH 2.0). After elution with methanol, evaporation, and dissolution in the mobile phase, the sample was introduced to the HPLC-ICP-MS system and the chromatographic peaks were assigned by adding standards. The species identified in urine were selenomethionine, trimethylselonium ion, and selenoadenosylmethionine. The last species was detected for the first time and our results suggest that selenomethionine might enter the metabolic pathway of its sulfur analog in the activated methylation cycle.

Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles.

The adsorption of a recombinant cutinase from Fusarium solani pisi onto the surface of 100 nm diameter poly(methyl methacrylate) (PMMA) latex particles was evaluated. Adsorption of cutinase is a fast process since more than 70% of protein molecules are adsorbed onto PMMA at time zero of experiment, irrespective of the tested conditions. A Langmuir-type model fitted both protein and enzyme activity isotherms at 25 degrees C. Gamma(max) increased from 1.1 to 1.7 mg m(-2) and U(max) increased from 365 to 982 U m(-2) as the pH was raised from 4.5 to 9.2, respectively. A decrease (up to 50%) in specific activity retention was observed at acidic pH values (pH 4.5 and 5.2) while almost no inactivation (eta(act) congruent with 87-94%) was detected upon adsorption at pH 7.0 and 9.2. Concomitantly, far-UV circular dichroism (CD) spectra evidenced a reduction in the alpha-helical content of adsorbed protein at acidic pH values while at neutral and alkaline pH the secondary structure of adsorbed cutinase was similar to that of native protein. Fluorescence anisotropy decays showed the release of some constraints to the local motion of the Trp69 upon protein adsorption at pH 8.0, probably due to the disruption of the tryptophan-alanine hydrogen bond when the tryptophan interacts with the PMMA surface. Structural data associated with activity measurements at pH 7.0 and 9.2 showed that cutinase adsorbs onto PMMA particles in an end-on orientation with active site exposed to solvent and full integrity of cutinase secondary structure. Hydrophobic interactions are likely the major contribution to the adsorption mechanism at neutral and alkaline pH values, and a higher amount of protein is adsorbed to PMMA particles with increasing temperature at pH 9.2. The maximum adsorption increased from 88 to 140 mg cutinase per g PMMA with temperature raising from 25 to 50 degrees C, at pH 9.2.