- Rate constant determination for the reaction of hydroxyl and glutathione thiyl radicals with glutathione in aqueous solution
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The techniques of pulse radiolysis, laser photolysis, and absorption spectroscopy have been used to investigate the glutathione disulfide radical anion formation over the pH range 7.0-13.0 in aqueous solution. Photolysis of the disulfide anion formed from the one-electron oxidation of reduced glutathione perturbs the disulfide anion/thiyl radical equilibrium, allowing the rate constant for. thiyl radical reaction with glutathione to be uniquely determined from the transient absorption bleach and subsequent pseudo-first-order recovery. These pH-dependent values were combined with measured disulfide equilibrium constants to calculate glutathione radical anion dissociation rate constants. From computer modeling of established mechanisms of the observed disulfide radical anion growths, pH-dependent rate constants for the reaction of hydroxyl radicals with glutathione to produce the thiyl radical were obtained. Utilizing literature ionization constants, values for hydroxyl and thiyl radical reactions with individual glutathione species were determined. The similarity of the measured values over the pH range 10-13 suggests that the rate constants for both the hydroxyl and oxide radical reaction with glutathione are essentially the same. These hydroxyl radical rate constants are contrasted with previously reported values determined using competition kinetics.
- Mezyk, Stephen P.
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- Synthesis of novel chiral bis-N-substituted-hydrazinecarboxamide receptors and probing their solution-phase recognition to chiral carboxylic guests by ESI-TOF/MS and tandem ESI-MS
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Seven novel bis-N-substituted-hydrazinecarboxamide receptors were synthesized in good to excellent yields by reacting chiral dicarbohydrazides, obtained from commercially available tartaric acid, with substituted aromatic isocyanates. The newly synthesized hydrazinecarboxamides formed structurally unique supramolecular aggregates, which have been confirmed by ESI-TOF/MS and tandem ESI-MS. They also showed molecular recognition to a selection of chiral carboxylic guests and oligopeptides, which mimic the backbone structure of the bacterial cell wall. The structures of the novel compounds were verified by various spectroscopic techniques including FTIR, 1H NMR, 13C NMR, ESI-TOF/MS, tandem ESI-MS, 2D ROESY NMR, and CD spectroscopy.
- Nour, Hany F.,Golon, Agnieszka,Islam, Tuhidul,Fernández-Lahore, Marcelo,Kuhnert, Nikolai
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- Convenient supported recyclable material based on dihydrolipoyl-residue for the reduction of disulfide derivatives
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A quantitative method for the reduction of disulfides, which uses a totally recyclable solid phase supported reducing agent, is reported. d,l-α-Lipoic acid was quantitatively condensed on a highly stable 100% PEG Aminomethyl-ChemMatrix resin that can swell in aqueous media as well as in organic solvents. Lipoic residue, subsequently reduced to its dihydrolipoyl form, was utilized as a reducing agent for highly valuable disulfide compounds.
- Bienvenu, Céline,Greiner, Jacques,Vierling, Pierre,Giorgio, Christophe Di
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- Dissecting the catalytic mechanism of trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modeling
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Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form trypanothione. Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing the entire kinetic profile. Conclusion: Trypanothione synthetase is affected by substrate and product inhibition. Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this parasite-specific enzyme.
- Leroux, Alejandro E.,Haanstra, Jurgen R.,Bakker, Barbara M.,Krauth-Siegel, R. Luise
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- Metabolic synthesis of clickable glutathione for chemoselective detection of glutathionylation
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Glutathionylation involves reversible protein cysteine modification that regulates the function of numerous proteins in response to redox stimuli, thereby altering cellular processes. Herein we developed a selective and versatile approach to identifying glutathionylation by using a mutant of glutathione synthetase (GS). GS wild-type catalyzes coupling of γGlu-Cys to Gly to form glutathione. We generated a GS mutant that catalyzes azido-Ala in place of Gly with high catalytic efficiency and selectivity. Transfection of this GS mutant (F152A/S151G) and incubation of azido-Ala in cells efficiently afford the azide-containing glutathione derivative, γGlu-Cys-azido-Ala. Upon H2O2 treatment, clickable glutathione allowed for selective and sensitive detection of glutathionylated proteins by Western blotting or fluorescence after click reaction with biotin-alkyne or rhodamine-alkyne. This approach affords the efficient metabolic tagging of intracellular glutathione with small clickable functionality, providing a versatile handle for characterizing glutathionylation.
- Samarasinghe, Kusal T. G.,Munkanatta Godage, Dhanushka N. P.,Vanhecke, Garrett C.,Ahn, Young-Hoon
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- Kinetic, spectroscopic and in silico characterization of the first step of the reaction between glutathione and selenite
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Reduction of dietary selenite (SeO3H?, SeO3H2) is an important process in vivo, which predominantly involves glutathione (GSH). Although the reaction between selenite and thiols has been studied extensively, its mechanism and the identification of products remain controversial. Herein, we present kinetic, spectroscopic and in silico data on the first step of the reaction between GSH and SeO32? in aqueous solutions of varying acidity. We found that the reaction reversibly produces glutathione-S-selenite (GS-SeO2?) absorbing at 259 nm in the UV spectrum. Assignment of the absorption maximum at 259 nm to GS-SeO2? was performed using TDDFT and mass spectrometry. GS-SeO2? undergoes protonation in acidic medium to form the corresponding conjugated acid, GS-SeO2H (pKa = 1.9 at 25 °C), which exhibits reduced absorption intensity at 259 nm. According to the kinetic data, the mechanism of GS-SeO2?(H+) formation includes two pathways: (i) nucleophilic substitution of HO-group in biselenite by the thiolate group of GSH, and (ii) nucleophilic substitution of HO-group in selenous acid by the thiol group of GSH. The complex GS-SeO2?(H+) is unstable in aqueous medium and undergoes hydrolysis to initial reactants, which is accelerated by an increase in alkalinity.
- Dereven'kov, Ilia A.,Hannibal, Luciana,Molodtsov, Pavel A.,Branzanic, Adrian M.V.,Silaghi-Dumitrescu, Radu,Makarov, Sergei V.
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- Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states
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Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover with a combination of NMR magnetization transfer, R2 relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically tuned to exchange on the millisecond time scale. The conformational changes of the protein backbone during catalysis were followed by 15N nuclear spin relaxation dispersion experiments. A conformational transition that is well described by a two-state process with an exchange rate corresponding to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions on the time scale of catalytic turnover were not observed for the enzyme in the resting states, implying that alternative conformers do not accumulate to significant concentrations. These results infer that the turnover rate in glutaredoxin is governed by formation of a productive enzyme-substrate encounter complex, and that catalysis proceeds by an induced fit mechanism rather than by conformer selection driven by intrinsic conformational dynamics.
- Jensen, Kristine Steen,Winther, Jakob R.,Teilum, Kaare
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- Characterization of nucleoside and DNA adducts formed by S-(1-Acetoxymethyl)glutathione and implications for dihalomethane - Glutathione conjugates
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S-(1-Acetoxymethyl)glutathione (GSCH2OAc) was synthesized and used as a model for the reaction of glutathione (GSH)-dihaloalkane conjugates with nucleosides and DNA. Previously, S-[1-(N2-deoxyguanosinyl)methyl]GSH had been identified as the major adduct formed in the reaction of GSCH2OAc with deoxyguanosine. GSCH2OAc was incubated with the three remaining deoxyribonucleosides to identify other possible adducts. Adducts to all three nucleosides were found using electrospray ionization mass spectrometry (ESI MS). The adduct of GSCH2OAc and deoxyadenosine was formed in yield of up to 0.05% and was identified as S-[1-(N7-deoxyadenosinyl)methyl]GSH. The pyrimidine deoxyribonucleoside adducts were formed more efficiently, resulting in yields of 1 and 2% for the GSCH2OAc adducts derived from thymidine and deoxycytidine, respectively, but their lability prevented their structural identification by 1H NMR. On the basis of the available UV spectra, we propose the structures S-[1-(N3-thymidinyl)methyl]GSH and S-[1-(N4-deoxycytidinyl)methyl]GSH. Because adduct degradation occurred most rapidly at alkaline and neutral pH values, an enzymatic DNA digestion procedure was developed for the rapid hydrolysis of DNA to deoxyribonucleosides at acidic pH. DNA digests were completed in less than 2 h with a two-step method, which consisted of a 15 min incubation of DNA with high concentrations of deoxyribonuclease II and phosphodiesterase II at pH 4.5, followed by incubation of resulting nucleotides with acid phosphatase. Analysis of the hydrolysis products by HPLC-ESI-MS indicated the presence of the thymidine adduct.
- Marsch, Glenn A.,Mundkowski, Ralf G.,Morris, Brent J.,Manier, M. Lisa,Hartman, Melanie K.,Guengerich, F. Peter
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- A New Synthesis of Glutathione via the Thiazoline Peptide
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A convenient synthesis of glutathione (GSH) by the use of minimal protecting groups was investigated.N-Formyl-L-2-amino-4-cyanobutyric acid ethyl ester was condensed with ethyl L-cysteinylglycinate to give (4R)-2--4-(ethoxycarbonylmethylcarbamoyl)-2-thiazoline.This compound was saponified in aqueous acetone at -15- -20 deg C and subsequently treated with dilute H2SO4 (pH 4) to yield formylglutathione, whose formyl group was then hydrolyzed with 0.5 M (1M = 1 mol dm-3) H2SO4 to give free GSH.For purification, this was changed to a copper thiolate, which was then decomposed with H2S to afford pure GSH.
- Ozawa, Yoichi,Tsuji, Toshiaki,Ariyoshi, Yasuo
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- Simple single-step single-enzyme synthesis of [14C]-GSH
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The tri-peptide [14C]-glutathione ([14C]-GSH) was synthesized in a single step by GSH synthetase catalyzed reaction of L-γ-glutamyl-L-cysteine and [14C]-glycine. Preparative reverse phase HPLC afforded [14C]-GSH in 30% yield and 98% purity. Preparation of GSH synthetase from E. coli via recombinant DNA and the interconversion of [14C]-GSH to the disulfide [14C]-GSSG for storage are discussed. Copyright
- De Keczer, Steve A.,Voronin, Tatyana,Yao, Jennifer,Zhang, Fang-Jie,Masjedizadeh, Mohammad R.
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- Acetone/Isopropanol Photoinitiating System Enables Tunable Disulfide Reduction and Disulfide Mapping via Tandem Mass Spectrometry
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Herein, we report the development of a new photochemical system which enables rapid and tunable disulfide bond reduction and its application in disulfide mapping via online coupling with mass spectrometry (MS). Acetone, a clean and electrospray ionization (ESI) compatible solvent, is used as the photoinitiator (1% volume) in the solvent system consisting of 1:1 alkyl alcohol and water. Under ultraviolet (UV) irradiation (～254 nm), the acetone/alcohol system produces hydroxyalkyl radicals, which are responsible for disulfide bond cleavage in peptides. Acetone/isopropanol is most suitable for optimizing the disulfide reduction products, leading to almost complete conversion in less than 5 s when the reaction is conducted in a flow microreactor. The flow microreactor device not only facilitates direct coupling with ESI-MS but also allows fine-tuning of the extent of disulfide reduction by varying the UV exposure time. Near full sequence coverage for peptides consisting of intra- or interchain disulfide bonds has been achieved from complete disulfide reduction and online tandem mass spectrometry (MS/MS) via low energy collision-induced dissociation. Coupling different degrees of partial disulfide reduction with ESI-MS/MS allows disulfide mapping as demonstrated for characterizing the three disulfide bonds in insulin.
- Adhikari, Sarju,Yang, Xiaoyue,Xia, Yu
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- Inhibition of thermus thermophilus HB8 thioredoxin activity by platinum(II)
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A 1 : 1 thioredoxin-Pt(bpy) complex 1 was prepared by adding [Pt(bpy)(en)]Cl2 (bpy = 2,2'-bipyridine, en = ethylenediamine) to Thermus thermophilus HB8 thioredoxin in pH 8 phosphate buffer. Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) and UV spectra of 1 indicate the formation of Pt(bpy)(cys-Ala-Pro-cys-containing peptide fragment). These findings suggest that the Pt(bpy)2+ unit binds to the active site of thioredoxin. The thioredoxin-platinum complex has no catalytic activity for the reduction of glutathione disulfide in the presence of NADPH and thioredoxin reductase, so that the platinum complex functions as an inhibitor. The Royal Society of Chemistry 2005.
- Kato, Masahiro,Yamamoto, Hitoshi,Okamura, Taka-Aki,Maoka, Nobuko,Masui, Ryoji,Kuramitsu, Seiki,Ueyama, Norikazu
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- Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation
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Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDOand rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein,we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans. The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 ? resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.
- Motl, Nicole,Skiba, Meredith A.,Kabil, Omer,Smith, Janet L.,Banerjee, Ruma
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- Adsorption and orientation of the physiological extracellular peptide glutathione disulfide on surface functionalized colloidal alumina particles
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Understanding the interrelation between surface chemistry of colloidal particles and surface adsorption of biomolecules is a crucial prerequisite for the design of materials for biotechnological and nanomedical applications. Here, we elucidate how tailoring the surface chemistry of colloidal alumina particles (d50 = 180 nm) with amino (-NH2), carboxylate (-COOH), phosphate (-PO3H2) or sulfonate (-SO3H) groups affects adsorption and orientation of the model peptide glutathione disulfide (GSSG). GSSG adsorbed on native, -NH2-functionalized, and -SO 3H-functionalized alumina but not on -COOH- and -PO3H 2-functionalized particles. When adsorption occurred, the process was rapid (≤5 min), reversible by application of salts, and followed a Langmuir adsorption isotherm dependent on the particle surface functionalization and ζ potential. The orientation of particle bound GSSG was assessed by the release of glutathione after reducing the GSSG disulfide bond and by ζ potential measurements. GSSG is likely to bind via the carboxylate groups of one of its two glutathionyl (GS) moieties onto native and -NH2-modified alumina, whereas GSSG is suggested to bind to -SO3H-modified alumina via the primary amino groups of both GS moieties. Thus, GSSG adsorption and orientation can be tailored by varying the molecular composition of the particle surface, demonstrating a step toward guiding interactions of biomolecules with colloidal particles.
- Meder, Fabian,Hintz, Henrik,Koehler, Yvonne,Schmidt, Maike M.,Treccani, Laura,Dringen, Ralf,Rezwan, Kurosch
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- Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells
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Density functional theory (DFT) was applied to study the thermodynamics and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31G(d) with the SMD solvation model can reliably predict the Gibbs free energy changes (ΔG) of thiol-Michael addition reactions with an error of less than 1 kcal·mol-1 compared with the experimental benchmarks. Taking advantage of this computational model, the first reversible reaction-based fluorescent probe was developed that can monitor the changes in glutathione levels in single living cells.
- Chen, Jianwei,Jiang, Xiqian,Carroll, Shaina L.,Huang, Jia,Wang, Jin
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- Target discovery of ebselen with a biotinylated probe
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Despite numerous studies on ebselen over the past decade, its cellular targets remain obscure. Here we synthesized a biotinylated ebselen probe (biotin-ebselen) and characterized ebselen-binding proteins via an efficient activity-based protein profiling (ABPP) method, which allowed for the robust identification of 462 targeted proteins in HeLa cells. This first work of global target profiling of ebselen will be helpful to re-design ebselen-based therapy appropriately in clinical trials.
- Chen, Zhenzhen,Jiang, Zhongyao,Chen, Nan,Shi, Qian,Tong, Lili,Kong, Fanpeng,Cheng, Xiufen,Chen, Hao,Wang, Chu,Tang, Bo
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- Reaction of COTC with glutathione: Structure of the putative glyoxalase I inhibitor
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(matrix presented) The structure of the active glyoxalase I inhibitor derived from the Streptomyces griseosporeus metabolite COTC 1 has been conclusively identified by means of total synthesis as 2c. Human glyoxalase I is competitively inhibited by 2c (Ki = 183 ± 6 μM) but is not inhibited by 1 itself.
- Huntley, C. Frederick M.,Hamilton, Diana S.,Creighton, Donald J.,Ganem, Bruce
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- A promiscuous glutathione transferase transformed into a selective thiolester hydrolase
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Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 × 107 M-1. The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a kcat of 0.0032 min -1 and a KM of 41 M. The previously reported catalysis of GSB has a kcat of 0.00078 min-1 and KM of 5 M. The kcat for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (kcat/K M)/kuncat for GS-2 is 3 × 106 M -1. The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate. The Royal Society of Chemistry 2006.
- Hederos, Sofia,Tegler, Lotta,Carlsson, Jonas,Persson, Bengt,Viljanen, Johan,Broo, Kerstin S.
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- The thioredoxin-thioredoxin reductase system can function in vivo as an alternative system to reduce oxidized glutathione in Saccharomyces cerevisiae
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Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E h′. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular Eh′. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.
- Tan, Shi-Xiong,Greetham, Darren,Raeth, Sebastian,Grant, Chris M.,Dawes, Ian W.,Perrone, Gabriel G.
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- Electrical 'Wiring' of Glutathione Reductase: an Efficient Method for the Reduction of Glutathione using Molecular Hydrogen as the Reductant
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The enzyme glutathione reductase is chemically modified to become electrically conductive, thus facilitating the reduction of oxidized glutathione to its reduced form using hydrogen as the reductant, and the modified enzyme and Pt colloid as catalysts.
- Willner, Itamar,Lapidot, Noa
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- The chemical basis of thiol addition to nitro-conjugated linoleic acid, a protective cell-signaling lipid
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Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.
- Turell, Lucía,Vitturi, Darío A.,Coiti?o, E. Laura,Lebrato, Lourdes,M?ller, Matías N.,Sagasti, Camila,Salvatore, Sonia R.,Woodcock, Steven R.,Alvarez, Beatriz,Schopfer, Francisco J.
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- Metal-dependent inhibition of glyoxalase II: A possible mechanism to regulate the enzyme activity
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Glyoxalase II (GLX2, EC 3.1.2.6., hydroxyacylglutathione hydrolase) is a metalloenzyme involved in crucial detoxification pathways. Different studies have failed in identifying the native metal ion of this enzyme, which is expressed with iron, zinc and/or manganese. Here we report that GloB, the GLX2 from Salmonella typhimurium, is differentially inhibited by glutathione (a reaction product) depending on the bound metal ion, and we provide a structural model for this inhibition mode. This metal-dependent inhibition was shown to occur in metal-enriched forms of the enzyme, complementing the spectroscopic data. Based on the high levels of free glutathione in the cell, we suggest that the expression of the different metal forms of GLX2 during Salmonella infection could be exploited as a mechanism to regulate the enzyme activity.
- Campos-Bermudez, Valeria A.,Morán-Barrio, Jorgelina,Costa-Filho, Antonio J.,Vila, Alejandro J.
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- Thiolation and Carboxylation of Glutathione Synergistically Enhance Its Lead-Detoxification Capabilities
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The natural tripeptide glutathione (GSH) is a ubiquitous compound harboring various biological tasks, among them interacting with essential and toxic metal ions. Yet, although weakly binding the poisonous metal lead (Pb), GSH poorly detoxifies it. β-Mercaptoaspartic acid is a new-to-nature novel amino acid that was found to enhance the Pb-detoxification capability of a synthetic cyclic tetrapeptide. Aiming to explore the advantages of noncanonical amino acids (ncAAs) of this nature, we studied the detoxification capabilities of GSH and three analogue peptides, each of which contains at least one ncAA that harbors both free carboxylate and thiolate groups. A thorough investigation that includes in vitro detoxification and mechanistic evaluations, metal-binding affinity, metal selectivity, and computational studies shows that these ncAAs are highly beneficial in additively enhancing Pb binding and reveals the importance of both high affinity and metal selectivity in synergistically reducing Pb toxicity in cells. Hence, such ncAAs join the chemical toolbox against Pb poisoning and pollution, enabling peptides to strongly and selectively bind the toxic metal ion.
- Sauser, Luca,Mohammed, Tagwa A.,Kalvoda, Tadeá?,Feng, Sheng-Jan,Spingler, Bernhard,Rulí?ek, Lubomír,Shoshan, Michal S.
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supporting information
p. 18620 - 18624
(2021/12/13)
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- Visible Light-Mediated Synthesis of Se?S Bond-Containing Peptides
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A visible light-initiated method has been developed for preparation of Se?S bond-containing peptides. The method is based on generation of sulfur-centered radical employing organic dye. The protocol is tolerant to unprotected peptides with “sensitive” amino acids. The stability of Se?S bond is evaluated in buffers at different pH (3.0–10.0) and also in the presence of oxidants and reducing agents. Additionally, the ability of Se?S bond to serve as an oxidation sensitive linker in biocompatible materials has been confirmed. (Figure presented.).
- Arsenyan, Pavel,Dimitrijevs, Pavels,Lapcinska, Sindija
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supporting information
p. 3968 - 3972
(2021/07/26)
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- Ethynylation of Cysteine Residues: From Peptides to Proteins in Vitro and in Living Cells
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Current approaches to introduce terminal alkynes for bioorthogonal reactions into biomolecules still present limitations in terms of either reactivity, selectivity, or adduct stability. We present a method for the ethynylation of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. The acetylene group is directly introduced onto the thiol group of cysteine and can be used for copper-catalyzed alkyne-azide cycloaddition (CuAAC) without further processing. Labeling proceeded with reaction rates comparable to or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab, and high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine reagents. Fine-tuning of the EBX reagents allows optimization of their reactivity and physical properties.
- Tessier, Romain,Nandi, Raj Kumar,Dwyer, Brendan G.,Abegg, Daniel,Sornay, Charlotte,Ceballos, Javier,Erb, Stéphane,Cianférani, Sarah,Wagner, Alain,Chaubet, Guilhem,Adibekian, Alexander,Waser, Jerome
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supporting information
p. 10961 - 10970
(2020/05/18)
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- Crystal-facet-dependent denitrosylation: Modulation of NO release from S-nitrosothiols by Cu2O polymorphs
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Nitric oxide (NO), a gaseous small molecule generated by the nitric oxide synthase (NOS) enzymes, plays key roles in signal transduction. The thiol groups present in many proteins and small molecules undergo nitrosylation to form the corresponding S-nitrosothiols. The release of NO from S-nitrosothiols is a key strategy to maintain the NO levels in biological systems. However, the controlled release of NO from the nitrosylated compounds at physiological pH remains a challenge. In this paper, we describe the synthesis and NO releasing ability of Cu2O nanomaterials and provide the first experimental evidence that the nanocrystals having different crystal facets within the same crystal system exhibit different activities toward S-nitrosothiols. We used various imaging techniques and time-dependent spectroscopic measurements to understand the nature of catalytically active species involved in the surface reactions. The denitrosylation reactions by Cu2O can be carried out multiple times without affecting the catalytic activity.
- Ghosh, Sourav,Roy, Punarbasu,Prasad, Sanjay,Mugesh, Govindasamy
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p. 5308 - 5318
(2019/05/29)
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- “Doubly Orthogonal” Labeling of Peptides and Proteins
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Herein, we report a cysteine bioconjugation methodology for the introduction of hypervalent iodine compounds onto biomolecules. Ethynylbenziodoxolones (EBXs) engage thiols in small organic molecules and cysteine-containing peptides and proteins in a fast and selective addition onto the alkynyl triple bond, resulting in stable vinylbenziodoxolone hypervalent iodine conjugates. The conjugation occurs at room temperature in an open flask under physiological conditions. The use of an azide-bearing EBX reagent enables a “doubly orthogonal” functionalization of the bioconjugate via strain-release-driven cycloaddition and Suzuki-Miyaura cross-coupling of the vinyl hypervalent iodine bond. We successfully applied the methodology on relevant and complex biomolecules, such as histone proteins. Through single-molecule experiments, we illustrated the potential of this doubly reactive bioconjugate by introducing a triplet-state quencher close to a fluorophore, which extended its lifetime by suppressing photobleaching. This work is therefore expected to find broad applications for peptide and protein functionalization. Understanding the molecular basis of life is essential in the search for new medicines. Chemical biology develops molecular tools for studying biological processes, setting the basis for new diagnostics and therapeutics, and relies heavily on the ability to selectively modify biomolecules. Two approaches have been especially fruitful: (1) selective modification of natural biomolecules and (2) selective reaction between non-natural functionalities in the presence of biomolecules (the so-called orthogonal bioconjugation). In our work, we contribute to both by transferring highly reactive hypervalent iodine reagents to cysteine residues in proteins and peptides. The obtained bioconjugates retain the reactive hypervalent bonds, which can be selectively functionalized via a metal-mediated reaction. Combined with a traditional azide tag, our approach allows a doubly orthogonal functionalization of biomolecules and is hence expected to be highly useful in chemical biology. Chemical biology develops molecular tools for studying biological processes, setting the basis for new diagnostics and therapeutics, and relies heavily on the ability to modify selectively biomolecules. In our work, we introduce hypervalent iodine bonds into peptides and proteins, via functionalization of cysteine, by using unique cyclic reagents developed in our group. The hypervalent bond can then be selectively modified in the presence of both natural and synthetic functional groups, opening new opportunities for applications in chemical biology.
- Tessier, Romain,Ceballos, Javier,Guidotti, Nora,Simonet-Davin, Raphael,Fierz, Beat,Waser, Jerome
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supporting information
p. 2243 - 2263
(2019/08/08)
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- Higher-energy collision-induced dissociation for the quantification by liquid chromatography/tandem ion trap mass spectrometry of nitric oxide metabolites coming from S-nitroso-glutathione in an in vitro model of the intestinal barrier
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Rationale: The potency of S-nitrosoglutathione (GSNO) as a nitric oxide (NO) donor to treat cardiovascular diseases (CVDs) has been highlighted in numerous studies. In order to study its bioavailability after oral administration, which represents the most convenient route for the chronic treatment of CVDs, it is essential to develop an analytical method permitting (i) the simultaneous measurement of GSNO metabolites, i.e. nitrite, S-nitrosothiols (RSNOs) and nitrate and (ii) to distinguish them from other sources (endogenous synthesis and diet). Methods: Exogenous GSNO was labeled with 15N, and the GS15NO metabolites after conversion into the nitrite ion were derivatized with 2,3-diaminonaphthalene. The resulting 2,3-naphthotriazole was quantified by liquid chromatography/tandem ion trap mass spectrometry (LC/ITMS/MS) in multiple reaction monitoring mode after Higher-energy Collision-induced Dissociation (HCD). Finally, the validated method was applied to an in vitro model of the intestinal barrier (monolayer of Caco-2 cells) to study GS15NO intestinal permeability. Results: A LC/ITMS/MS method based on an original transition (m/z 171 to 156) for sodium 15N-nitrite, GS15NO and sodium 15N-nitrate measurements was validated, with recoveries of 100.8 ± 3.8, 98.0 ± 2.7 and 104.1 ± 3.3%, respectively. Intra- and inter-day variabilities were below 13.4 and 12.6%, and the limit of quantification reached 5 nM (signal over blank = 4). The permeability of labeled GS15NO (10–100 μM) was evaluated by calculating its apparent permeability coefficient (Papp). Conclusions: A quantitative LC/ITMS/MS method using HCD was developed for the first time to selectively monitor GS15NO metabolites. The assay allowed evaluation of GS15NO intestinal permeability and situated this drug candidate within the middle permeability class according to FDA guidelines. In addition, the present method has opened the perspective of a more fundamental work aiming at studying the fragmentation mechanism leading to the ion at m/z 156 in HCD tandem mass spectrometry in the presence of acetonitrile.
- Yu, Haiyan,Bonetti, Justine,Gaucher, Caroline,Fries, Isabelle,Vernex-Loset, Lionel,Leroy, Pierre,Chaimbault, Patrick
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- Method for manufacturing reduced glutathione
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A method of producing reduced glutathione by electrolytic reduction of oxidized glutathione using a cathode cell and an anode cell separated from each other by a separating membrane, including using a cathode having a metal cathode surface, and, as a cathode solution, an aqueous oxidized glutathione solution having a pH adjusted to higher than 3.0 and 5.0 or below by adding a base, which is added with the same metal as the metal of the cathode surface, a metal salt thereof, or a metal oxide thereof.
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Page/Page column 5
(2018/10/30)
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- Celastrol binds to its target protein via specific noncovalent interactions and reversible covalent bonds
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Celastrol is one of the most studied natural products. Our studies show for the first time that celastrol can bind to its target protein via specific noncovalent interactions that position celastrol next to the thiol group of the reactive cysteine for reversible covalent bond formation. Such specific noncovalent interactions confer celastrol binding specificity and demonstrate the feasibility of improving the efficacy and selectivity of celastrol for therapeutic applications.
- Zhang, Duo,Chen, Ziwen,Hu, Chaochao,Yan, Siwei,Li, Zhuoer,Lian, Baohuan,Xu, Yang,Ding, Rong,Zeng, Zhiping,Zhang, Xiao-kun,Su, Ying
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supporting information
p. 12871 - 12874
(2018/11/30)
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- Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry
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Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254?nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (–SOCH3) at one cysteine residue and a thiol (–SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (–32?Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (–SCH2OH) and methyl sulfoxide (–S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS2 CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS3 CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations. [Figure not available: see fulltext.].
- Durand, Kirt L.,Tan, Lei,Stinson, Craig A.,Love-Nkansah, Chasity B.,Ma, Xiaoxiao,Xia, Yu
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p. 1099 - 1108
(2017/05/24)
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- Tetrabutylammonium Fluoride as a Mild and Versatile Reagent for Cleaving Boroxazolidones to Their Corresponding Free α-Amino Acids
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Protection of α-amino acids with 9-borabicyclo[3.3.1]nonane (9-BBN) to give their corresponding boroxazolidones is highly attractive, as it concurrently masks both the amino and the carboxylic acid functionalities. However, the harsh methods required for deprotection of these boroxazolidones have limited their use. Herein, we report that tetrabutylammonium fluoride serves as a mild and versatile reagent that can be used to cleave boroxazolidones to their corresponding free α-amino acids. The reaction conditions were explored, including the use of various nucleophilic fluoride sources, solvents, and reaction temperatures. Nucleophilic fluoride sources comprising an ammonium cation proved superior to other countercations. The scope of the reaction was extended to the cleavage of B,B-diphenyl- and B,B-diethyl boroxazolidone complexes. Furthermore, a wide range of α-amino acid side-chain functionalities were shown to be compatible, including acids, esters, amides, thiols, thioethers, alkynes, phenols, basic heterocycles, and important biorelevant molecules such as glutathione, (S)-adenosyl-l-homocysteine, and l-biocytin.
- Poulie, Christian B. M.,Bunch, Lennart
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supporting information
p. 1475 - 1478
(2017/04/01)
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- Protection of Endogenous Thiols against Methylmercury with Benzimidazole-Based Thione by Unusual Ligand-Exchange Reactions
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Organomercurials, such as methylmercury (MeHg+), are among the most toxic materials to humans. Apart from inhibiting proteins, MeHg+ exerts its cytotoxicity through strong binding with endogenous thiols cysteine (CysH) and glutathione (GSH) to form MeHgCys and MeHgSG complexes. Herein, it is reported that the N,N-disubstituted benzimidazole-based thione 1 containing a N?CH2CH2OH substituent converts MeHgCys and MeHgSG complexes to less toxic water-soluble HgS nanoparticles (NPs) and releases the corresponding free thiols CysH and GSH from MeHgCys and MeHgSG, respectively, in solution by unusual ligand-exchange reactions in phosphate buffer at 37 °C. However, the corresponding N-substituted benzimidazole-based thione 7 and N,N-disubstituted imidazole-based thione 3, in spite of containing a N?CH2CH2OH substituent, failed to convert MeHgX (X=Cys, and SG) to HgS NPs under identical reaction conditions, which suggests that not only the N?CH2CH2OH moiety but the benzimidazole ring and N,N-disubstitution in 1, which leads to the generation of a partial positive charge at the C2 atom of the benzimidazole ring in 1:1 MeHg-conjugated complex of 1, are crucial to convert MeHgX to HgS NPs under physiologically relevant conditions.
- Banerjee, Mainak,Karri, Ramesh,Chalana, Ashish,Das, Ranajit,Rai, Rakesh Kumar,Rawat, Kuber Singh,Pathak, Biswarup,Roy, Gouriprasanna
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supporting information
p. 5696 - 5707
(2017/04/28)
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- Reactivity of 9-aminoacridine drug quinacrine with glutathione limits its antiprion activity
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Quinacrine—the drug based on 9-aminoacridine—failed in clinical trials for prion diseases, whereas it was active in in vitro studies. We hypothesize that aromatic nucleophilic substitution at C9 could be contributing factor responsible for this failure be
- ?afa?ík, Martin,Mo?ko, Tibor,Zawada, Zbigniew,?afa?íková, Eva,Dra?ínsky, Martin,Holada, Karel,?ebestík, Jaroslav
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p. 932 - 942
(2017/05/19)
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- Mass spectrometry-based assay for the rapid detection of thiol-containing natural products
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Natural products are privileged scaffolds due to their high propensity to possess bioactivity. To expedite discovery of thiol-containing compounds, we devised a selective solid-supported reagent for their immobilization, followed by cleavage of a photocleavable linker to yield stable natural product conjugates for direct detection by mass spectrometry. Importantly, the natural products can also be tracelessly released to yield the native structures for chemical and biological evaluation.
- Capehart, Stacy L.,Carlson, Erin E.
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supporting information
p. 13229 - 13232
(2016/11/17)
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- Converting disulfide bridges in native peptides to stable methylene thioacetals
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Disulfide bridges play a crucial role in defining and rigidifying the three-dimensional structure of peptides. However, disulfides are inherently unstable in reducing environments. Consequently, the development of strategies aiming to circumvent these deficiencies-ideally with little structural disturbance-are highly sought after. Herein, we report a simple protocol converting the disulfide bond of peptides into highly stable methylene thioacetal. The transformation occurs under mild, biocompatible conditions, enabling the conversion of unprotected native peptides into analogues with enhanced stability. The developed protocol is applicable to a range of peptides and selective in the presence of a multitude of potentially reactive functional groups. The thioacetal modification annihilates the reductive lability and increases the serum, pH and temperature stability of the important peptide hormone oxytocin. Moreover, it is shown that the biological activities for oxytocin are retained.
- Kourra,Cramer
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p. 7007 - 7012
(2016/11/23)
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- OXYLIPIN-PEPTIDE CONJUGATED MEDIATORS THAT PROMOTE RESOLUTION OF INFECTION, ORGAN PROTECTION AND TISSUE REGENERATION
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A family of bioactive compounds identified in self-resolving inflammatory exudates is disclosed. The compounds give UV chromophores characteristic of a conjugated triene double bond system coupled to an auxochrome allylic to the triene. Further elucidation of the compounds reveals that they have an oxylipin backbone conjugated to a peptide or amino acid moiety via an auxochrome. In some embodiments the auxochrome is sulfur. However, the auxochrome may be NH, CH2 or O. The compounds have potent bioactivity, in vitro, and, in vivo, including promoting resolution of infection, stimulating macrophage phagocytosis of bacteria; protecting tissues from neutrophil mediated damage, promoting tissue repair and regeneration and preventing or limiting second organ reflow/reperfusion damage.
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- A mild and selective protecting and reversed modification of thiols
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One selective thiol-protecting study has been investigated for a wide range of thiols including general thiols and thiols containing multiple functional groups. The reactions of bromomaleimides and thiols under the mild condition afforded the protected products in excellent yields. The thiols can be recovered very quickly using dithiothreitol (DTT) under the mild condition.
- Li, Xiangmin,Li, Hongxian,Yang, Wei,Zhuang, Jinchen,Li, Hao,Wang, Wei
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supporting information
p. 2660 - 2663
(2016/06/01)
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- Online Investigation of Aqueous-Phase Electrochemical Reactions by Desorption Electrospray Ionization Mass Spectrometry
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Electrochemistry (EC) combined with mass spectrometry (MS) is a powerful tool for elucidation of electrochemical reaction mechanisms. However, direct online analysis of electrochemical reaction in aqueous phase was rarely explored. This paper presents the online investigation of several electrochemical reactions with biological relevance in the aqueous phase, such as nitrosothiol reduction, carbohydrate oxidation, and carbamazepine oxidation using desorption electrospray ionization mass spectrometry (DESI-MS). It was found that electroreduction of nitrosothiols [e.g.; nitrosylated insulin B (13-23)] leads to free thiols by loss of NO, as confirmed by online MS analysis for the first time. The characteristic mass shift of 29 Da and the reduced intensity provide a quick way to identify nitrosylated species. Equally importantly, upon collision-induced dissociation (CID), the reduced peptide ion produces more fragment ions than its nitrosylated precursor ion (presumably the backbone fragmentation cannot compete with the facile NO loss for the precursor ion), thus facilitating peptide sequencing. In the case of saccharide oxidation, it was found that glucose undergoes electro-oxidation to produce gluconic acid at alkaline pH, but not at neutral and acidic pHs. Such a pH-dependent electrochemical behavior was also observed for disaccharides such as maltose and cellobiose. Upon electrochemical oxidation, carbamazepine was found to undergo ring contraction and amide bond cleavage, which parallels the oxidative metabolism observed for this drug in leucocytes. The mechanistic information of these redox reactions revealed by EC/DESI-MS would be of value in nitroso-proteome research and carbohydrate/drug metabolic studies.
- Lu, Mei,Liu, Yong,Helmy, Roy,Martin, Gary E.,Dewald, Howard D.,Chen, Hao
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p. 1676 - 1685
(2015/09/22)
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- Reaction of hydrogen sulfide with disulfide and Sulfenic acid to form the strongly Nucleophilic Persulfide
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Background: Hydrogen sulfide (H2S) modulates physiological processes in mammals. Results: The reactivity of H2S toward disulfides (RSSR) and albumin sulfenic acid (RSOH) to form persulfides (RSSH) was assessed. Conclusion: H2S is less reactive than thiols. Persulfides have enhanced nucleophilicity. Significance: This kinetic study helps rationalize the contribution of the reactions with oxidized thiol derivatives toH2S biology.
- Cuevasanta, Ernesto,Lange, Mike,Bonanata, Jenner,Coiti?o, E. Laura,Ferrer-Sueta, Gerardo,Filipovic, Milos R.,Alvarez, Beatriz
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p. 26866 - 26880
(2015/11/17)
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- The crystal structure of a homodimeric Pseudomonas glyoxalase I enzyme reveals asymmetric metallation commensurate with half-of-sites activity
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The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni2+ ion, whereas the other contains two inactivating Zn2+ ions. Enzymological experiments prompted by the binuclear Zn2+ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn2+/Co2+ -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.
- Bythell-Douglas, Rohan,Suttisansanee, Uthaiwan,Flematti, Gavin R.,Challenor, Michael,Lee, Mihwa,Panjikar, Santosh,Honek, John F.,Bond, Charles S.
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p. 541 - 544
(2015/10/12)
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- Vicinal disulfide constrained cyclic peptidomimetics: A turn mimetic scaffold targeting the norepinephrine transporter
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Loopy peptides: Peptide turn mimetics of a clinically relevant norepinephrine reuptake inhibitor were developed employing a high-throughput synthesis approach to generate peptide thioesters, with subsequent cyclization through native chemical ligation. The vicinal disulfide constrained cyclic peptidomimetics (see scheme) show high structural and functional similarity to the parent peptide, though with superior metabolic stability. Copyright
- Brust, Andreas,Wang, Ching-I. A.,Daly, Norelle L.,Kennerly, Joe,Sadeghi, Mahsa,Christie, Macdonald J.,Lewis, Richard J.,Mobli, Mehdi,Alewood, Paul F.
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supporting information
p. 12020 - 12023
(2013/12/04)
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- Facile dimethylarsenic exchange and pyramidal inversion in its cysteine and glutathione adducts
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Rapid thiolate exchange of dimethylarsonium, Me2As+, is observed between two different thiolate species in solution. NMR is used to characterize the equilibrium constants for interthiol transfer as well the rapid intra molecular conf
- Bohle, D. Scott,Gu, Yuxuan
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p. 2578 - 2581
(2013/06/05)
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- BINARY AND TERTIARY GALVANIC PARTICULATES AND METHODS OF MANUFACTURING AND USE THEREOF
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The present invention relates to galvanic particulates, their methods of manufacture and uses in treatments are described. The galvanic particulates may be binary or tertiary galvanic particulates, for example, containing multiple layers or phases of conductive materials.
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- Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties
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Background: Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance. Methods: The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR). Results: Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S-nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR. Conclusions and general significance: Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.
- Arias, Diego G.,Regner, Erika L.,Iglesias, Alberto A.,Guerrero, Sergio A.
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p. 1859 - 1866
(2013/01/15)
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- S-nitrosothiol chemistry at the single-molecule level
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SNO patrol: S-Nitrosothiols (RSNO) are important molecules involved in cell signaling, which control physiological processes such as vasodilation and bronchodilation. By using the protein pore α-hemolysin as a nanoreactor, the biological chemistry of RSNO has been investigated at the single-molecule level (see scheme). Copyright
- Choi, Lai-Sheung,Bayley, Hagan
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supporting information; scheme or table
p. 7972 - 7976
(2012/08/29)
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- Controllable synthesis of PbSe nanocubes in aqueous phase using a quasi-biosystem
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By coupling two biochemical processes of reduction of Na 2SeO3 with detoxification of Pb2+ in a quasi-biosystem, water-dispersed PbSe nanocubes with good monodispersity were controllably synthesized with different sizes at low temperature (90°C) under mild conditions. The crystallization mechanism and the nature of bio-molecules influenced on the crystallization process were investigated.
- Cui, Ran,Gu, Yi-Ping,Zhang, Zhi-Ling,Xie, Zhi-Xiong,Tian, Zhi-Quan,Pang, Dai-Wen
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supporting information; experimental part
p. 3713 - 3716
(2012/06/15)
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- Antitumor quinol PMX464 is a cytocidal anti-trypanosomal inhibitor targeting trypanothione metabolism
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Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class ("quinols") and identified several with potent trypanocidal activity (EC50 2), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)2, TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and L-cysteine bound in a 2:1 ratio with PMX464 with Kd values of 6 and 27 μM, respectively, whereas T(SH)2 bound more tightly in a 1:1 ratio with a Kd value of 430 nM. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)2 is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.
- Koenig, Janine,Wyllie, Susan,Wells, Geoffrey,Stevens, Malcolm F.,Wyatt, Paul G.,Fairlamb, Alan H.
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experimental part
p. 8523 - 8533
(2012/03/26)
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- Fluorescence turn-on assay for glutathione reductase activity based on a conjugated polyelectrolyte with multiple carboxylate groups
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In this work, an anionic conjugated polyelectrolyte (CPE) that features four carboxylate groups on each repeat unit, PPE-(COOK)4, has been designed and synthesized by integrating the two synthetic strategies for conjugated poly(phenylene ethynylene) and dendritic polyamidoamine. Through the installation of multiple functional groups, the proposed CPE has been greatly improved in many aspects, such as the good water solubility, slight aggregation, and relatively high quantum yield. Thanks to the close resemblance in chemical structures between the polymer's pendant groups and EDTA, the resulting PPE-(COOK)4 has also been found to show stronger complexation with metal ions, and has further been demonstrated to be an excellent fluorescent probe for Cu2+ with both high sensitivity and remarkable specificity. Moreover, by taking advantage of the distinct fluorescence recovery rates of PPE-(COOK)4/Cu2+ caused by GSH and by GSSG, a novel fluorescence turn-on assay for glutathione reductase (GR) has successfully been developed. With a minimum detectable enzyme concentration of 0.2 mU/mL, this newly proposed GR activity assay is highly sensitive and robust as compared to most spectrophotometric and fluorescent methods. The Royal Society of Chemistry 2010.
- Fan, Hongliang,Zhang, Tao,Lv, Shaowu,Jin, Qinhan
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body text
p. 10901 - 10907
(2011/09/20)
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- Purification and kinetics of bovine kidney cortex glutathione reductase
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Glutathione reductase was purified 34806-fold with a final yield of 85% from the bovine kidney cortex. Some molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme obeys 'branched' mechanism: KmNADPH 18 ± 3 μM and KmGSSG 65 ± 5 μM were determined.
- Tandogan, Berivan,Nuray Ulusu
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experimental part
p. 667 - 674
(2011/12/03)
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- Crystal structure of an S-formylglutathione hydrolase from pseudoalteromonas haloplanktis TAC125
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S-formylglutathione hydrolases (FGHs) constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes, catalyzing the hydrolysis of S-formylglutathione to formic acid and glutathione. While a large number of functional studies have been reported on these enzymes, few structural studies have so far been carried out. In this article we report on the functional and structural characterization of PhEst, a FGH isolated from the psychrophilic bacterium Pseudoalteromonas haloplanktis. According to our functional studies, this enzyme is able to efficiently hydrolyze several thioester substrates with very small acyl moieties. By contrast, the enzyme shows no activity toward substrates with bulky acyl groups. These data are in line with structural studies which highlight for this enzyme a very narrow acyl-binding pocket in a typical α/β-hydrolase fold. PhEst represents the first cold-adapted FGH structurally characterized to date; comparison with its mesophilic counterparts of known three-dimensional structure allowed to obtain useful insights into molecular determinants responsible for the ability of this psychrophilic enzyme to work at low temperature.
- Alterio, Vincenzo,Aurilia, Vincenzo,Romanelli, Alessandra,Parracino, Antonietta,Saviano, Michele,D'Auria, Sabato,de Simone, Giuseppina
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body text
p. 669 - 677
(2011/11/04)
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- Protein S-thiolation by glutathionylspermidine (Gsp): The role of Escherichia coli Gsp synthetase/amidase in redox regulation
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Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated byH 2O2 oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA and GspSA/glutaredoxin null mutants toH2O2 support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli.
- Chiang, Bing-Yu,Chen, Tzu-Chieh,Pai, Chien-Hua,Chou, Chi-Chi,Chen, Hsuan-He,Ko, Tzu-Ping,Hsu, Wen-Hung,Chang, Chun-Yang,Wu, Whei-Fen,Wang, Andrew H.-J.,Lin, Chun-Hung
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experimental part
p. 25345 - 25353
(2011/04/17)
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- Reaction mechanisms of allicin and allyl-mixed disulfides with proteins and small thiol molecules
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Allylsulfides from garlic are chemopreventive agents. Entering cells they are expected to initially interact with glutathione. Accordingly, reaction mechanisms of the product, S-allylthio-glutathione, with model proteins and thiols were analyzed in cell f
- Miron, Talia,Listowsky, Irving,Wilchek, Meir
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body text
p. 1912 - 1918
(2010/07/04)
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- Bisbenzamidine derivatives for use as antioxidant
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The invention is directed to the use of bisbenzamidine compounds of Formula (I) as antioxidant and as neuroprotective agents, wherein R1, R2, R3, R4 and L have the meaning defined in the claims.
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- Kinetics and mechanism of thermal decomposition of kynurenines and biomolecular conjugates: Ramifications for the modification of mammalian eye lens proteins
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Thermal degradation reactions of kynurenine (KN), 3-hydroxykynurenine (3OHKN), and several adducts of KN, to amino acids and reduced glutathione (GSH) have been studied at physiological temperature. These compounds are all implicated in age-related mammalian eye lens cataract formation at the molecular level. The main reaction pathway for both KN and 3OHKN is deamination viaβ-elimination to carboxyketoalkenes CKA and 3OHCKA. These reactions show a weak pH dependence below pH values of ～8, and a strong pH dependence above this value. The 3OHKN structure deaminates at a faster rate than KN. A mechanism for the deamination reaction is proposed, involving an aryl carbonyl enol/enolate ion, that is strongly supported by the structural, kinetic, and pH data. The degradation of Lys, His, Cys and GSH adducts of the CKA moieties was also studied. The Lys adduct was found to be relatively stable over 200 h at 37 °C, while significant degradation was observed for the other adducts. The results are discussed in terms of known post-translational modification reactions of the lens proteins and compared to incubation studies involving KN and related compounds in the presence of proteins.
- Kopylova, Lyudmila V.,Snytnikova, Olga A.,Chernyak, Elena I.,Morozov, Sergey V.,Forbes, Malcolm D. E.,Tsentalovich, Yuri P.
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experimental part
p. 2958 - 2966
(2011/02/25)
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