Multiple roles of cysteine in biocatalysis

In biology, sulfur frequently occurs in the form of cysteine, an amino acid that fulfills a wide range of different functions in proteins including disulfide formation, metal-binding, electron donation, hydrolysis, and redox-catalysis. The 'redox-chameleon' sulfur appears in several oxidation states in vitro, each of them exhibiting specific reactivity, redox-activity, and metal-binding properties. While cysteine-peptidases rely on reduced cysteine to catalyze hydrolytic reactions, many redox-enzymes use distinctively different cysteine redox-couples for exchange, electron, atom, and radical transfer reactions. Although cysteine and cystine can still be considered as the most abundant forms of cysteine in vivo, other modifications such as cysteine acids and sulfur-centered radicals are becoming increasingly important in biochemical research. As such, the biochemistry of sulfur remains a source of continuous investigation and excitement.     

Generation of a cysteine sulfinic acid analog for incorporation in peptides using solid phase peptide synthesis

The sulfinic acid analog of aspartic acid, cysteine sulfinic acid, introduces a sulfur atom that perturbs the acidity and oxidation properties of aspartic acid. Cysteine sulfinic acids are often introduced in peptides and proteins by oxidation of cysteine, but this method is limited as all cysteine residues are oxidized and cysteine residues are often oxidized to sulfonic acids. To provide the foundation for the specific incorporation of cysteine sulfinic acids in peptides and proteins, we synthesized a 9-fluorenylmethyloxycarbonyl (Fmoc) benzothiazole sulfone analog. Oxidation conditions to generate the sulfone were examined and oxidation of the Fmoc-protected sulfide (3) with NbC in hydrogen peroxide provided the corresponding sulfone (4) in the highest yield and purity. Reduction with sodium borohydride generated the cysteine sulfinic acid (5) suggesting this approach may be an efficient method to incorporate a cysteine sulfinic acid in biomolecules. A model tripeptide bearing a cysteine sulfinic acid was synthesized using this approach. Future studies are aimed at using this method to incorporate cysteine sulfinic acids in peptide hormones and proteins for use in the study of biological function.     

Isotope-coded chemical reporter and acid-cleavable affinity reagents for monitoring protein sulfenic acids

We have developed an approach that allows relative quantification of protein sulfenic acids using a pair of light and heavy isotope labled probes, DAz-2 and d₆-DAz-2. In conjunction with a new complementary acid-cleavable linker, Yn-ACL, we demonstrate that tagged peptides are successfully labeled, enriched, and fully characterized by LC-MS/MS analysis. Overall, this method can be applied to map sites of cysteine oxidation and compare protein sulfenylation in normal and disease states.     

Novel findings on the copper catalysed oxidation of cysteine

Summary The oxidation of cysteine (RSH) has been studied by using O₂, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200M Cu^II to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-Cu^I-SR) is responsible for this absorbance. In the absence of O₂ the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of Cu^II enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-Cu^I-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.Abbreviations RSH cysteine - RS^– cysteine in the thiolate form - RS^· thiyl radical of cysteine - RSSR cystine - Cyt c cytochrome c - SOD superoxide dismutase - NBT nitro blue tetrazolium - NBF nitro blue formazan - DTNB 5,5-dithiobis-2-nitrobenzoic acid - DTPA diethylenetriaminepentaacetic acid Dedicated to prof. A. Ballio ob the occasion of his 75th birthday.     

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

Formation of 8-S-l-cysteinylguanosine from 8-bromoguanosine and cysteine

When 8-bromoguanosine was incubated with cysteine at pH 7.4 and 37 °C, a previously unidentified product was formed as a major product in addition to guanosine. The product was identified as a cysteine substitution derivative of guanosine at the 8 position, 8-S-l-cysteinylguanosine. The reaction was accelerated under mildly basic conditions. The cysteine adduct of guanosine was fairly stable and decomposed with a half-life of 193 h at pH 7.4 and 37 °C. Similar results were observed for incubation of 8-bromo-2′-deoxyguanosine with cysteine. The results suggest that 8-bromoguanine in nucleosides, nucleotides, RNA, and DNA can react with thiols resulting in stable adducts.     

Differential Effects of Methionine and Cysteine Oxidation on [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Cultured Hippocampal Neurons

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca²⁺]_i in hippocampal neurons. We investigated the effects of H₂O₂ and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB)—a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H₂O₂, 1 mM Ch-T, and 500 μM DTNB, induced an sustained elevation of [Ca²⁺]_i by 76.1 ± 3.9%, 86.5 ± 5.0%, and 24.4 ± 3.2% over the basal level, respectively. The elevation induced by H₂O₂ and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca²⁺]_i, but only partially reduced the effects of H₂O₂ and Ch-T on [Ca²⁺]_i, the reductions were 44.6 ± 4.2% and 29.6 ± 6.1% over baseline, respectively. The elevation of [Ca²⁺]_i induced by H₂O₂ and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca²⁺]_i mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.     

Antioxidant enzymes as redox-based biomarkers: a brief review

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208]     

Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50 ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29 ± 0.29% for cysteine to 3.65 ± 0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.     

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO₂H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.     

Comparative reactivity analysis of small-molecule thiol surrogates

Targeted covalent inhibitors represent an increasingly popular approach to modulate challenging drug targets. Since covalent and non-covalent interactions are both contributing to the affinity of these compounds, evaluation of their reactivity is a key-step to find feasible warheads. There are well-established HPLC- and NMR-based kinetic assays to tackle this task, however, they use a variety of cysteine-surrogates including cysteamine, cysteine or acetyl-cysteine and GSH. The diverse nature of the thiol sources often makes the results incomparable that prevents compiling a comprehensive knowledge base for the design of covalent inhibitors. To evaluate kinetic measurements from different sources we performed a comparative analysis of the different thiol surrogates against a designed set of electrophilic fragments equipped with a range of warheads. Our study included seven different thiol models and 13 warheads resulting in a reactivity matrix analysed thoroughly. We found that the reactivity profile might be significantly different for various thiol models. Comparing the different warheads, we concluded that – in addition to its human relevance - glutathione (GSH) provided the best estimate of reactivity with highest number of true positives identified.     

Inhibitory Effects of Cysteine and Cysteine Derivatives on Germination of Sporangiospores and Hyphal Growth of Different Zygomycetes

The in vitro antifungal activity of cysteine (d- and l-cysteine) and its four derivatives (l-cysteine-methyl-ester, N-acetyl-cysteine, N-isobutyryl-d-cysteine, and N-isobutyryl-l-cysteine) were investigated on 20 fungal isolates representing 16 genera (Absidia, Actinomucor, Backusella, Gilbertella, Micromucor, Mortierella, Mucor, Mycotypha, Phycomyces, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, Thamnostylum, Umbellopsis, and Zygorynchus). The inhibitory potential of different concentrations of these compounds, ranging from 0.625 to 10 mM, were investigated on the germination of sporangiospores as well as on hyphal extension, using broth microdilution method and agar plate test. Treatment with cysteine and its derivatives resulted in a strong inhibition in most studied strains. At 10 mM of compounds, complete blockage of growth was observed for some isolates. Sensitive species exhibited severe changes in colony morphology in the presence of 10 mM l-cysteine, N-acetyl-cysteine, and N-isobutyryl-l-cysteine. Microscopic observations revealed that 10 mM N-acetyl-cysteine induced dramatic modifications in the structural organization of the hyphae. Results suggest that cysteine and its derivatives have a therapeutic potential against fungal infections caused by Zygomycetes species.     

The functional role of cysteine residues for c-Abl kinase activity

S-glutathionylation, the formation of mixed disulfides of glutathione with cysteine residues of proteins, is a broadly observed physiological modification that occurs in response to oxidative stress. Since cysteine residues are particularly susceptible to oxidative modification by reactive oxygen species, S-glutathionylation can protect proteins from irreversible oxidation. In this study, we show that the kinase activity of the non-receptor tyrosine kinase c-Abl is inhibited by in vitro thiol modification; specifically, the cysteine residues of c-Abl are modified by S-glutathionylation and by thiol alkylating agents such as 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid and N-ethylmaleimide. Modification of cysteine residues of c-Abl tyrosine kinase using glutathione disulfide and thiol alkylating agents corresponds to a concomitant loss of kinase activity. We also demonstrate that S-glutathionylation of c-Abl can be reversed using a physiological system involving glutaredoxin and this reversal restores c-Abl kinase activity. To our knowledge, these are the first data to show S-glutathionylation of c-Abl, and this modification may represent a mechanism of regulation of c-Abl kinase activity in cells under oxidative stress.     

Crystal structure of serine acetyl transferase from Brucella abortus and its complex with coenzyme A

Brucella abortus is the major cause of premature foetal abortion in cattle, can be transmitted from cattle to humans, and is considered a powerful biological weapon. De novo cysteine biosynthesis is one of the essential pathways reported in bacteria, protozoa, and plants. Serine acetyltransferase (SAT) initiates this reaction by catalyzing the formation of O-acetylserine (OAS) using l-serine and acetyl coenzyme A as substrates. Here we report kinetic and crystallographic studies of this enzyme from B. abortus. The kinetic studies indicate that cysteine competitively inhibits the binding of serine to B. abortus SAT (BaSAT) and noncompetitively inhibits the binding of acetyl coenzyme A. The crystal structures of BaSAT in its apo state and in complex with coenzyme A (CoA) were determined to 1.96 Å and 1.87 Å resolution, respectively. BaSAT was observed as a trimer in a size exclusion column; however, it was seen as a hexamer in dynamic light scattering (DLS) studies and in the crystal structure, indicating it may exist in both states. The complex structure shows coenzyme A bound to the C-terminal region, making mostly hydrophobic contacts from the center of the active site extending up to the surface of the protein. There is no conformational difference in the enzyme between the apo and the complexed states, indicating lock and key binding and the absence of an induced fit mechanism.     

An assay to quantitate reducible cysteines from nanograms of GST-fusion proteins

Cysteine residues in proteins are targets of numerous post-translational modifications and play important roles in protein structure and enzymatic function. Consequently, understanding the full biochemistry of proteins depends on determining the oxidation state and availability of the residues to be modified. We have developed a highly sensitive assay that accurately determines the number of unmodified cysteine residues in GST-fusion proteins. Only picomoles of protein are required for each reaction, which are carried out in 96-well glutathione-coated plates. Free unmodified cysteine residues are labeled and quantified using biotin and HRP-conjugated streptavidin. Our assay can be used to quantify reactions targeting sulfhydryl groups in proteins. We demonstrate this assay using full-length and truncation mutants of the SNARE proteins syntaxin1A, SNAP-25B, and synaptobrevin2, which have 0–4 cysteines. We are able to accurately determine the number of cysteine residues in each protein and follow the modification of these cysteines by oxidation and reaction with NEM (N-ethylmaleimide). This assay is as simple as running an ELISA or Western blot and, because of its high resolution, should allow detailed analysis of the chemistry of cysteine residues in proteins.     

Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase

Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize. Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain. The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M. thermophila and possibly form an operon. When M. thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.     

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.     

The inhibitory effect of l-cysteine and its derivatives on glycolysis in Ehrlich ascites tumour cells

(1) l-Cysteine inhibits aerobic glycolysis and restores the Pasteur effect in Ehrlich ascites tumour cells or in their supernatants, while d-cysteine has no effect on this process. (2) Other compounds which have configuration l at the α-carbon and a thiol group in the β-position (penicillamine) or restore them in vivo (3-mercaptopyruvate, cystine or l-serine together with l-homocysteine) also show inhibitory properties. (3) dl-Homocysteine with a free thiol group in the γ-position, reduced glutathione, methionine and products of cysteine oxidation (cysteic acid, taurine) do not inhibit tumour aerobic glycolysis. (4) Glycolysis of normal tissue supernatants (mouse liver and muscle) is not sensitive to the inhibitory effect of cysteine. (5) Metabolic studies showing a cysteine-induced decrease in ATP content, coupled with cross-over of the pyruvate and 2-phosphoenolpyruvate concentrations in Ehrlich ascites tumour cells, indicate that tumour pyruvate kinase is an enzyme sensitive to cysteine inhibition. (6) Enzymatic studies carried out both after preincubation of Ehrlich ascites tumour cells with cysteine or during direct action of this substance on tumour and normal tissue supernatants indicate the presence of a cysteine-sensitive isoenzyme besides the normal cysteine-insensitive pyruvate kinase in tumour material.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.