• Bulletin of the Korean Chemical Society
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• v.33 no.12
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• pp.4169-4172
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• 2012

To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the ${K_m}^{CDNB}$ value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

• Molecules and Cells
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• v.26 no.3
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• pp.228-235
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• 2008

Thiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. Thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues.

• Journal of the Korean Chemical Society
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• v.50 no.5
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• pp.362-368
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• 2006

SH group modifying chemicals were used to characterize the eight cysteine residues of cabbage PLD. 5,5-dithiobis(2-nitrobenzoate)(DTNB) was used to titrate the SH group of cysteine residues . Based on the optical density at 412nm due to the reduced DTNB, 4 SH groups are found to be present in a native PLD while 8 SH groups in the denatured PLD whose tertiary structure was perturbed by 8M urea. The results imply that among the 8 cysteine residues of PLD, the half(4) are exposed on the surface whereas the other half are present at the interior of the enzyme tertiary structure. The PLD was inactivated by SH modifying reagents such as p-chloromercuribenzoate(PCMB), iodoacetate, iodoacetamide, and N-ethylmaleimide. At the addition of dithiothreitol(DTT) only the PCMB inhibited PLD activity was recovered reversibly. The micro-environment of the exposed SH group of cysteine residues was examined with various disulfide compounds with different functional groups and we found that anionic or neutral disulfides appear to be more effective than the positively charged cystamine for inactivating the PLD activity. The effect of redox state of cysteine residues on the PLD activity was further explored with H2O2. The oxidation of SH groups by H2O2 inhibited the PLD activity more than 70%, which was mostly recovered by DTT. From these results, we could confirm chemically that all the cysteine residues of PLD are present as in their reduced SH forms and the 4 SH groups exposed on the surface of the enzyme may play important roles in the regulation of PLD activity.

• Mass Spectrometry Letters
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• v.3 no.1
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• pp.10-14
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• 2012

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.

• The Journal of Natural Sciences
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• v.17 no.1
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• pp.13-29
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• 2006

Most redox-active proteins have thiol-bearing cysteine residues that are sensitive to oxidation. Cysteine thiols oxidized to sulfenic acid are generally unstable, either forming a disulfide with a nearby thiol or being further oxidized to a stable sulfinic acid, which have been viewed as an irreversible protein modification. However, recent studies showed that cysteine residues of certain thiol peroxidases (Prxs) undergo reversible oxidation to sulfinic acid and the reduction reaction is catalyzed by sulfiredoxin (Srx1). Specific Cys residues of various other proteins are also oxidized to sulfinic acid ($Cys-So_2H$). Srxl is considered one of the oxidant proteins with a role in signaling through catalytic reduction of oxidative modification like in the reduction of glutathionylation, a post-translational, oxidative modification that occurs on numerous proteins. In this study, the role of sulfiredoxin in cellular processes, was investigated by studying its interaction with other proteins. Through the yeast two-hybrid system (Y2HS) technique, we have found that Ams1 is a potential and novel interacting protein partner of Srxl. $\alpha$-mannosidase (Ams1) is a resident vacuolar hydrolase which aids in recycling macromolecular components of the cell through hydrolysis of terminal, non-reducing $\alpha$-D-mannose residues. It forms an oligomer in the cytoplasm and under nutrient rich condition and is delivered to the vacuole by the Cytoplasm to Vacuole (Cvt) pathway. Aside from the role of Srxl as a catalyst in the reduction of cysteine sulfenic acid groups, it may play a completely new function in the cellular process as indicated by its interaction with Ams1 of the yeast Saccharomyces cerevisiae.

• BMB Reports
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• v.38 no.4
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• pp.474-480
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• 2005

Curcumin, a major active component of turmeric, has been identified as an inhibitor of the transcriptional activity of activator protein-1 (AP-1). Recently, it was also found that curcumin and synthetic curcumin derivatives can inhibit the binding of Jun-Fos, which are the members of the AP-1 family, to DNA. However, the mechanism of this inhibition by curcumin and its derivatives was not disclosed. Since the binding of Jun-Fos dimer to DNA can be modulated by redox control involving conserved cysteine residues, we studied whether curcumin and its derivatives inhibit Jun-Fos DNA binding activity via these residues. However, the inhibitory mechanism of curcumin and its derivatives, unlike that of other Jun-Fos inhibitors, was found to be independent of these conserved cysteine residues. In addition, we investigated whether curcumin derivatives can inhibit AP-1 transcriptional activity in vivo using a luciferase assay. We found that, among the curcumin derivatives examined, only inhibitors shown to inhibit the binding of Jun-Fos to DNA by Electrophoretic Mobility Shift Assay (EMSA) inhibited AP-1 transcriptional activity in vivo. Moreover, RT-PCR revealed that curcumin derivatives, like curcumin, downregulated c-jun mRNA in JB6 cells. These results suggest that the suppression of the formation of DNA-Jun-Fos complex is the main cause of reduced AP-1 transcriptional activity by curcuminoids, and that EMSA is a suitable tool for identifying inhibitors of transcriptional activation.

• BMB Reports
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• v.35 no.2
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• pp.178-185
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• 2002

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine $Cys^{50}$, $Cys^{83}$, and $Cys^{230}$, and $lys^{81}$ residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the $Cys^{50}$ and $Cys^{230}$ mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the $Cys^{50}$ and $Cys^{230}$ mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the $Cys^{50}$ and $Cys^{230}$ residues are structurally important.

• Bulletin of the Korean Chemical Society
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• v.22 no.1
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• pp.77-83
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• 2001

To gain further insight into the relationship between structure and function of glutathione S-transferase (GST), the four cysteine mutants, C14S, C47S, C101S and C169S, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized glutathione (GSH). The catalytic activities of the four mutant enzymes were characterized with five different substrates as well as by their binding to four different inhibitors. Cys14 seems to participate in the catalytic reaction of GST by stabilizing the conformation of the active-site loop, not in the GSH binding directly. The substitution of Cys47 with serine significantly reduces the affinity of GSH binding, although it does not prevent GSH binding. On the other hand, the substitution of Cys101 with serine appears to change the binding affinity of electrophilic substrate by inducing a conformational change of the $\alpha-helix$ D. Cys169 seems to be important for maintaining the stable conformation of the enzyme. In addition, all four cysteine residues are not needed for the steroid isomerase activity of human glutathione S-transferase P1-1.

• Animal cells and systems
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• v.2 no.2
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• pp.243-248
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• 1998

We have partially sequenced glutaminyl cyclases from several mammalian and one avian species and found that the two cysteine residues of the human glutaminyl cyclase are completely conserved. The mammalian glutaminyl cyclase has been reported to possess reactive thiols (Busby, Jr, et aI., 1987, J BioI Chern 262, 8532-8536). Mutagenesis of these cysteine residues, however, resulted in only a slight decrease in enzyme activity. Likewise, the recombinant human enzyme was completely resistant to attempted chemical modification of the putative reactive thiols. Although the human glutaminyl cyclase did not appear to have reactive thiols, it was sensitive to diethylpyrocarbonate and acetylimidazole, indicating the presence of functionally important histidine and tyrosine residues which could act as acid/base catalysts. Almost identical deuterium solvent isotope effect (1.2 vs 1.3) upon the reaction by the human and papaya enzymes, respectively, provides an evidence both animal and plant glutaminyl cyclases catalyze pyroglutamyl-peptide formation by intramolecular cyclization.

• BMB Reports
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• v.34 no.2
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• pp.139-143
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• 2001

Thioredoxin (Trx) is a redox protein possessing conserved sequence Cys-Gly-Pro-Cys in ail organisms. Trx acts as an electron donor of many proteins including thioredoxin peroxidase (TPx). Yeast Trx 2 has two redox active cysteine residues at positions 31 and 34. To investigate the redox activity of each cysteine, we generated mutants C31S, C34S, and C31S/C34S using site directed mutagenesis and examined the redox activity of Trx variants as an electron donor for yeast TPx enzymes. None of the three Cysmutated Trx proteins was active as a redox protein in the 5', 5'-dithiobis-(2-dinitrobenzoic acid) reduction under the condition of the presence of NADPH and thioredoxin reductase, and in the thioredoxin dependent peroxidase activity of yeast TPx II. C34S enhanced the glutamine synthetase protection activity of yeast TPx I, even though 100 times more protein was needed to exhibit the same activity to WT. The formation of a mixed disulfide intermediate between Trx and TPx II subunits was analyzed by SDS-PAGE. The mixed dieter form of TPx II was found only for C34S. These results suggest that Cys-31 more effectively acts as an electron donor for TPx enzymes.