The Expression and Activity of Rhodanese, 3-Mercaptopyruvate Sulfurtransferase, Cystathionine γ-Lyase in the Most Frequently Chosen Cellular Research Models.

This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese (TST, EC: 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC: 2.8.1.2) and cystathionine γ-lyase (CTH, EC: 4.4.1.1) in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line (NMuNG, ATCC: CRL-1636), mouse mammary gland tumor (4T1, ATCC: CRL-2539), mouse fibroblast (MEF, ATCC: SCRC-1008), mouse melanoma (B16-F1, ATCC: CRL-6323), human colorectal adenocarcinoma (Caco-2, ATCC: HTB-37), human embryonic kidney (HEK-293, ATCC: CRL-1573), human osteosarcoma (MG-63, ATCC: CRL-1427) and rat myocardium (H9c2, ATCC: CRL-1446). Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them.

Unraveling the role of thiosulfate sulfurtransferase in metabolic diseases.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, is a mitochondrial enzyme which catalyzes the transfer of sulfur in several molecular pathways. After its initial identification as a cyanide detoxification enzyme, it was found that its functions also include sulfur metabolism, modification of iron­sulfur clusters and the reduction of antioxidants glutathione and thioredoxin. TST deficiency was shown to be strongly related to the pathophysiology of metabolic diseases including diabetes and obesity. This review summarizes research related to the enzymatic properties and functions of TST, to then explore the association between the effects of TST on mitochondria and development of diseases such as diabetes and obesity.

Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA--A Possible Intermediate in Its Selenation?

The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)-a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s2 group of mnm5s2U and of cmnm5s2U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm5ges2U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH+ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm5s2U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.

Dimethyl trisulfide: A novel cyanide countermeasure.

In the present studies, the in vitro and in vivo efficacies of a novel cyanide countermeasure, dimethyl trisulfide (DMTS), were evaluated. DMTS is a sulfur-based molecule found in garlic, onion, broccoli, and similar plants. DMTS was studied for effectiveness as a sulfur donor-type cyanide countermeasure. The sulfur donor reactivity of DMTS was determined by measuring the rate of the formation of the cyanide metabolite thiocyanate. In experiments carried out in vitro in the presence of the sulfurtransferase rhodanese (Rh) and at the experimental pH of 7.4, DMTS was observed to convert cyanide to thiocyanate with greater than 40 times higher efficacy than does thiosulfate, the sulfur donor component of the US Food and Drug Administration-approved cyanide countermeasure Nithiodote® In the absence of Rh, DMTS was observed to be almost 80 times more efficient than sodium thiosulfate in vitro The fact that DMTS converts cyanide to thiocyanate more efficiently than does thiosulfate both with and without Rh makes it a promising sulfur donor-type cyanide antidote (scavenger) with reduced enzyme dependence in vitro The therapeutic cyanide antidotal efficacies for DMTS versus sodium thiosulfate were measured following intramuscular administration in a mouse model and expressed as antidotal potency ratios (APR = LD50 of cyanide with antidote/LD50 of cyanide without antidote). A dose of 100 mg/kg sodium thiosulfate given intramuscularly showed only slight therapeutic protection (APR = 1.1), whereas the antidotal protection from DMTS given intramuscularly at the same dose was substantial (APR = 3.3). Based on these data, DMTS will be studied further as a promising next-generation countermeasure for cyanide intoxication.

Glutathione-garlic sulfur conjugates: slow hydrogen sulfide releasing agents for therapeutic applications.

Natural organosulfur compounds (OSCs) from Allium sativum L. display antioxidant and chemo-sensitization properties, including the in vitro inhibition of tumor cell proliferation through the induction of apoptosis. Garlic water- and oil-soluble allyl sulfur compounds show distinct properties and the capability to inhibit the proliferation of tumor cells. In the present study, we optimized a new protocol for the extraction of water-soluble compounds from garlic at low temperatures and the production of glutathionyl-OSC conjugates during the extraction. Spontaneously, Cys/GSH-mixed-disulfide conjugates are produced by in vivo metabolism of OSCs and represent active molecules able to affect cellular metabolism. Water-soluble extracts, with (GSGaWS) or without (GaWS) glutathione conjugates, were here produced and tested for their ability to release hydrogen sulfide (H2S), also in the presence of reductants and of thiosulfate:cyanide sulfurtransferase (TST) enzyme. Thus, the TST catalysis of the H2S-release from garlic OSCs and their conjugates has been investigated by molecular in vitro experiments. The antiproliferative properties of these extracts on the human T-cell lymphoma cell line, HuT 78, were observed and related to histone hyperacetylation and downregulation of GAPDH expression. Altogether, the results presented here pave the way for the production of a GSGaWS as new, slowly-releasing hydrogen sulfide extract for potential therapeutic applications.

Selenomodification of tRNA in archaea requires a bipartite rhodanese enzyme.

5-Methylaminomethyl-2-selenouridine (mnm(5)Se(2)U) is found in the first position of the anticodon in certain tRNAs from bacteria, archaea and eukaryotes. This selenonucleoside is formed in Escherichia coli from the corresponding thionucleoside mnm(5)S(2)U by the monomeric enzyme YbbB. This nucleoside is present in the tRNA of Methanococcales, yet the corresponding 2-selenouridine synthase is unknown in archaea and eukaryotes. Here we report that a bipartite ybbB ortholog is present in all members of the Methanococcales. Gene deletions in Methanococcus maripaludis and in vitro activity assays confirm that the two proteins act in trans to form in tRNA a selenonucleoside, presumably mnm(5)Se(2)U. Phylogenetic data suggest a primal origin of seleno-modified tRNAs.

Selective effects of diallyl disulfide, a sulfane sulfur precursor, in the liver and Ehrlich ascites tumor cells.

The present in vivo studies demonstrated that diallyl disulfide (DADS), occurring in garlic, elevated hepatic sulfane sulfur level and activities of gamma-cystathionase and 3-mercaptopyruvate sulfotransferase in healthy mice but did not affect the hepatic glutathione level. DADS efficiently corrected the concentrations of glutathione and sulfane sulfur, and ameliorated gamma-cystathionase activity that had been lowered in the livers of Ehrlich ascites tumor-bearing mice. In Ehrlich ascites tumor cells, diallyl disulfide did not alter bound sulfane sulfur level, sulfotransferases activity or glutathione level. These data indicate that this compound is capable of acting efficiently and selectively only in the liver and can be used for hepatoprotection during chemotherapy.

Common themes and variations in the rhodanese superfamily.

The rhodanese homology domain is a ubiquitous fold found in several phylogenetically related proteins encoded by eubacterial, archeal, and eukaryotic genomes. Although rhodanese-like proteins share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese superfamily. The variability occurs at different levels including sequence, active site loop length, presence of a critical catalytic Cys residue, and domain arrangement. Even within the same genome, multiple genes encode rhodanese-like proteins presenting with variably arranged rhodanese domain(s): as single or tandem domain(s), or combined with other protein domain(s). Given the highly variable organization of the rhodanese domain(s) and the context where it is found, here we review the structural organization and function of the rhodanese-like proteins. The overview of the most recent findings about rhodanese allow us to depict a superfamily of versatile proteins relying on persulfide chemistry to accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to sulfur metabolism and progression of cell cycle.

Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.

Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities.

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.

Tissue-specific bioenergetic effects and increased enzymatic activities following acute sublethal peroral exposure to cyanide in the mallard duck.

Protection of wildlife and in particular migratory birds, which are protected by the Migratory Bird Treaty Act, from cyanide waste in and around gold mining operations is an important environmental issue. We have investigated the bioenergetic effects of sublethal peroral cyanide exposure using the mallard duck (Anus platyrhynchos) as a model migratory bird. At cyanide concentrations well below levels considered safe by the mining industry and some regulatory agencies (50 ppm weak acid dissociable (WAD) cyanide) significant depletions of heart, liver, and brain tissue ATP levels were observed. Tissue ATP levels were restored to normal by 24 hr postexposure. Rhodanese and 3-mercaptopyruvate sulfurtransferase activities were determined in these tissues both for basal activity and post-cyanide exposure. Only brain tissue showed increased enzymatic activity following cyanide exposure, suggesting tissue-specific regulation of these enzymatic activities. These studies suggest that 50 ppm WAD cyanide is not a safe level of cyanide in water where avian wildlife exposure can occur.

Active site modifications quench intrinsic fluorescence of rhodanese by different mechanisms.

Beef liver rhodanese can be modified covalently at the active site (Cys-247) either reversibly or irreversibly by sulfur, selenium, iodoacetate, and hydrogen peroxide. Each derivative shows an intrinsic fluorescence lower than that of the free enzyme. The reaction of rhodanese with iodoacetate or hydrogen peroxide is time-dependent and accompanied by enzyme inactivation, by the loss of one or two sulfhydryl groups, respectively, by quenching and bathochromic shift of fluorescence, and by an absorbance perturbation in the near UV. The latter findings are indicative for a displacement of some tryptophyl side chains from hydrophobic to hydrophilic environment. The fluorescence decays of the various rhodanese derivatives can be fitted by a double-exponential function with two lifetimes: a shorter one of 1-1.7 ns and a longer one of 2.8-4.6 ns. The S-loaded and Se-loaded rhodanese samples have proportionally shorter lifetimes and lower quantum yields. No such proportionality was observed for the iodoacetate-treated and for the hydrogen peroxide treated enzyme. These findings indicate that two different quenching mechanisms are operating in rhodanese derivatives, a long-range energy transfer from tryptophan to persulfide (or sulfoselenide) group and a static quenching accompanying a conformational change of the protein after modification of the active site.

Differences in the binding of sulfate, selenate and thiosulfate ions to bovine liver rhodanese, and a description of a binding site for ammonium and sodium ions. An X-ray diffraction study.

The binding of sulfate, selenate and thiosulfate by the sulfur-transferase rhodanese (EC 2.8.1.1) in the crystalline state has been studied by X-ray analysis at resolutions between 0.23 nm and 0.4 nm. The three ions appear to occupy a common site between the N eta atoms of Arg-29 and the main-chain NH group of Glu-148 at the surface of the enzyme molecule. A second binding site for the three ions is situated at the entrance to the active centre, between the side chains of Arg-186 and Lys-249. Selenate and thiosulfate are bound equally well at both anion-binding sites. Sulfate, however, binds better at the first position, near Arg-29, than at the second site near Arg-186. In the complex of sulfur-rhodanese with thiosulfate, the outer sulfur atom of the anion near the active centre points towards the extra sulfur atom which is bound as a persulfide to the S gamma of the essential Cys-247. The distance between the outer sulfur atom of the thiosulfate ion and the persulfide sulfur atom appears to be about 0.3 nm. The thiosulfate difference Fourier also shows a distinct, localized conformational change involving residues 71, 72 and 249. This is the result of the replacement of an ammonium ion in the sulfate and selenate media by a sodium ion in the sodium thiosulfate solution. Rhodanese is apparently able to accomodate ions with different radii at this cation-binding site by minor structural alterations.