Characterization of a sulfurtransferase from Arabidopsis thaliana.

A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants.     

The crystal structure of Leishmania major 3-mercaptopyruvate sulfurtransferase. A three-domain architecture with a serine protease-like triad at the active site

Leishmania major 3-mercaptopyruvate sulfurtransferase is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis, Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack, whereas Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes benzoyl-Arg-p-nitroanilide, an activity that is sensitive to the presence of the serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone, which also lowers 3-mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The L. major 3-mercaptopyruvate sulfurtransferase is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506-binding protein class of peptidylprolyl cis/trans-isomerase. This domain may be involved in mediating protein folding and sulfurtransferase-protein interactions.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition.

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues

The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl(2) L(-1) for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3-mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium.     

3-Mercaptopyruvate sulfurtransferase of Leishmania contains an unusual C-terminal extension and is involved in thioredoxin and antioxidant metabolism

Cytosolic 3-mercaptopyruvate sulfurtransferases (EC ) of Leishmania major and Leishmania mexicana have been cloned, expressed as active enzymes in Escherichia coli, and characterized. The leishmanial single-copy genes predict a sulfurtransferase that is structurally peculiar in possessing a C-terminal domain of some 70 amino acids. Homologous genes of Trypanosoma cruzi and Trypanosoma brucei encode enzymes with a similar C-terminal domain, suggesting that this feature, not known in any other sulfurtransferase, is a characteristic of trypanosomatid parasites. Short truncations of the C-terminal domain resulted in misfolded inactive proteins, demonstrating that the domain plays some key role in facilitating correct folding of the enzymes. The leishmanial recombinant enzymes exhibited high activity toward 3-mercaptopyruvate and catalyzed the transfer of sulfane sulfur to cyanide to form thiocyanate. They also used thiosulfate as a substrate and reduced thioredoxin as the accepting nucleophile, the latter being oxidized. The enzymes were expressed in all life cycle stages, and the expression level was increased under peroxide or hypo-sulfur stress. The results are consistent with the enzymes having an involvement in the synthesis of sulfur amino acids per se or iron-sulfur centers of proteins and the parasite's management of oxidative stress.     

Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases

Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase.     

Affinity labeling of a catalytic site, cysteine(247), in rat mercaptopyruvate sulfurtransferase by chloropyruvate as an analog of a substrate

A bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (EC 2.8.1.2), is inactivated by 3-chloropyruvate, an analog of 3-mercaptopyruvate serving as a sulfur-donor and -acceptor substrate. To elucidate a reaction mechanism of the enzyme, the inactivation kinetic studies using 3-chloropyruvate were carried out. However, 3-chloropyruvate cannot be mixed with 3-mercaptopyruvate, 2-mercaptoethanol and thiosulfate because these substrates decompose 3-chloropyruvate. Thus, 3-mercaptopyruvate sulfurtransferase was incubated with 3-chloropyruvate, and then the remaining activity was measured separately in the assay system containing 3-mercaptopyruvate and 2-mercaptoethanol. The inactivation kinetics was analyzed by Kitz and Wilson method (J. Biol. Chem. 237 (1962) 3245-3248). The inactivation of mercaptopyruvate sulfurtransferase by 3-chloropyruvate proceeded in one-on-one manner and exhibited pseudo first-order kinetics with k(inact) = 0.068 +/- 0.003 min(-1) and K(I) = 4.0 +/- 0.2 mM (n = 3, mean +/- S.D.). Further, SH titration using DTNB revealed that MST was inactivated by 3-chloropyruvate in a 1:1 stoichiometry. Site-directed mutagenesis for binding sites of 3-mercaptopyruvate (Arg(187)-->Gly or Arg(196)-->Gly) (J. Biol. Chem. 271 (1996) 27395-27401) did not critically affect the inactivation. These findings suggest that 3-chloropyruvate behaves as an affinity label and directly tags the catalytic site, Cys(247). An ESI-LC/Q-TOF mass spectrometric study suggests that a pyruvate adduct is formed at Cys(247), which mimics a reaction intermediate.     

Potential therapeutic advantage of ribose-cysteine in the inhibition of astrocytoma cell proliferation

It has been observed that astrocyte and astrocytoma cells differ in their response to d-ribose-l-cysteine (RibCys) in the culture medium. RibCys, a prodrug of l-cysteine, elevates the level of cysteine and glutathione in both astrocytoma and astrocyte cultures. It also affects the activity of two sulfurtransferases, 3-mercaptopyruvate sulfurtransferase and rhodanese, involved in the metabolism of sulfane sulfur-containing compounds and in consequence exerts an effect on the level of sulfane sulfur. Under conditions, in which the raised level of sulfane sulfur was accompanied by an elevated activity of 3-mercaptopyruvate sulfurtransferase, the proliferation of the human astrocytome U373 line was decreased. The experiments were simultaneously performed with murine astrocytes to compare the behavior of normal cells under similar conditions. In murine astrocytes, RibCys was capable of increasing cellular proliferation, and was accompanied by a diminished level of sulfane sulfur and unchanged activity of the two sulfurtransferases. Thus, RibCys might offer a therapeutic advantage in the inhibition of astrocytoma cell proliferation. Besides, in the absence of oxidative stress, measured as the ratio of GSH/GSSG, the obtained results confirm that the fall in the level of sulfane sulfur is associated with increasing proliferation of cells, whereas a rise in the level causes a decrease in the proliferation of U373 cells.     

Solution structure of a single-domain thiosulfate sulfurtransferase from Arabidopsis thaliana

We describe the three-dimensional structure of the product of Arabidopsis thaliana gene At5g66040.1 as determined by NMR spectroscopy. This protein is categorized as single-domain sulfurtransferase and is annotated as a senescence-associated protein (sen1-like protein) and ketoconazole resistance protein (http://arabidopsis.org/info/genefamily/STR_genefamily.html). The sequence of At5g66040.1 is virtually identical to that of a protein from Arabidopsis found by others to confer ketoconazole resistance in yeast. Comparison of the three-dimensional structure with those in the Protein Data Bank revealed that At5g66040.1 contains an additional mobile beta-hairpin not found in other rhodaneses that may function in binding specific substrates. This represents the first structure of a single-domain plant sulfurtransferase. The enzymatically active cysteine-containing domain belongs to the CDC25 class of phosphatases, sulfide dehydrogenases, and stress proteins such as senescence specific protein 1 in plants, PspE and GlpE in bacteria, and cyanide and arsenate resistance proteins. Versions of this domain that lack the active site cysteine are found in other proteins, such as phosphatases, ubiquitin hydrolases, and sulfuryltransferases.     

Methods for in situ visualization and assay of sulfurtransferases

The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied. Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm). This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations. This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase. In addition, a refinement of the thiosulfate reductase assay technique is reported.     

Steady-state kinetics of 3-mercaptopyruvate sulfurtransferase from bovine kidney

Mammalian 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2), purified to apparent homogeneity by a new procedure, was studied by steady-state kinetic methods. The enzyme-catalyzed transfer of a sulfur atom from 3-mercaptopyruvate either to 2-mercaptoethanol or to a second molecule of 3-mercaptopyruvate was found to proceed by a sequential formal mechanism. An overall mechanism incorporating both of these transfers was shown to be capable of generating all of the initial velocity and product inhibition behavior observed.     

Enzymatic activity of the Arabidopsis sulfurtransferase resides in the C-terminal domain but is boosted by the N-terminal domain and the linker peptide in the full-length enzyme

Sulfurtransferases/rhodaneses are a group of enzymes widely distributed in plants, animals, and bacteria that catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. Sulfurtransferases (STs) consist of two globular domains of nearly identical size and conformation connected by a short linker sequence. In plant STs this linker sequence is exceptionally longer than in sequences from other species. The Arabidopsis ST1 protein (AJ131404) contains five cysteine residues: one residue is universally conserved in all STs and considered to be catalytically essential; a second one, closely located in the primary sequence, is conserved only in sequences from eukaryotic species. Of the remaining three cysteine residues two are conserved in the so far known plant STs and one is unique to the Arabidopsis ST1. The aim of our study was to investigate the role of the two-domain structure, of the unique plant linker sequence and of each cysteine residue. The N- and C-terminal domains of the Arabidopsis ST1, the full-length protein with a shortened linker sequence and several point-mutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The C-terminal domain itself displayed ST activity which could be increased by adding the separately prepared N-terminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wild-type activity, probably because of a drastically reduced protein stability. The replacement of each cysteine residue resulted in mutant forms which differed significantly in their stability, in the specific ST activities, and in their kinetic parameters which were determined for 3-mercaptopyruvate as well as thiosulfate as sulfur substrates: mutation of the putative active site cysteine (C332) essentially abolished activity; for C339 a crucial role at least for the turnover of thiosulfate could be identified.     

The effect of three α-keto acids on 3-mercaptopyruvate sulfurtransferase activity

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three α-keto acids (α-ketobutyrate, α-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the α-keto acid. However, it has also been proposed that α-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three α-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three α-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant K_i was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I₅₀, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three α-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanisms of the α-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate. © 1996 John Wiley & Sons, Inc.     

Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein

Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3) encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesis-related genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal pathogens.     

Differential expression of Arabidopsis sulfurtransferases under various growth conditions.

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyse the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. Neither the in vivo sulfur donors nor the acceptors of Str could be clearly identified in any of the organisms investigated so far. In Arabidopsis thaliana 20 Str proteins have been identified and grouped according to sequence homology. To investigate their respective in vivo function, Arabidopsis plants were grown in sterile hydroponic cultures at different sulfate (50, 500, and 1500 microM) and phosphate (0.1 and 1mM) concentrations, and in medium supplemented with 1mM thiosulfate. Northern blot analysis revealed the differential expression of the Str investigated. Thiosulfate Str activity was significantly increased at low sulfate concentrations in the medium. The Str mRNA levels were highly dependent on the developmental stage of the Arabidopsis plants. The expression of most Str analysed increased with progressing plant age in parallel with increasing 3-mercaptopyruvate and thiosulfate Str activities. The Str investigated were differentially expressed in a light/dark cycle whereas Str enzyme activities were not affected by the light conditions. The results indicate that each Str is regulated in a different way and plays an individual specific role in the plant metabolism.     

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.     

Arabidopsis thaliana plants possess a specific farnesylcysteine lyase that is involved in detoxification and recycling of farnesylcysteine

In plants, prenylated proteins are involved in actin organization, calcium-mediated signal transduction, and many other biological processes. Arabidopsis thaliana mutants lacking functional protein prenyltransferase genes have also revealed roles for prenylated proteins in phytohormone signaling and meristem development. However, to date, the turnover of prenylated plant proteins and the fate of the prenylcysteine (PC) residue have not been described. We have detected an enzyme activity in Arabidopsis plants that metabolizes farnesylcysteine (FC) to farnesal, which is subsequently reduced to farnesol. Unlike its mammalian ortholog, Arabidopsis FC lyase exhibits specificity for FC over geranylgeranylcysteine (GGC), and recognizes N-acetyl-FC (AFC). FC lyase is encoded by a gene on chromosome 5 of the Arabidopsis genome (FCLY, At5g63910) and is ubiquitously expressed in Arabidopsis tissues and organs. Furthermore, T-DNA insertions into the FCLY gene cause significant decreases in FC lyase activity and an enhanced response to abscisic acid (ABA) in seed germination assays. The effects of FCLY mutations on ABA sensitivity are even greater in the presence of exogenous FC. These data suggest that plants possess a specific FC detoxification and recycling pathway.