Cysteine Desulfhydrases Research Articles

Identification, characterization, and application of a d-cysteine desulfhydrase from rice seed (Oryza sativa L.)

Cysteine desulfhydrases decompose cysteine to produce pyruvate, ammonium, and hydrogen sulfide. Using d-cysteine (D-cys) as a substrate, an enzyme with this activity was purified from rice seeds and identified at the native protein level. MALDI-TOF-MS analysis of its tryptic peptides revealed a 426 amino acid protein encoded by the OsDCD1 gene (Os02g0773300). Recombinant OsDCD1 (rOsDCD1) was expressed in Escherichia coli cells and purified as a single protein by column chromatography. Gel filtration column chromatography indicated that the native enzyme was a homodimer. The enzyme exhibited maximum catalytic activity at approximately pH 7.5 and 40 °C and was stable at pH 5.5–7.5 and < 37 °C. Kinetics analysis indicated Km and Vmax values for D-cys of 136 μM and 45.5 μmol/min/mg protein, respectively. In contrast, l-cysteine (L-cys) acted as an inhibitor with mixed non-competitive inhibition. Based on the substrate specificity of rOsDCD1, the amount of D-cys in rice flour was quantified. Even in the presence of up to 1 mM L-cys, the quantification of low concentrations of D-cys was unaffected. We demonstrate for the first time that the amount of D-cys in rice flour varies in the range of 0.76–0.93 μmol/g depending on the variety.

Glutathione catabolism by Enterobacteriaceae species to hydrogen sulphide adversely affects the viability of host systems in the presence of 5'fluorodeoxyuridine.

Reduced glutathione (GSH) plays an essential role in relieving oxidative insult from the generation of free radicals via normal physiological processes. However, GSH can be exploited by bacteria as a signalling molecule for the regulation of virulence. We describe findings arising from a serendipitous observation that when GSH and Escherichia coli were incubated with 5′fluorodeoxyuridine (FUdR)‐synchronised populations of Caenorhabditis elegans, the nematodes underwent rapid death. Death was mediated by the production of hydrogen sulphide mainly through the action of tnaA, a tryptophanase‐encoding gene in E. coli. Other Enterobacteriaceae species possess similar cysteine desulfhydrases that can catabolise l‐cysteine‐containing compounds to hydrogen sulphide and mediate nematode killing when worms had been pre‐treated with FUdR. When colonic epithelial cell lines were infected, hydrogen sulphide produced by these bacteria in the presence of GSH was also able to inhibit ATP synthesis in these cells particularly when cells had been treated with FUdR. Therefore, bacterial production of hydrogen sulphide could act in concert with a commonly used genotoxic cancer drug to exert host cell impairment. Hydrogen sulphide also increases bacterial adhesion to the intestinal cells. These findings could have implications for patients undergoing chemotherapy using FUdR analogues that could result in intestinal damage.

Cloning and Characterization of a gene Encoding True D-cysteine Desulfhydrase from Oryza sativa

Hydrogen sulfide (H2S) has been regarded as the third gasotransmitter and plays an active role in multiple signaling events of plants and animals. Cysteine desulfhydrases (CDes), including both D- and L-cysteine desulfhydrases (D/L-CDes) that degrade L- or D-cysteine into H2S, pyruvate, and ammonium, are considered the key enzymes responsible for endogenous H2S generation in plants. Several D-CDes are homologous to 1-aminocyclopropane-1-carboxylate deaminase (ACCD) and possess both ACCD and D-CDes activities, thus not a real specific D-CDes. However, little attention had been paid to true D-CDes and little information has been known about this protein in plants. In this study, a putative D-CDes transcript was cloned and characterized from Oryza sativa which encodes a protein with 423 amino acids possessing D-CDes activity and named as OsDCD1. Neither activities of ACCD nor O-acetyl-L-serine (thiol) lyase (OASTL) can be detected from OsDCD1 recombinant protein. For D-Cys, the Km of OsDCD1 is 0.13 ± 0.01 mM and the Vm is 111.55 ± 1.91 units mg−1 of protein. The pH-optimum and temperature-optimum of the OsDCD1 are 8.5 and 35°C, respectively. By site-directed mutagenesis, mutation of S357E or S357E/T589L almost fully abolished the D-CDes activity of OsDCD1, while the T389L mutant retained only partial D-CDes activity by 3.7%, indicating these two amino acid residues play critical roles for the maintenance of OsDCD1 activity. Besides, subcellular localization analysis in rice protoplast revealed that the OsDCD1 localizes in the chloroplast but not mitochondria, which is different from DCD1 in Arabidopsis. The qRT-PCR analysis further showed that the abundance of OsDCD1 transcript was widely regulated by different hormones and chemical reagents we used. In general, our results provided evidence that OsDCD1 is a potentially important endogenous H2S producing enzyme in rice, which may play an important role in plant growth regulators and chemical stimuli.

Complete genome sequence of Raoultella sp. strain X13, a promising cell factory for the synthesis of CdS quantum dots.

A novel cadmium-resistant bacterium, Raoultella sp. strain X13, recently isolated from heavy metal-contaminated soil, and this strain can synthesize CdS quantum dots using cadmium nitrate [Cd(NO4)2] and l-cysteine. Biomineralization of CdS by strain X13 can efficiently remove cadmium from aqueous solution. To illuminate the molecular mechanisms for the biosynthesis of CdS nanoparticle, the complete genome of Raoultella sp. strain X13 was sequenced. The whole genome sequence comprises a circular chromosome and a circular plasmid. Cysteine desulfhydrase smCSE has been previously found to be associated with the synthesis of CdS quantum dots. Bioinformatics analysis indicated that the genome of Raoultella sp. strain X13 encodes five putative cysteine desulfhydrases and all of them are located in the chromosome. The genome information may help us to determine the molecular mechanisms of the synthesis of CdS quantum dots and potentially enable us to engineer this microorganism for applications in biotechnology.

Light regulates hydrogen sulfide signalling during skoto- and photo-morphogenesis in foxtail millet.

Signal transduction mediated by photoreceptors regulates many physiological processes during plant growth and development including seed germination, flowering and photosynthesis, which are also regulated by hydrogen sulfide (H2S). However, studies of the connection between the vital environmental factors - light and the significant endogenous gasotransmitter - H2S, is lacking. Here, the seedlings of foxtail millet were used to reveal the mechanism of light regulation in H2S generation. Results showed that seedling hypocotyl elongation was promoted by H2S, but inhibited by HA under dark or white light condition. H2S contents in hypocotyl increased at first under red, blue or white light then decreased, and the duration of increase under white light was longer than under red or blue light. The activity of cysteine desulfhydrases, which catalyse H2S generation, was increased by red light but decreased by blue and white light. The expressions of cysteine desulfhydrases coding genes LCD1 and LCD2 were promoted by red or white light, but inhibited by blue light. In contrast, DES gene was promoted by white light but inhibited by red or blue light. In addition, the activities of LCDs were regulated by the phosphorylation mediated by photoreceptors PHYB and CRY1/CRY2. Finally, there are two pathways of light regulating H2S production, including a rapid process that involves the modification of phosphorylation on LCDs protein mediated by photoreceptors directly or indirectly, as well as a slower process that involves in regulating the expressions of LCDs and DES genes. This discovery has potential value for the application of H2S in agricultural production protecting the crops from unsuited light condition.

Nitrate reductase-dependent NO production is involved in H 2 S-induced nitrate stress tolerance in tomato via activation of antioxidant enzymes

Abstract Hydrogen sulfide (H2S) has been proposed as the third gasotransmitter after nitric oxide (NO) and carbon monoxide in animals and plants. In this study, the effect of H2S under excess nitrate stress in tomato root was studied. The results showed that excess nitrate inhibited the growth of tomato seedlings, while application of the H2S donor NaHS efficiently alleviated the growth inhibition, and decreased the lipid peroxidation, hydrogen peroxide (H2O2), and protein carbonyl contents, as well as the reactive oxygen species accumulation, especially 100 μM NaHS. Treatment with NaHS under nitrate stress increased the endogenous H2S and l -cysteine desulfhydrases (LCD) activity. Moreover, treatment with NaHS induced NO production and the synthesis of NO was mediated through nitrate reductase, but not through nitric oxide synthase in the presence or absence of nitrate. Besides, exogenous NaHS increased antioxidant enzyme genes (SOD, POD, CAT, and APX) expression, reducing the oxidative stress caused by excess nitrate. The alleviating effects of lipid peroxidation, H2O2 contents, and enhanced antioxidant enzyme activities by NaHS and NO donor (sodium nitroprusside, SNP), were reversed by 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the specific scavenger of NO. Together, above results suggested that NR-dependent NO production by NaHS was involved in the alleviation of nitrate stress by enhancing antioxidant enzyme activities in tomato root.

Hydrogen sulfide mediates ion fluxes inducing stomatal closure in response to drought stress in Arabidopsis thaliana

In many plant species, hydrogen sulfide (H2S) triggers stomatal closure, which is produced mainly by two classes of enzymes, cysteine desulfhydrases (CDes) and O-acetyl-L-serine (thiol) lyases (OASTLs). Stomatal movement is accompanied by several ion fluxes across the plasma membranes of guard cells. In this paper, we detected the fluxes of H+, Ca2+, K+ and Cl− in guard cells of wild-type Arabidopsis thaliana and the mutants associated with H2S production (lcd, OE-LCD, des, OE-DES, oastl-a1, oastl-a2, oastl-b and oastl-c), using a non-invasive micro-test technique. The results showed that endogenous H2S induced a transmembrane K+ efflux, and Ca2+ and Cl− influxes, while not affecting the flow of H+. Furthermore, the K+ channel was the main osmolyte responder during the regulation of stomatal movement by H2S in response to drought stress. Finally, the two classes of enzymes produced H2S, CDes and OASTLs, played different roles in regulating stomatal movements. Thus, H2S mediates ion fluxes inducing stomatal closure in response to drought stress in Arabidopsis thaliana.

Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli

Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability.

The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601.

Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis.

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated expressions of two cysteine desulfhydrases, and exogenous H2S donor (sodium hydrosulfide, NaHS) and H2S scavenger (hypotaurine, HT) pre-treated plants were used to dissect the involvement of H2S in plant stress responses. The cysteine desulfhydrases overexpressing plants and NaHS pre-treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and biotic stress resistance, while cysteine desulfhydrases knockdown plants and HT pre-treated plants displayed lower endogenous H2S level and decreased stress resistance. Moreover, H2S upregulated the transcripts of multiple abiotic and biotic stress-related genes, and inhibited reactive oxygen species (ROS) accumulation. Interestingly, MIR393-mediated auxin signaling including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3) was transcriptionally regulated by H2S, and was related with H2S-induced antibacterial resistance. Moreover, H2S regulated 50 carbon metabolites including amino acids, organic acids, sugars, sugar alcohols, and aromatic amines. Taken together, these results indicated that cysteine desulfhydrase and H2S conferred abiotic stress tolerance and biotic stress resistance, via affecting the stress-related gene expressions, ROS metabolism, metabolic homeostasis, and MIR393-targeted auxin receptors.

Hydrogen sulfide improves drought resistance in Arabidopsis thaliana

Hydrogen sulfide (H2S) plays a crucial role in human and animal physiology. Its ubiquity and versatile properties have recently caught the attention of plant physiologists and biochemists. Two cysteine desulfhydrases (CDes), l-cysteine desulfhydrase and d-cysteine desulfhydrase, were identified as being mainly responsible for the degradation of cysteine in order to generate H2S. This study investigated the expression regulation of these genes and their relationship to drought tolerance in Arabidopsis. First, the expression pattern of CDes in Arabidopsis was investigated. The expression levels of CDes gradually increased in an age-dependent manner. The expression of CDes was significantly higher in stems and cauline leaves than in roots, rosette leaves and flowers. Second, the protective effect of H2S against drought was evaluated. The expression pattern of CDes was similar to the drought associated genes induced by dehydration, and H2S fumigation was found to stimulate further the expression of drought associated genes. Drought also significantly induced increased H2S production, a process that was reversed by re-watering. In addition, seedlings after treatment with NaHS (a H2S donor) showed a higher survival rate and displayed a significant reduction in the size of the stomatal aperture compared to the control. These findings provide evidence that H2S, as a gasotransmitter, improves drought resistance in Arabidopsis.

The interconversion of ACC deaminase and d-cysteine desulfhydrase by directed mutagenesis

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.

Characterization of Cysteine‐Degrading and H2S‐Releasing Enzymes of Higher Plants ‐ From the Field to the Test Tube and Back

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.

Biosynthesis of sulfur volatile compounds in broccoli seedlings stored under anaerobic conditions

Abstract Sterile broccoli seedlings were used to investigate the effects of anaerobic atmosphere on biosynthesis and emission of sulfur volatile compounds which are responsible for off-odors in controlled and modified atmosphere stored broccoli. The identification of methanethiol, dimethyl sulfide, dimethyl disulfide and hydrogen sulfide in the head space of anaerobically stored broccoli seedlings confirmed that undesirable odors in broccoli are of plant origin. Evidence in support of the enzymatic origin of these volatiles was also obtained by using aminooxyacetic acid which is a potential inhibitor of pyridoxal phosphate-dependent enzymes; the production of sulfur volatiles was delayed and reduced by more than 95%. However, an anaerobic atmosphere had no inducible effect on the activities of cystine lyase, S-alkylcysteine lyase and cysteine desulfhydrases. These pyridoxal phosphate-dependent enzymes are thought to be involved in the production of volatile sulfur compounds by catalyzing the respective breakdown of cystine, cysteine and S-methyl- l -cysteine. The results suggest that these enzymes are not limiting factors in the production of sulfur volatiles in broccoli. However, the analysis of the contents of free sulfur amino acids and their derivatives showed an important increase in potential substrates for the synthesis of volatile sulfur compounds such as cysteine, methionine and S-methylcysteine as a result of anaerobic treatment. This increase usually preceeded or corresponded with the increased emission of sulfur volatiles. Results obtained by monitoring individual free amino acid content variation as a function of storage time, under anaerobic conditions, suggested enhanced conversions among sulfur amino acids. Other sources of sulfur amino acids, such as protein degradation and de novo synthesis, might explain the increase in these amino acids under anaerobiosis.

Effects of Anaerobic Atmosphere on the Metabolism of Sulfur Volatile Compounds Produced by Broccoli

Objectionable off-odors are produced by broccoli (Brassica oleracea, L.) when it is held under anaerobic conditions. These off-odors were attributed to sulfur volatile compounds mainly methanethiol (MT) and hydrogen sulfide. The present study was undertaken to investigate the effects of anaerobic conditions on the metabolism and emission of sulfur volatiles by broccoli. Inhibition assays using aminooxyacetic acid (AOA)—a potential inhibitor of pyridoxal-phosphate-dependent enzymes-confirmed the enzymatic origin of these volatiles. However, anaerobic atmosphere had no inducible effect on the enzymes cystine lyase, cysteine desulfhydrases and S-alkylcysteine lyase. These pyridoxal-phosphate-dependent enzymes thought to catalyze the respective degradation of cystine, cysteine and S-methyl-L-cysteine to sulfur volatiles showed no significant activity increase. Storage of sterile broccoli seedlings under anaerobic atmosphere resulted in an important increase of the content of sulfur amino acids that corresponded to an increased emission of sulfur volatiles. Cysteine and methionine content increased particularly at 24 hours and decreased later. Whereas, S-methyl-L-cysteine content increase was more obvious after 48 hours. The results suggest a possible involvement of the pathways for synthesis and breakdown of sulfur amino acids via methionine.