Bacterial Isothiocyanate Biosynthesis by Rhodanese-Catalyzed Sulfur Transfer onto Isonitriles.

Natural products bearing isothiocyanate (ITC) groups are an important group of specialized metabolites that play various roles in health, nutrition, and ecology. Whereas ITC biosynthesis via glucosinolates in plants has been studied in detail, there is a gap in understanding the bacterial route to specialized metabolites with such reactive heterocumulene groups, as in the antifungal sinapigladioside from Burkholderia gladioli. Here we propose an alternative ITC pathway by enzymatic sulfur transfer onto isonitriles catalyzed by rhodanese-like enzymes (thiosulfate:cyanide sulfurtransferases). Mining the B. gladioli genome revealed six candidate genes (rhdA-F), which were individually expressed in E. coli. By means of a synthetic probe, the gene products were evaluated for their ability to produce the key ITC intermediate in the sinapigladioside pathway. In vitro biotransformation assays identified RhdE, a prototype single-domain rhodanese, as the most potent ITC synthase. Interestingly, while RhdE also efficiently transforms cyanide into thiocyanate, it shows high specificity for the natural pathway intermediate, indicating that the sinapigladioside pathway has recruited a ubiquitous detoxification enzyme for the formation of a bioactive specialized metabolite. These findings not only elucidate an elusive step in bacterial ITC biosynthesis but also reveal a new function of rhodanese-like enzymes in specialized metabolism.

Thiosulfate sulfurtransferase deficiency promotes oxidative distress and aberrant NRF2 function in the brain.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1) was discovered as an enzyme that detoxifies cyanide by conversion to thiocyanate (rhodanide) using thiosulfate as substrate; this rhodanese activity was subsequently identified to be almost exclusively located in mitochondria. More recently, the emphasis regarding its function has shifted to hydrogen sulfide metabolism, antioxidant defense, and mitochondrial function in the context of protective biological processes against oxidative distress. While TST has been described to play an important role in liver and colon, its function in the brain remains obscure. In the present study, we therefore sought to address its potential involvement in maintaining cerebral redox balance in a murine model of global TST deficiency (Tst-/- mice), primarily focusing on characterizing the biochemical phenotype of TST loss in relation to neuronal activity and sensitivity to oxidative stress under basal conditions. Here, we show that TST deficiency is associated with a perturbation of the reactive species interactome in the brain cortex secondary to altered ROS and RSS (specifically, polysulfide) generation as well as mitochondrial OXPHOS remodeling. These changes were accompanied by aberrant Nrf2-Keap1 expression and thiol-dependent antioxidant function. Upon challenging mice with the redox-active herbicide paraquat (25 mg/kg i.p. for 24 h), Tst-/- mice displayed a lower antioxidant capacity compared to wildtype controls (C57BL/6J mice). These results provide a first glimpse into the molecular and metabolic changes of TST deficiency in the brain and suggest that pathophysiological conditions associated with aberrant TST expression and/or activity renders neurons more susceptible to oxidative stress-related malfunction.

Identification and characterization of a small molecule that activates thiosulfate sulfurtransferase and stimulates mitochondrial respiration.

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the Escherichia coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption.

The isolation of rumen enterococci strains along with high potential utilizing cyanide.

Cyanogenic glycosides in forage species and the possibility of cyanide (CN) poisoning can have undesirable effects on ruminants. The literature estimates that unknown rumen bacteria with rhodanese activity are key factors in the animal detoxification of cyanogenic glycosides, as they are capable of transforming CN into the less toxic thiocyanate. Therefore, identifying these bacteria will enhance our understanding of how to improve animal health with this natural CN detoxification process. In this study, a rhodanese activity screening assay revealed 6 of 44 candidate rumen bacterial strains isolated from domestic buffalo, dairy cattle, and beef cattle, each with a different colony morphology. These strains were identified as belonging to the species Enterococcus faecium and E. gallinarum by 16S ribosomal DNA sequence analysis. A CN-thiocyanate transformation assay showed that the thiocyanate formation capacity of the strains after a 12 h incubation ranged from 4.42 to 25.49 mg hydrogen CN equivalent/L. In addition, thiocyanate degradation resulted in the production of ammonia nitrogen and acetic acid in different strains. This study showed that certain strains of enterococci substantially contribute to CN metabolism in ruminants. Our results may serve as a starting point for research aimed at improving ruminant production systems in relation to CN metabolism.

Yohimbine Alleviates Oxidative Stress and Suppresses Aerobic Cysteine Metabolism Elevated in the Rat Liver of High-Fat Diet-Fed Rats.

Yohimbine is a small indole alkaloid derived from the bark of the yohimbe tree with documented biological activity, including anti-inflammatory, erectile dysfunction relieving, and fat-burning properties. Hydrogen sulfide (H2S) and sulfane sulfur-containing compounds are regarded as important molecules in redox regulation and are involved in many physiological processes. Recently, their role in the pathophysiology of obesity and obesity-induced liver injury was also reported. The aim of the present study was to verify whether the mechanism of biological activity of yohimbine is related to reactive sulfur species formed during cysteine catabolism. We tested the effect of yohimbine at doses of 2 and 5 mg/kg/day administered for 30 days on aerobic and anaerobic catabolism of cysteine and oxidative processes in the liver of high-fat diet (HFD)-induced obese rats. Our study revealed that HFD resulted in a decrease in cysteine and sulfane sulfur levels in the liver, while sulfates were elevated. In the liver of obese rats, rhodanese expression was diminished while lipid peroxidation increased. Yohimbine did not influence sulfane sulfur and thiol levels in the liver of obese rats, however, this alkaloid at a dose of 5 mg decreased sulfates to the control level and induced expression of rhodanese. Moreover, it diminished hepatic lipid peroxidation. It can be concluded that HFD attenuates anaerobic and enhances aerobic cysteine catabolism and induces lipid peroxidation in the rat liver. Yohimbine at a dose of 5 mg/kg can alleviate oxidative stress and reduce elevated concentrations of sulfate probably by the induction of TST expression.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

Uba1: A Potential Ubiquitin-like Activator Protein of Urm1 in Toxoplasma gondii.

We had shown in our previous study that TgUrm1 (ubiquitin-related Modifier 1) was involved in the regulation of anti-oxidant stress in Toxoplasma gondii by conjugating with TgAhp1. It is generally believed that Urm1 binds to target proteins through a mechanism involving Uba (ubiquitin-like activator protein). Here, we identified the TgUrm1-exclusive ubiquitin-like activator-TgUba1, which was located in the cytoplasm of Toxoplasma. TgUba1 contained three domains, including the atrophin-1 domain (ANT1), the E1-like domain (AD), and the rhodanese homology domain (RHD). We explored the interaction of TgUba1 with TgUrm1, and the AD domain was essential for the interaction of the two proteins. The TgUba1 knockout and complementary mutants were obtained based on CRISPR/Cas9 gene editing technology. The knockout of TgUba1 attenuated parasite proliferation and virulence in mice, but not invasion and egress processes, revealing the pivotal role played by TgUba1 in T. gondii survival. Meanwhile, the conjugate band of TgUrm1 was significantly reduced under oxidative stress stimulation without TgUba1, indicating that TgUba1 enhanced the targeted conjugation ability of TgUrm1 in response to oxidative stress, especially under diamide (Dia) stimulation. Furthermore, eleven TgUba1-interacting proteins were identified by proximity-based protein labeling techniques, relating them to ubiquitin-like modifications, anti-oxidative stress and metabolic regulation processes. In conclusion, TgUba1 was essential for T. gondii survival and might be a potential ubiquitin-like activator protein for TgUrm1.

Thiosulfate-Cyanide Sulfurtransferase a Mitochondrial Essential Enzyme: From Cell Metabolism to the Biotechnological Applications.

Thiosulfate: cyanide sulfurtransferase (TST), also named rhodanese, is an enzyme widely distributed in both prokaryotes and eukaryotes, where it plays a relevant role in mitochondrial function. TST enzyme is involved in several biochemical processes such as: cyanide detoxification, the transport of sulfur and selenium in biologically available forms, the restoration of iron-sulfur clusters, redox system maintenance and the mitochondrial import of 5S rRNA. Recently, the relevance of TST in metabolic diseases, such as diabetes, has been highlighted, opening the way for research on important aspects of sulfur metabolism in diabetes. This review underlines the structural and functional characteristics of TST, describing the physiological role and biomedical and biotechnological applications of this essential enzyme.

Thiosulfate sulfurtransferase prevents hyperglycemic damage to the zebrafish pronephros in an experimental model for diabetes.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate ß-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.

Rhodaneses minimize the accumulation of cellular sulfane sulfur to avoid disulfide stress during sulfide oxidation in bacteria.

Heterotrophic bacteria and human mitochondria often use sulfide: quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) to oxidize sulfide to sulfite and thiosulfate. Bioinformatic analysis showed that the genes encoding RHOD domains were widely presented in annotated sqr-pdo operons and grouped into three types: fused with an SQR domain, fused with a PDO domain, and dissociated proteins. Biochemical evidence suggests that RHODs facilitate the formation of thiosulfate and promote the reaction between inorganic polysulfide and glutathione to produce glutathione polysulfide. However, the physiological roles of RHODs during sulfide oxidation by SQR and PDO could only be tested in an RHOD-free host. To test this, 8 genes encoding RHOD domains in Escherichia coli MG1655 were deleted to produce E. coli RHOD-8K. The sqrCp and pdoCp genes from Cupriavidus pinatubonensis JMP134 were cloned into E. coli RHOD-8K. SQRCp contains a fused RHOD domain at the N-terminus. When the fused RHOD domain of SQRCp was inactivated, the cells oxidized sulfide into increased thiosulfate with the accumulation of cellular sulfane sulfur in comparison with cells containing the intact sqrCp and pdoCp. The complementation of dissociated DUF442 minimized the accumulation of cellular sulfane sulfur and reduced the production of thiosulfate. Further analysis showed that the fused DUF442 domain modulated the activity of SQRCp and prevented it from directly passing the produced sulfane sulfur to GSH. Whereas, the dissociated DUF442 enhanced the PDOCp activity by several folds. Both DUF442 forms minimized the accumulation of cellular sulfane sulfur, which spontaneously reacted with GSH to produce GSSG, causing disulfide stress during sulfide oxidation. Thus, RHODs may play multiple roles during sulfide oxidation.

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18.

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain-containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain-containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD-Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3-STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.

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.

Integrative Analyses Reveal Tstd1 as a Potential Modulator of HDL Cholesterol and Mitochondrial Function in Mice.

High-density lipoprotein (HDL) cholesterol levels are closely associated with human health and diseases. To identify genes modulating plasma HDL levels, we integrated HDL measurements and multi-omics data collected from diverse mouse cohorts and combined a list of systems genetics methods, including quantitative trait loci (QTL) mapping analysis, mediation analysis, transcriptome-wide association analysis (TWAS), and correlation analysis. We confirmed a significant and conserved QTL for plasma HDL on chromosome 1 and identified that Tstd1 liver transcript correlates with plasma HDL in several independent mouse cohorts, suggesting Tstd1 may be a potential modulator of plasma HDL levels. Correlation analysis using over 70 transcriptomics datasets in humans and mice revealed consistent correlations between Tstd1 and genes known to be involved in cholesterol and HDL regulation. Consistent with strong enrichment in gene sets related to cholesterol and lipoproteins in the liver, mouse strains with high Tstd1 exhibited higher plasma levels of HDL, total cholesterol and other lipid markers. GeneBridge using large-scale expression datasets identified conserved and positive associations between TSTD1/Tstd1 and mitochondrial pathways, as well as cholesterol and lipid pathways in human, mouse and rat. In summary, we identified Tstd1 as a new modulator of plasma HDL and mitochondrial function through integrative systems analyses, and proposed a new mechanism of HDL modulation and a potential therapeutic target for relevant diseases. This study highlights the value of such integrative approaches in revealing molecular mechanisms of complex traits or diseases.

Hypertension and Aging Affect Liver Sulfur Metabolism in Rats.

Hypertension and age are key risk factors for cardiovascular morbidity and mortality. Hydrogen sulfide (H2S), a gaseous transmitter, contributes significantly to regulating arterial blood pressure and aging processes. This study evaluated the effects of hypertension and aging on the hepatic metabolism of sulfur-containing compounds, the activity of the enzymes involved in sulfur homeostasis, and the liver's ability to generate H2S. Livers isolated from 16- and 60-week-old normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR) were used to evaluate gene expression using RT-PCR, and the activity of enzymes participating in H2S metabolism, including thiosulfate sulfurtransferase (rhodanese; TST), cystathionine gamma-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST). The levels of cysteine, cystine, reduced and oxidized glutathione were measured using RP-HPLC. SHR livers from both age groups showed a higher capacity to generate H2S than livers from WKY. The gene expression and activity of enzymes involved in sulfur metabolism differed between WKY and SHR, and between the age groups. For example, 16-week-old SHR had significantly higher activity of TST than 16-week-old WKY. Furthermore, differences between younger and older WKY rats in the expression and/or activity of TST and MPST were present. In conclusion, our study shows that arterial hypertension and aging affect hepatic sulfur metabolism and H2S production in rats. These findings pave the way for interventional studies evaluating a potential causal relation between liver sulfur metabolism, hypertension and aging.

Structure of TBC1D23 N-terminus reveals a novel role for rhodanese domain.

Members of the Tre2-Bub2-Cdc16 (TBC) family often function to regulate membrane trafficking and to control signaling transductions pathways. As a member of the TBC family, TBC1D23 is critical for endosome-to-Golgi cargo trafficking by serving as a bridge between Golgi-bound golgin-97/245 and the WASH/FAM21 complex on endosomal vesicles. However, the exact mechanisms by which TBC1D23 regulates cargo transport are poorly understood. Here, we present the crystal structure of the N-terminus of TBC1D23 (D23N), which consists of both the TBC and rhodanese domains. We show that the rhodanese domain is unlikely to be an active sulfurtransferase or phosphatase, despite containing a putative catalytic site. Instead, it packs against the TBC domain and forms part of the platform to interact with golgin-97/245. Using the zebrafish model, we show that impacting golgin-97/245-binding, but not the putative catalytic site, impairs neuronal growth and brain development. Altogether, our studies provide structural and functional insights into an essential protein that is required for organelle-specific trafficking and brain development.

Glutaredoxin-like protein (GLP)-a novel bacteria sulfurtransferase that protects cells against cyanide and oxidative stresses.

The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.

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.

Selenite uptake by outer membrane porin ExtI and its involvement in the subcellular localization of rhodanese-like lipoprotein ExtH in Geobacter sulfurreducens.

Selenite reduction is a key step in the biogeochemical cycle of selenium-an essential trace element for life. A variety of bacteria can transform selenite into elemental selenium nanoparticles on the cell surface via anaerobic respiration or detoxification processes. However, the proteins associated with the uptake of selenite for these processes are poorly understood. In this study, we investigated the role of an outer membrane porin-like protein, ExtI, in selenite permeation in Geobacter sulfurreducens. We demonstrated that selenite uptake and selenium nanoparticle formation were impaired in an extI-deficient strain. A putative rhodanese-like lipoprotein is encoded by an extH gene located immediately upstream of extI in the genome. We showed that ExtH is translocated into inner and outer membranes and that extI deficiency exclusively affects the localization of ExtH in the outer membrane. Coelution of ExtI and ExtH during gel filtration analysis of the outer membrane fraction of wild-type cells suggests a direct protein-protein interaction between them. Taken together, these results lead us to propose a physiological role for ExtI as a selenite channel associated with ExtH in the outer membrane.

MdRDH1, a HSP67B2-like rhodanese homologue plays a positive role in maintaining redox balance in Musca domestica.

Rhodanese homology domains (RHODs) are the structural modules of ubiquitous tertiary that occur in three major evolutionary phyla. Despite the versatile and important physiological functions of RHODs containing proteins, little is known about their invertebrate counterparts. A novel HSP67B2-like single-domain rhodanese homologue, MdRDH1 from Musca domestica, whose expression can be induced by bacterial infection or oxidative stress. Silencing MdRDH1 through RNAi causes important accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), and increases mortality in the larvae treated with bacterial invasion. The E. coli with MdRDH1 and the mutant MdRDH1C135A are transformed, with significant rhodanese activity of the recombinant protein of MdRDH1 in vitro found, without no detection of enzyme activity of the mutant MdRDH1C135A, revealing that catalytic Cys135 in the active-site loop is essential in the sulfurtransferase activity of MdRDH1. When oxidative stress is insulted by phenazine methosulfate (PMS), the MdRDH1 transformed E. coli shows enhanced survival rates compared with those bacteria transformed with MdRDH1C135A. Our research indicates that MdRDH1 confers oxidative stress tolerance, thus rendering evidence for the idea that rhodanese family genes play a critical role in antioxidant defenses. This paper yields novel insights into the potential antioxidative and immune functions of HSP67B2-like rhodanese homologues in invertebrate.

Hydrogen sulfide formation in experimental model of acute pancreatitis.

Acute pancreatitis (AP) is a disease defined as acute or chronic inflammatory process of the pancreas characterized by premature activation of digestive enzymes within the pancreatic acinar cells and causing pancreatic auto-digestion. In mammalian tissues, H2S is synthesized endogenously from L-cysteine in regulated enzymatic pathways catalyzed by pyridoxal phosphate-dependent enzymes: cystathionine beta - synthase (CBS), gamma - cystathionase (CTH) and cysteine aminotransferase (CAT) coupled with 3-mercaptopyruvate sulfurtransferase (MPST). In the mitochondria, hydrogen sulfide is oxidized to sulfite, which is then converted to thiosulfate (sulfane sulfur-containing compound) by thiosulfate sulfurtransferase (rhodanese; TST). The activity and the expression of CBS, CTH, MPST, and TST have been determined in vivo in pancreas of control rats, rats with acute pancreatitis and sham group. Levels of low-molecular sulfur compounds such as reduced and oxidized glutathione, cysteine, cystine and cystathionine were also determined. The study showed the significant role of MPST in H2S metabolism in pancreas. Stress caused by the surgery (sham group) and AP cause a decrease in H2S production associated with a decrease of MPST activity and expression. Markedly higher level of cysteine in the AP pancreas may be caused by a reduced rate of cysteine consumption in reaction catalyzed by MPST but it can also be a sign of the processes of proteolysis occurring in the changed tissue.