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.

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.

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.

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.

Evidence That Non-Syndromic Familial Tall Stature Has an Oligogenic Origin Including Ciliary Genes.

Human growth is a complex trait. A considerable number of gene defects have been shown to cause short stature, but there are only few examples of genetic causes of non-syndromic tall stature. Besides rare variants with large effects and common risk alleles with small effect size, oligogenic effects may contribute to this phenotype. Exome sequencing was carried out in a tall male (height 3.5 SDS) and his parents. Filtered damaging variants with high CADD scores were validated by Sanger sequencing in the trio and three other affected and one unaffected family members. Network analysis was carried out to assess links between the candidate genes, and the transcriptome of murine growth plate was analyzed by microarray as well as RNA Seq. Heterozygous gene variants in CEP104, CROCC, NEK1, TOM1L2, and TSTD2 predicted as damaging were found to be shared between the four tall family members. Three of the five genes (CEP104, CROCC, and NEK1) belong to the ciliary gene family. All genes are expressed in mouse growth plate. Pathway and network analyses indicated close functional connections. Together, these data expand the spectrum of genes with a role in linear growth and tall stature phenotypes.

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.

Motor control and cognition deficits associated with protein carbamoylation in food (cassava) cyanogenic poisoning: Neurodegeneration and genomic perspectives.

A case-control design determined whether konzo, an upper motoneuron disease linked to food (cassava) toxicity was associated with protein carbamoylation and genetic variations. Exon sequences of thiosulfate sulfurtransferase (TST) or mercaptopyruvate sulfurtransferase (MPST), plasma cyanide detoxification rates, and 2D-LC-MS/MS albumin carbamoylation were assessed in 40 children [21 konzo-affected and 19 putatively healthy controls, mean (SD) age: 9.2 (3.0) years] subjected to cognition and motor testing using the Kaufman Assessment Battery and the Bruininks/Oseretsky Test, respectively. Konzo was significantly associated with higher levels of carbamoylated peptides 206-219 (LDELRDEGKASSAK, pep1) after adjusting for age, gender, albumin concentrations and BUN [regression coefficient: 0.03 (95%CI:0.02-0.05), p = 0.01]. Levels of pep1 negatively correlated with performance scores at all modalities of motor proficiency (r = 0.38 to 0.61; all p < 0.01) or sequential processing (memory)(r = - 0.59, p = 0.00) and overall cognitive performance (r = - 0.48, p = 0.00) but positively with time needed for cyanide detoxification in plasma (r = 0.33, p = 0.04). Rare potentially damaging TST p.Arg206Cys (rs61742280) and MPST p.His317Tyr (rs1038542246) heterozygous variants were identified but with no impact on subject phenotypes. Protein carbamoylation appears to be a reliable marker for cassava related neurodegeneration.

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.

Effect of glycosaminoglycans accumulation on the non-oxidative sulfur metabolism in mouse model of Sanfilippo syndrome, type B.

Lack of the N-alpha-acetylglucosaminidase gene is responsible for the occurrence of a rare disease - the Sanfilippo syndrome, type B. The result of this gene knock-out is accumulation of glycosaminoglycans (GAGs) - more specifically heparan sulfate - a sulfate rich macromolecule. The sulfur oxidative pathway is involved in the sulfate groups' turnover in the cells. In contrast, the non-oxidative sulfur pathway leads mostly to formation of sulfane sulfur-containing compounds. The aim of our research was to observe an interaction between MPS IIIB and non-oxidative sulfur metabolism. In this work, we examined selected tissues (livers, kidneys, hearts and spleens) of 3 month old mice with confirmed accumulation of GAGs. The activity and expression of three sulfurtransferases (components of non-oxidative sulfur metabolism): rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase was determined, as well as the sulfane sulfur level and the level of other low molecular sulfur-containing compounds (reduced and oxidized glutathione, cysteine and cystine). In all tested tissues, the sulfane sulfur and/or sulfurtransferases' activities, as well as the cysteine content, underwent statistically significant changes. These correlations were also related to the sex of the tested animals. The obtained results indicated that accumulation of incompletely degraded GAGs in the tissues had affected the non-oxidative sulfur metabolism.

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.

Molecular identification and characterization of rhodaneses from the insect herbivore Pieris rapae.

The association of cabbage white butterflies (Pieris spec., Lepidoptera: Pieridae) with their glucosinolate-containing host plants represents a well-investigated example of the sequential evolution of plant defenses and insect herbivore counteradaptations. The defensive potential of glucosinolates, a group of amino acid-derived thioglucosides present in plants of the Brassicales order, arises mainly from their rapid breakdown upon tissue disruption resulting in formation of toxic isothiocyanates. Larvae of P. rapae are able to feed exclusively on glucosinolate-containing plants due to expression of a nitrile-specifier protein in their gut which redirects glucosinolate breakdown to the formation of nitriles. The release of equimolar amounts of cyanide upon further metabolism of the benzylglucosinolate-derived nitrile suggests that the larvae are also equipped with efficient means of cyanide detoxification such as ß-cyanoalanine synthases or rhodaneses. While insect ß-cyanoalanine synthases have recently been identified at the molecular level, no sequence information was available of characterized insect rhodaneses. Here, we identify and characterize two single-domain rhodaneses from P. rapae, PrTST1 and PrTST2. The enzymes differ in their kinetic properties, predicted subcellular localization and expression in P. rapae indicating different physiological roles. Phylogenetic analysis together with putative lepidopteran rhodanese sequences indicates an expansion of the rhodanese family in Pieridae.

Thiosulfate sulfurtransferase-like domain-containing 1 protein interacts with thioredoxin.

Rhodanese domains are structural modules present in the sulfurtransferase superfamily. These domains can exist as single units, in tandem repeats, or fused to domains with other activities. Despite their prevalence across species, the specific physiological roles of most sulfurtransferases are not known. Mammalian rhodanese and mercaptopyruvate sulfurtransferase are perhaps the best-studied members of this protein superfamily and are involved in hydrogen sulfide metabolism. The relatively unstudied human thiosulfate sulfurtransferase-like domain-containing 1 (TSTD1) protein, a single-domain cytoplasmic sulfurtransferase, was also postulated to play a role in the sulfide oxidation pathway using thiosulfate to form glutathione persulfide, for subsequent processing in the mitochondrial matrix. Prior kinetic analysis of TSTD1 was performed at pH 9.2, raising questions about relevance and the proposed model for TSTD1 function. In this study, we report a 1.04 Å resolution crystal structure of human TSTD1, which displays an exposed active site that is distinct from that of rhodanese and mercaptopyruvate sulfurtransferase. Kinetic studies with a combination of sulfur donors and acceptors reveal that TSTD1 exhibits a low Km for thioredoxin as a sulfane sulfur acceptor and that it utilizes thiosulfate inefficiently as a sulfur donor. The active site exposure and its interaction with thioredoxin suggest that TSTD1 might play a role in sulfide-based signaling. The apical localization of TSTD1 in human colonic crypts, which interfaces with sulfide-releasing microbes, and the overexpression of TSTD1 in colon cancer provide potentially intriguing clues as to its role in sulfide metabolism.

Analysis of Streptomyces coelicolor M145 genes SCO4164 and SCO5854 encoding putative rhodaneses.

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.

A single-domain rhodanese homologue MnRDH1 helps to maintain redox balance in Macrobrachium nipponense.

Rhodaneses are known to catalyze in vitro the transfer of a sulfane sulfur atom from thiosulfate to cyanide with concomitant formation of thiocyanate, however, their biological functions remain speculative despite the main role is considered as detoxifying cyanide especially in animal livers. In this study, we characterized a single-domain rhodanese homologue, MnRDH1, from Macrobrachium nipponense. We found MnRDH1 with the highest expression in hemocytes. Upon Aeromonas hydrophila challenge, expression of MnRDH1 was up-regulated in various tissues, including hepatopancreas, gill, intestine and hemocytes. RNAi knockdown of MnRDH1 led to rapid increases of malondialdehyde content, which reveals that MnRDH1 deficiency causes oxidative stress. The expression of MnRDH1 in hepatopancreas was significantly increased in response to the doxorubicin-induced oxidative stress, indicating the gene is oxidative stress inducible. We transformed E. coli with MnRDH1 and the mutant MnRDH1C75A, and found significant rhodanese activity of the recombinant protein of MnRDH1 in vitro, but detected no enzyme activity of the mutant MnRDH1C75A. When under the oxidative insult by H2O2, the MnRDH1 transformed E. coli had significantly enhanced survival rates compared to those bacteria transformed with MnRDH1C75A. In conclusion, our study demonstrates that rhodanese in M. nipponense confers oxidative stress tolerance, and thus renders an evidence for the notion that rhodanese family genes act a critical role in antioxidant defenses.