Persulfide Dioxygenase Research Articles

Hsa-miR-29c-3p regulates biological function of colorectal cancer by targeting SPARC.

Colorectal cancer (CRC) is the most common type of behavioral cancers, miRNAs play a critical role in cancer development and progression. In the present study, we downloaded the original data from Gene Expression Omnibus (GEO) and conduct data analysis. has-mir-29c-3p mimic, inhibitor, negative control or si-SPARC (secreted protein acidic, rich in cysteine) were transfected into HCT116 cells, respectively. Quantitative real time PCR (qRT-PCR) was used to measure has-mir-29c-3p and SPARC mRNA expressions, western blot was used to detect ACAA1 (acetyl-CoA acyltransferase 1), ACOX1 (acyl-CoA oxidase 1), COL1A1(collagen, type I, alpha-1), COL1A2 (collagen, type I, alpha-2), COL4A1 (collagen, type IV, alpha-1), COL5A2 (collagen, type V, alpha-2), COL12A1 (collagen, type XII, alpha-1), CPT2 (carnitine palmitoyltransferase 2), ETHE1 (persulfide dioxygenase), HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthase 2), SPARC, SQRDL (sulfide quinone oxidoreductase), and TST (thiosulfate sulfurtransferase) protein expression. CCK-8 and wound healing assay were employed to verify cell proliferation and migration. The luciferase reporter assay data made sure the target correlation of has-mir-29c-3p and SPARC. Firstly, we found that the expression of has-mir-29c-3p was lower in CRC tissues than in their paired corresponding non-cancerous tissues and there was significant inversed correlation between has-mir-29c-3p and SPARC. Overexpression of has-mir-29c-3p reduced cell proliferation and migration. SPARC was identified as a direct target of has-mir-29c-3p, whose silencing reduced cell proliferation and migration. These data showed that has-mir-29c-3p regulates CRC cell functions through regulating SPARC expression. Taken together, has-mir-29c-3p may function as an oncogenic miRNA targeting SPARC, targeted modulation of has-mir-29c-3p expression may became a potential strategy for the treatment.

Cupriavidus necator H16 Uses Flavocytochrome c Sulfide Dehydrogenase To Oxidize Self-Produced and Added Sulfide.

Production of sulfide (H2S, HS-, and S2-) by heterotrophic bacteria during aerobic growth is a common phenomenon. Some bacteria with sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) can oxidize self-produced sulfide to sulfite and thiosulfate, but other bacteria without these enzymes release sulfide into the medium, from which H2S can volatilize into the gas phase. Here, we report that Cupriavidus necator H16, with the fccA and fccB genes encoding flavocytochrome c sulfide dehydrogenases (FCSDs), also oxidized self-produced H2S. A mutant in which fccA and fccB were deleted accumulated and released H2S. When fccA and fccB were expressed in Pseudomonas aeruginosa strain Pa3K with deletions of its sqr and pdo genes, the recombinant rapidly oxidized sulfide to sulfane sulfur. When PDO was also cloned into the recombinant, the recombinant with both FCSD and PDO oxidized sulfide to sulfite and thiosulfate. Thus, the proposed pathway is similar to the pathway catalyzed by SQR and PDO, in which FCSD oxidizes sulfide to polysulfide, polysulfide spontaneously reacts with reduced glutathione (GSH) to produce glutathione persulfide (GSSH), and PDO oxidizes GSSH to sulfite, which chemically reacts with polysulfide to produce thiosulfate. About 20.6% of sequenced bacterial genomes contain SQR, and only 3.9% contain FCSD. This is not a surprise, since SQR is more efficient in conserving energy because it passes electrons from sulfide oxidation into the electron transport chain at the quinone level, while FCSD passes electrons to cytochrome c The transport of electrons from the latter to O2 conserves less energy. FCSDs are grouped into three subgroups, well conserved at the taxonomic level. Thus, our data show the diversity in sulfide oxidation by heterotrophic bacteria.IMPORTANCE Heterotrophic bacteria with SQR and PDO can oxidize self-produced sulfide and do not release H2S into the gas phase. C. necator H16 has FCSD but not SQR, and it does not release H2S. We confirmed that the bacterium used FCSD for the oxidation of self-produced sulfide. The bacterium also oxidized added sulfide. The common presence of SQRs, FCSDs, and PDOs in heterotrophic bacteria suggests the significant role of heterotrophic bacteria in sulfide oxidation, participating in sulfur biogeochemical cycling. Further, FCSDs have been identified in anaerobic photosynthetic bacteria and chemolithotrophic bacteria, but their physiological roles are unknown. We showed that heterotrophic bacteria use FCSDs to oxidize self-produced sulfide and extraneous sulfide, and they may be used for H2S bioremediation.

Sulfide production and oxidation by heterotrophic bacteria under aerobic conditions

Sulfide (H2S, HS− and S2−) oxidation to sulfite and thiosulfate by heterotrophic bacteria, using sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO), has recently been reported as a possible detoxification mechanism for sulfide at high levels. Bioinformatic analysis revealed that the sqr and pdo genes were common in sequenced bacterial genomes, implying the sulfide oxidation may have other physiological functions. SQRs have previously been classified into six types. Here we grouped PDOs into three types and showed that some heterotrophic bacteria produced and released H2S from organic sulfur into the headspace during aerobic growth, and others, for example, Pseudomonas aeruginosa PAO1, with sqr and pdo did not release H2S. When the sqr and pdo genes were deleted, the mutants also released H2S. Both sulfide-oxidizing and non-oxidizing heterotrophic bacteria were readily isolated from various environmental samples. The sqr and pdo genes were also common in the published marine metagenomic and metatranscriptomic data, indicating that the genes are present and expressed. Thus, heterotrophic bacteria actively produce and consume sulfide when growing on organic compounds under aerobic conditions. Given their abundance on Earth, their contribution to the sulfur cycle should not be overlooked.

Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation

Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.

Sulfide Oxidation and Transfer Catalyzed by a Bacterial Persulfide Dioxygenase Fused to a Rhodanese

Hydrogen sulfide (H2S) is the third eukaryotic gaseous signaling molecule that evokes a broad range of physiological effects. H2S levels are governed by the rates of both production and clearance. In eukaryotes, sulfide clearance occurs primarily via the mitochondrial sulfide oxidation pathway which consists of the enzymes sulfide: quinone oxidoreductase (SQR), persulfide dioxygenase (PDO also known as ETHE1), rhodanese and sulfite oxidase. Recently, bacterial proteins that are fusions between PDO and rhodanese have been identified. Existence of such a fusion suggests that their mitochondrial counterparts in higher organisms may interact and that characterization of such fusion proteins will facilitate the modeling of interactions between human mitochondrial PDO and rhodanese and aid in elucidating the metabolic intermediates in the sulfide oxidation pathway. Here we have characterized a bacterial PDO‐rhodanese fusion (PRF) protein from Burkholderia phytofirmans which displays 1.8‐fold higher PDO activity and 4.8‐fold lower Km for GSSH as compared to human PDO, and rhodanese activity comparable to other bacterial rhodaneses. Additionally, the rhodanese domain catalyzed the formation of GSSH and sulfite from thiosulfate and glutathione, but no detectable sulfur transfer activity was observed for the reverse reaction. Similar observation were made for the stand‐alone rhodanese domain; however, several fold increases in specific activity and Km values for thiosulfate and glutathione were observed for the various sulfur transfer reactions, suggesting that the PDO domain modulates the activity of the rhodanese domain. Combined, these results suggest that the fusion is poised to produce sulfite as its main product. Bioinformatic analysis suggests that this enzyme may be involved in sulfur assimilation pathways rather than in sulfide oxidation as in mammals. Spectral changes were observed with the ferric form of the enzyme in the presence of Na2S. Further analysis of this interaction displayed that Na2S binds with moderate affinity resulting in polysulfide production and suggesting a potentially novel role for non‐heme iron proteins in polysulfide production. To obtain structural insights into its mechanism, the x‐ray crystal structure of PRF co‐crystallized with thiosulfate was determined at 1.79 Å resolution. Notably, in this structure the persulfidated form of the rhodanese domain active site cysteine, C314, was captured.Support or Funding InformationNational Institutes of Health (GM112455 to RB) and National Institute of General Medical Sciences Training Grant support (T32GM008353 to NM)

Protein polysulfidation-dependent persulfide dioxygenase activity of ethylmalonic encephalopathy protein 1

Reactive persulfide species such as glutathione persulfide (GSSH) are highly abundant biomolecules. Persulfide dioxygenase (also called ethylmalonic encephalopathy protein 1, ETHE1) reportedly metabolizes GSSH to GSH with simultaneous oxygen consumption. How ETHE1 activity is regulated is still unclear, however. In this study, we describe the possible role of protein polysulfidation in the catalytic activity of ETHE1. We first found that ETHE1 catalyzed the persulfide dioxygenase reaction mostly for glutathione polysulfides, GS-(S)n-H, as well as for GSSH, but not for other endogenous persulfides such as cysteine and homocysteine persulfides/polysulfides. We then developed a novel method to detect protein polysulfidation and named it the polyethylene glycol-conjugated maleimide-labeling gel shift assay (PMSA). PMSA analysis indicated that most cysteine residues in ETHE1 were polysulfidated. Site-directed mutagenesis of cysteine residues in ETHE1 combined with liquid chromatography tandem mass spectrometry for polysulfidation determination surprisingly indicated that the Cys247 residue was important for polysulfidation of other Cys residues and that the C247S mutant possessed no persulfide dioxygenase activity. These results suggested that ETHE1 is a major enzyme regulating endogenous GSSH/GS-(S)n-H and that its activity is controlled by polysulfidation of the Cys247 residue.

Recombinant Escherichia coli with sulfide:quinone oxidoreductase and persulfide dioxygenase rapidly oxidises sulfide to sulfite and thiosulfate via a new pathway.

Many heterotrophic bacteria contain sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) genes. It is unclear how these enzymes cooperate to oxidise sulfide in bacteria. Cupriavidus pinatubonensis JMP134 contains a gene cluster of sqr and pdo, and their functions were analysed in Escherichia coli. Recombinant E. coli cells with SQR and PDO rapidly oxidised sulfide to thiosulfate and sulfite. The SQR also contains a DUF442 domain that was shown to have rhodanese activities. E. coli cells with PDO and SQR-C94S, an active site mutant of the rhodanese domain, oxidised sulfide to thiosulfate with transitory accumulation of polysulfides. Cellular and enzymatic evidence showed that DUF442 speeds up the reaction of polysulfides with glutathione to produce glutathione persulfide (GSSH). Thus, SQR oxidises sulfide to polysulfides; rhodanese enhances the reaction of polysulfides with glutathione to produce GSSH; PDO oxidises GSSH to sulfite; sulfite spontaneously reacts with polysulfides to generate thiosulfate. The pathway is different from the proposed mitochondrial pathway because it has polysulfides, that is, disulfide and trisulfide, as intermediates. The data demonstrated that heterotrophic bacteria with SQR and PDO can rapidly oxidise sulfide to thiosulfate and sulfite, providing the foundation for using heterotrophic bacteria with SQR and PDO for sulfide bioremediation.

Untargeted Metabolomics Analysis Reveals a Link between ETHE1-Mediated Disruptive Redox State and Altered Metabolic Regulation.

Defects in the gene encoding the persulfide dioxygenase ETHE1 are known to cause the severe inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of known clinical characteristics, the molecular mechanisms underlying the ETHE1 deficiency are still obscure. Herein, to further analyze the molecular phenotype of the disease, we applied an untargeted metabolomics approach on cultivated fibroblasts of EE patients for pinpointing alterations in metabolite levels. Metabolites, as direct signatures of biochemical functions, can decipher biochemical pathways involved in the cellular phenotype of patient cells. Using liquid chromatography-mass spectrometry-based untargeted metabolomics, we identified 18 metabolites that have altered levels in fibroblasts from EE patients. Our data demonstrate disrupted redox state in EE patient cells, which is reflected by significantly decreased level of reduced glutathione. Furthermore, the down-regulation of several intermediate metabolites such as the redox cofactors NAD(+) and NADH as well as Krebs cycle intermediates revealed clear alteration in metabolic regulation. Pantothenic acid and several amino acids exhibited decreased levels, whereas the β-citrylglutamate with a putative role in brain development had an increased level in the EE patient cells. These observations indicate the severe impact of ETHE1 deficiency on cellular physiology and redox state, meanwhile suggesting targets for experimental studies on novel treatment options for the devastating metabolic disorder.

Staphylococcus aureus CstB Is a Novel Multidomain Persulfide Dioxygenase-Sulfurtransferase Involved in Hydrogen Sulfide Detoxification.

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that the cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus and is under the transcriptional control of the persulfide sensor CstR and H2S. Here, we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (ethylmalonic encephalopathy protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstB(PDO)) and Cys408 in the C-terminal rhodanese domain (CstB(Rhod)) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity, which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both of these activities require Cys408. CstB protects S. aureus against H2S toxicity, with the C201S and C408S cstB genes being unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S misregulation, in concert with the other enzymes encoded by the cst operon.

Crystal structure of human persulfide dioxygenase: structural basis of ethylmalonic encephalopathy.

The ethylmalonic encephalopathy protein 1 (ETHE1) catalyses the oxygen-dependent oxidation of glutathione persulfide (GSSH) to give persulfite and glutathione. Mutations to the hETHE1 gene compromise sulfide metabolism leading to the genetic disease ethylmalonic encephalopathy. hETHE1 is a mono-iron binding member of the metallo-β-lactamase (MBL) fold superfamily. We report crystallographic analysis of hETHE1 in complex with iron to 2.6 Å resolution. hETHE1 contains an αββα MBL-fold, which supports metal-binding by the side chains of an aspartate and two histidine residues; three water molecules complete octahedral coordination of the iron. The iron binding hETHE1 enzyme is related to the ‘classical’ di-zinc binding MBL hydrolases involved in antibiotic resistance, but has distinctive features. The histidine and aspartate residues involved in iron-binding in ETHE1, occupy similar positions to those observed across both the zinc 1 and zinc 2 binding sites in classical MBLs. The active site of hETHE1 is very similar to an ETHE1-like enzyme from Arabidopsis thaliana (60% sequence identity). A channel leading to the active site is sufficiently large to accommodate a GSSH substrate. Some of the observed hETHE1 clinical mutations cluster in the active site region. The structure will serve as a basis for detailed functional and mechanistic studies on ETHE1 and will be useful in the development of selective MBL inhibitors.

P161 - Human persulfide dioxygenase and a bacterial variant fused to rhodanese

P161 - Human persulfide dioxygenase and a bacterial variant fused to rhodanese

Characterization of Patient Mutations in Human Persulfide Dioxygenase (ETHE1) Involved in H2S Catabolism

Hydrogen sulfide (H(2)S) is a recently described endogenously produced gaseous signaling molecule that influences various cellular processes in the central nervous system, cardiovascular system, and gastrointestinal tract. The biogenesis of H(2)S involves the cytoplasmic transsulfuration enzymes, cystathionine β-synthase and γ-cystathionase, whereas its catabolism occurs in the mitochondrion and couples to the energy-yielding electron transfer chain. Low steady-state levels of H(2)S appear to be controlled primarily by efficient oxygen-dependent catabolism via sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1), rhodanese, and sulfite oxidase. Mutations in the persulfide dioxgenase, i.e. ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared with the wild-type enzyme. The D196N mutation results in a 2-fold higher K(m) for the substrate, glutathione persulfide.

P66 Characterization of wild type and patient-described mutant forms of Human Persulfide Dioxygenase, ETHE1

Hydrogen sulfide (H 2 S) is an endogenously produced gaseous signaling molecule with beneficial effects in various cellular processes in the central nervous system, cardiovascular system and gastrointestinal tract. The biogenesis of H 2 S involves the enzymes of the transsulfuration pathway cystathionine b-synthase and g-cystathionase. H 2 S oxidation and removal occurs in the mitochondria and the electrons from this oxidation are used in electron transfer chain to generate energy. Steady-state levels of H 2 S are controlled by its biogenesis in the transsulfuration pathway and its efficient oxygen-dependent catabolism in the mitochondria by a 3-enzyme pathway including sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1) and a sulfur transferase. Mutations in the persulfide dioxgenase, ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared to the wild-type enzyme. The D196N mutation results in a 2-fold higher K M for the substrate, glutathione persulfide (GSSH).