ArsC Protein Research Articles

Biochemical, molecular and in silico characterization of arsenate reductase from Bacillus thuringiensis KPWP1 tolerant to salt, arsenic and a wide range of pH.

The present study characterized aresenate reductase of Bacillus thuringiensis KPWP1, tolerant to salt, arsenate and a wide range of pH during growth. Interestingly, it was found that arsC, arsB and arsR genes involved in arsenate tolerance are distributed in the genome of strain KPWP1. The inducible arsC gene was cloned, expressed and the purified ArsC protein showed profound enzyme activity with the KM and Kcat values as 25µM and 0.00119s-1, respectively. In silico studies revealed that in spite of 19-26% differences in gene sequences, the ArsC proteins of Bacillus thuringiensis, Bacillus subtilis and Bacillus cereus are structurally conserved and ArsC structure of strain KPWP1 is close to nature. Docking and analysis of the binding site showed that arsenate ion interacts with three cysteine residues of ArsC and predicts that the ArsC from B. thuringiensis KPWP1 reduces arsenate by using the triple Cys redox relay mechanism.

SpxA2, encoding a regulator of stress resistance in Bacillus anthracis, is controlled by SaiR, a new member of the Rrf2 protein family.

Spx, a member of the ArsC (arsenate reductase) protein family, is conserved in Gram-positive bacteria, and interacts with RNA polymerase to activate transcription in response to toxic oxidants. In Bacillus anthracis str. Sterne, resistance to oxidative stress requires the activity of two paralogues, SpxA1 and SpxA2. Suppressor mutations were identified in spxA1 mutant cells that conferred resistance to hydrogen peroxide. The mutations generated null alleles of the saiR gene and resulted in elevated spxA2 transcription. The saiR gene resides in the spxA2 operon and encodes a member of the Rrf2 family of transcriptional repressors. Derepression of spxA2 in a saiR mutant required SpxA2, indicating an autoregulatory mechanism of spxA2 control. Reconstruction of SaiR-dependent control of spxA2 was accomplished in Bacillus subtilis, where deletion analysis uncovered two cis-elements within the spxA2 regulatory region that are required for repression. Mutations to one of the sequences of dyad symmetry substantially reduced SaiR binding and SaiR-dependent repression of transcription from the spxA2 promoter in vitro. Previous studies have shown that spxA2 is one of the most highly induced genes in a macrophage infected with B. anthracis. The work reported herein uncovered a key regulator, SaiR, of the Spx system of stress response control.

Phylogenetic analysis of gammaproteobacterial arsenate reductase proteins specific to enterobacteriaceae family, signifying arsenic toxicity

This study focuses on the phylogenetic analysis of all the ArsC protein sequences, obtained from similarity search against Gammaproteobacteria, and also studies the role of Gammaproteobacterial family in arsenic toxicity. The ars gene provides arsenic tolerance for microbial cell system and encodes for an arsenate reductase (ArsC), which is essential for arsenate resistance that converts arsenate into arsenite. Phylogenetic analysis offers an opportunity to understand the evolutionary relationship between organisms of interest. The phylogenetic experiment was set up for all possible ArsC sequences in class Gammaproteobacteria. The results suggested a wide similarity between ArsC sequences in the species of Enterobacteriaceae family rather than other families in Gammaproteobacteria. The three evolutionary clades revealed a role of Enterobacteriaceae species, which has the capability to code ArsC protein. Further phylogenetic analysis of ArsC crystal structure sequences has also shown the separate cluster of Enterobacter species. The overall phylogeny of the ArsC protein sequences suggests the species of Enterobacteriaceae family express more among all family of Gammaproteobacteria. This study could be advantageous to emphasize the importance of Enterobacteriaceae in arsenic toxicity.

Structural Basis for Cyclization Specificity of Two Azotobacter Type III Polyketide Synthases: A SINGLE AMINO ACID SUBSTITUTION REVERSES THEIR CYCLIZATION SPECIFICITY

Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min⁻¹ mg protein⁻¹) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min⁻¹ mg protein⁻¹) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (∆arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.

Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP.

Computational identification and analysis of arsenate reductase protein in Cronobacter sakazakii ATCC BAA-894 suggests potential microorganism for reducing arsenate

This study focuses a bioinformatics-based prediction of arsC gene product arsenate reductase (ArsC) protein in Cronobacter sakazakii BAA-894 strain. A protein structure-based study encloses three-dimensional structural modeling of target ArsC protein, was carried out by homology modeling method. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in protein. The ten probable ligand binding sites were predicted for target protein structure and highlighted the common binding residues between target and template protein. It has been first time identified that modeled ArsC protein structure in C. sakazakii was structurally and functionally similar to well-characterized ArsC protein of Escherichia coli because of having same structural motifs and fold with similar protein topology and function. Investigation revealed that ArsC from C. sakazakii can play significant role during arsenic resistance and potential microorganism for bioremediation of arsenic toxicity.

1H, 13C and 15N resonance assignments of the arsenate reductase from Synechocystis sp. strain PCC 6803

Arsenate reductases (ArsC) are a group of enzymes that play essential roles in biological arsenic detoxification pathways by catalyzing the intracellular reduction of arsenate to arsenite, which is subsequently extruded from the cells by specific transport systems. The ArsC protein from cyanobacterium Synechocystis sp. strain PCC 6803 (SynArsC) is related to the thioredoxin-dependent ArsC family, but uses the glutathione/glutaredoxin system for arsenate reduction. Therefore, it is classified to a novel thioredoxin/glutaredoxin hybrid arsenate reductase family. Herein we report the chemical shift assignments of (1)H, (13)C and (15)N atoms for the reduced form of SynArsC, which provides a starting point for further structural analysis and elucidation of its enzymatic mechanism.

Functional analysis of a chromosomal arsenic resistance operon in Pseudomonas fluorescens strain MSP3.

We reported earlier about the detection of a chromosomally located arsenic operon (arsRBC) in a gram-negative bacterium Pseudomonas fluorescens strain MSP3, which showed resistance to elevated levels of sodium arsenate and sodium arsenite. The genes for arsenic resistance were cloned into the HindIII site of pBluescript vector producing three clones MSA1, MSA2 and MSI3 conferring resistance to sodium arsenate and arsenite salts. They were further sub-cloned to delineate the insert size and the sub-clones were designated as MSA11, MSA12 and MSI13. The sub-clone pMSA12 (2.6 kb) fragment was further packaged into EcoRI-PstI site of M13mp19 and sequenced. Nucleotide sequencing revealed the presence of three open reading frames homologous to the arsR, arsB and arsC genes of arsenic resistance. Three cistrons of the ars operon encoded polypeptides ArsR, ArsB and ArsC with molecular weights ranging approximately 12, 37and 24 kDa, respectively. These polypeptides were visualized on SDS-PAGE stained with Coomassie blue and measured in a densitometer. The arsenic resistance operon (arsRBC) of strain MSP3 plasmid pMSA12 consists of 3 genes namely, arsR--encoding a repressor regulatory protein, arsB--the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells and arsC--a small cytoplasmic polypeptide required for arsenate resistance only, not for arsenite resistance. ArsB protein is believed to use the cell membrane potential to drive the efflux of intracellular arsenite ions. ArsC encodes for the enzyme arsenate reductase which reduces intracellular As(V) (arsenate) to more toxic As(III) (arsenite) and is subsequently extruded from the cell. The arsenate reductase activity was present in the soluble cytoplasmic fraction in E. coli clones. In the context of specified function of the arsenic operon encoded proteins, uptake and efflux mechanisms were studied in the wild strain and the arsenate/arsenite clones. The cell free filtrates of the arsenate clones (MSA11 and MSA12) obtained from P. fluorescens containing the arsC gene showed that arsenate reduction requires glutathione reductase, glutathione (GSH), glutaredoxin and ArsC protein. The protein was purified in an active form and a spectrophotometric assay was developed in which the oxidation of NADPH was coupled to reduction of arsenate. The molecular weights and the location of the polypeptides were obtained from Coomassie stained SDS-PAGE of extracellular and intracellular fractions of the cells.

Secondary structure and fold homology of the ArsC protein from the Escherichia coli arsenic resistance plasmid R773.

Resistance to several toxic anions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells. In this paper, we report the determination of the overall fold for ArsC, a 16 kDa protein of the ars operon involved in the reduction of arsenate to arsenite, using multidimensional, multinuclear NMR. The protein is found to contain large regions of extensive mobility, particularly in the active site. A model fold, computed on the basis of a preliminary set of NOEs, was found to be structurally homologous to E. coli glutaredoxin, thiol transferases, and glutathione S-transferase. Some kinship to the structure of low molecular weight tyrosine phosphatases, based on rough topological similarity but more so on the basis of a common anion-binding-loop motif H-CX(n)R, was also detected. Although functional, secondary, and tertiary structural homology is observed with these molecules, no significant homology in primary structure was detected. The mobilities of the active site of ArsC and of other enzymes are discussed.

Identification of an essential cysteinyl residue in the ArsC arsenate reductase of plasmid R773.

The ArsC protein encoded by the arsenical resistance operon of plasmid R773 catalyzes the reduction of arsenate to arsenite in Escherichia coli. The reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. The ArsC arsenate reductase has two cysteinyl residues, Cys12 and Cys106. By a combination of random and site-specific mutagenesis, Cys12 was altered to four other amino acid residues. Cells expressing any of those arsC genes were sensitive to arsenate. The ArsCC12S protein was purified and found to be catalytically inactive. Cys106 was altered separately to seryl, glycyl, and valyl residues. Cells expressing arsCC106S, arsCC106G, and arsCC106V genes retained arsenate resistance, and the purified C106S and C106G proteins had reductase activity. Both wild-type ArsC and C106S proteins were inactivated by iodoacetate. In the native enzyme only Cys12 was alkylate by iodoacetate; Cys106 was alkylated only if the enzyme was first denatured. In the presence of the substrate, arsenate, or competitive inhibitors, phosphate or sulfate, the rate of alkylation was reduced. Reductase activity was inhibited by N-ethylmaleimide and could be protected by arsenate. These results suggest Cys12 is an active-site residue essential for catalysis by the arsenate reductase.

Mechanisms of metalloregulation of an anion-translocating ATPase.

The ars (arsenical resistance) operon cloned from R-factor R773 has five genes that encode two repressor proteins, ArsR and ArsD, and three structural proteins, ArsA, ArsB, and ArsC. The ArsA and ArsB proteins form a membrane-bound pump that functions as an oxyanion-translocating ATPase. The substrates of the pump are the oxyanions arsenite or antimonite. The ArsC protein is an arsenate reductase that reduces arsenate to arsenite, which is subsequently pumped out of the cell. This review deals with the mechanism of transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA protein, the catalytic subunit of the pump. The chemical nature of the inducer plays an important role in regulation. In solution arsenite or antimonite exist as oxyanions and reacts with the cysteines in proteins. In both transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA ATPase, the ability of As(III) and Sb(III) to interact with the cysteines of the proteins, involves their action as effector.

Crystallization and Preliminary X-ray Diffraction Analysis of the ArsC Protein from the Escherichia coli Arsenical Resistance Plasmid, R773

Diffraction data to 3·0 Å resolution were collected on crystals of ArsC protein from the conjugative resistance factor R773 which mediates arsenical resistance in the Gram negative bacterium Escherichia coli. The crystal system is tetragonal, a = 116·2 Å, c = 145·0 Å and the space group is either P 41212 or P43212. The most probable range for the contents of the asymmetric unit is four eight ArsC molecules (Mr = 15,811).

Properties of the arsenate reductase of plasmid R773.

Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

Arsenate reduction mediated by the plasmid‐encoded ArsC protein is coupled to glutathione

Resistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenite. A genetic analysis was performed to identify the source of reducing potential in vivo. In addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenite was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC-catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.

Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase.

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.

Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 consists of three genes, arsR (encoding the repressor regulatory protein), arsB (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsC (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). ArsC has now been shown to be an arsenate reductase, converting intracellular arsenate [As(V)] to arsenite [As(III)], which is then exported from the cells by an energy-dependent efflux process. The arsenate reductase activity was found in the soluble cytoplasmic fraction in Escherichia coli (and not associated with the periplasmic fraction or the sedimentable cell envelope). Purified ArsC protein coupled in vitro with thioredoxin plus dithiothreitol (but not 2-mercaptoethanol or reduced glutathione) to reduce arsenate to arsenite.

Plasmid-encoded resistance to arsenic and antimony

Resistance determinants to the toxic oxyanionic salts of arsenic and antimony are found on plasmids of both gram-negative and gram-positive organisms. In most cases these provide resistance to both the oxyanions of +III oxidation state, antimonite and arsenite, and the +V oxidation state, arsenate. In both gram-positive and -negative bacteria, resistance is correlated with efflux of the anions from cells. The determinant from the plasmid R773, isolated from a gram-negative organism, has been studied in detail. It encodes an oxyanion-translocating ATPase with three subunits, a catalytic subunit, the ArsA protein, a membrane subunit, the ArsB subunit, and a specificity factor, the ArsC protein. The first two form a membrane-bound complex with arsenite-stimulated ATPase activity. The determinants from gram-positive bacteria have only the arsB and arsC genes and encode an efflux system without the participation of an ArsA homologue.

Molecular analysis of an anion pump: Purification of the ArsC protein

The ars operon of resistance plasmid R773 encodes an anion-translocating ATPase which catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. Although both arsenite and arsenate contain arsenic, they have different chemical properties. In the absence of the arsC gene the pump transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. The complex neither transports nor provides resistance to arsenate, the oxyanion of the +V oxidation state of arsenic. The arsC gene encodes a 16-kDa polypeptide, the ArsC protein, which alters the substrate specificity of the pump to allow for recognition and transport of the alternate substrate arsenate. The arsC gene was cloned behind a strong promoter and expressed at high levels. The ArsC protein was purified and crystallized.

Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon

The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals. The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC. The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities. Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription. The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb). This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein. Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes. In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein.