Arsenical pumps in prokaryotes and eukaryotes.
Explore the source record for details and available documents.
Adenosine Triphosphatases↗
SEARCH · PubMed Health
Results for “Arsenite Transporting ATPases”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme.
BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion. RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite. The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A. However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.
Arsenates↗
Sensing antimonite and arsenite at the subattomole level with genetically engineered bioluminescent bacteria.
A highly sensitive and selective optical sensing system for antimonite has been developed using genetically engineered bacteria. The basis of this system is the ability of certain bacteria to survive in environments that are contaminated with antimonite, arsenite, and arsenate. The survival is conferred to the bacteria by the ars operon, which consists of five genes that code for three structural proteins, ArsA, ArsB, and ArsC, and two regulatory proteins, ArsD and ArsR. ArsA, ArsB, and ArsC form a protein pump system that extrudes antimonite, arsenite, and arsenate once these anions reach the cytoplasm of the bacterium. A method was developed for monitoring antimonite and arsenite by using a single plasmid that incorporates the regulatory gene of the extrusion system, arsR, and the genes of bacterial luciferase, luxA and luxB. In the designed plasmid, ArsR regulates the expression of bacterial luciferase in a manner that is dependent on the concentration of antimonite and arsenite in the sample. Thus, the bioluminescence emitted by luciferase can be related to the concentration of antimonite and arsenite in the sample. Concentrations for antimonite and arsenite in the order of 10(-5) M, which corresponds to subattomole levels, can be detected. This bacterial-based sensing system is highly selective for antimonite and arsenite.
Adenosine Triphosphatases↗
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.
Adenosine Triphosphatases↗
Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase.
An arsenate (As(V)) reductase has been partially purified from human liver. Its apparent molecular mass is approximately 72 kDa. The enzyme required a thiol and a heat stable cofactor for activity. The cofactor is less than 3 kDa in size. The thiol requirement can be satisfied by dithiothreitol (DTT). However, the extent of stimulation of reductase activity by glutathione, thioredoxin, or reduced lipoic acid was negligible compared to that of DTT. The heat stable cofactor does not appear to be Cu(2+), Mn(2+), Zn(2+), Mg(2+), or Ca(2+). The enzyme does not reduce monomethylarsonic acid (MMA(V)). The isolation and characterization of this enzyme demonstrates that in humans, the reduction of arsenate to arsenite is enzymatically catalyzed and is not solely the result of chemical reduction by glutathione as has been proposed in the past.
Adenosine Triphosphatases↗
Molecular evolution of an arsenate detoxification pathway by DNA shuffling.
Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo. Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determined arsenate resistance from plasmid pl258 encoded by genes arsR, arsB, and arsC was evolved in Escherichia coli. Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance. Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome. In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal. The integrated ars operon had 13 mutations. Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance. While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12-fold. These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation. These mechanisms may be difficult to explain and are likely to be overlooked by rational design.
Adenosine Triphosphatases↗
Expression cloning for arsenite-resistance resulted in isolation of tumor-suppressor fau cDNA: possible involvement of the ubiquitin system in arsenic carcinogenesis.
Arsenic is a human carcinogen whose mechanism of action is unknown. Previously, this laboratory demonstrated that arsenite acts as a comutagen by interfering with DNA repair, although a specific DNA repair enzyme sensitive to arsenite has not been identified. A number of stable arsenite-sensitive and arsenite-resistant sublines of Chinese hamster V79 cells have now been isolated. In order to gain understanding of possible targets for arsenite's action, one arsenite-resistant subline, As/R28A, was chosen as a donor for a cDNA expression library. The library from arsenite-induced As/R28A cells was transfected into arsenite-sensitive As/S5 cells, and transfectants were selected for arsenite-resistance. Two cDNAs, asr1 and asr2, which confer arsenite resistance to arsenite-hypersensitive As/S5 cells as well as to wild-type cells, were isolated. asr1 shows almost complete homology with the rat fau gene, a tumor suppressor gene which contains a ubiquitin-like region fused to S30 ribosomal protein. Arsenite was previously shown to inhibit ubiquitin-dependent proteolysis. These results suggest that the tumor suppressor fau gene product or some other aspect of the ubiquitin system may be a target for arsenic toxicity and that disruption of the ubiquitin system may contribute to the genotoxicity and carcinogenicity of arsenite.
Amino Acid Sequence↗
Purine nucleoside phosphorylase: a fortuitous cytosolic arsenate reductase?
Explore the source record for details and available documents.
Arsenates↗
Purine nucleoside phosphorylase as a cytosolic arsenate reductase.
The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII.
Animals↗
Reduction of arsenate to arsenite in hepatic cytosol.
After finding that rat liver mitochondria reduce arsenate (AsV) to the more toxic arsenite (AsIII), it was of interest to know if other cell fractions also carried out this process. Postmitochondrial supernatant (PMSN), isolated from rat liver, reduced AsV to AsIII only in the presence of a thiol. Dithiothreitol (DTT) supported the reduction much more effectively than glutathione. Separation of PMSN into microsome and cytosol revealed that the AsV reducing activity resided in the cytosol. AsV-like oxyanions, e.g., phosphate (P(i)) and vanadate, as well as mercurial thiol reagents inhibited the cytosolic AsV reducing activity, indicating the involvement of a P(i)-utilizing SH enzyme. On searching for a reduction partner, it was found unexpectedly that oxidized pyridine nucleotides (NAD(+) or NADP(+)), but not their reduced forms, increased AsIII formation. Some other purine nucleotide derivatives (e.g., AMP, GMP), but not pyrimidine nucleotides, also increased the rate 2-3-fold. Examination of the effect of nucleosides and nucleobases on AsV reduction yielded dramatic results: purine nucleosides (inosine or guanosine) increased the reduction 80-100-fold, whereas purine bases (hypoxanthine or guanine) decreased it 80-90%. Although the retentate obtained by ultrafiltration of cytosol was almost inactive, its AsV reductase activity could be regained by adding the filtrate or inosine or guanosine to the retentate, indicating that endogenous purine nucleosides were essential for AsV reduction by the cytosol. The hepatic cytosol of mice, hamsters, guinea pigs, and rabbits also exhibited AsV reductase activities in the presence of DTT, which were dramatically enhanced by inosine. Thus, the hepatic cytosol of all tested species can reduce AsV to AsIII. The reduction requires the presence of an appropriate thiol as well as a purine nucleoside (inosine or guanosine) and is inhibited by thiol reagents, the AsV analogue phosphate, and purine bases. Characterization of this AsV reductase activity led us to identification of a cytosolic AsV reductase, which is presented in the accompanying paper.
Animals↗
Bacillus macyae sp. nov., an arsenate-respiring bacterium isolated from an Australian gold mine.
A strictly anaerobic arsenate-respiring bacterium isolated from a gold mine in Bendigo, Victoria, Australia, belonging to the genus Bacillus is described. Cells are Gram-positive, motile rods capable of respiring with arsenate and nitrate as terminal electron acceptors using a variety of substrates, including acetate as the electron donor. Reduction of arsenate to arsenite is catalysed by a membrane-bound arsenate reductase that displays activity over a broad pH range. Synthesis of the enzyme is regulated; maximal activity is obtained when the organism is grown with arsenate as the terminal electron acceptor and no activity is detectable when it is grown with nitrate. Mass of the catalytic subunit was determined to be approximately 87 kDa based on ingel activity stains. The closest phylogenetic relative, based on 16S rRNA gene sequence analysis, is Bacillus arseniciselenatis, but DNA-DNA hybridization experiments clearly show that strain JMM-4(T) represents a novel Bacillus species, for which the name Bacillus macyae sp. nov. is proposed. The type strain is JMM-4(T) (=DSM 16346(T)=JCM 12340(T)).
Acetates↗
The ArsR protein is a trans-acting regulatory protein.
The arsR gene encodes the regulatory protein of the plasmid-encoded arsenical resistance operon. A series of in-frame fusions was constructed between the C-terminally truncated arsR gene and the coding region for the mature form of beta-lactamase (blaM). Fusions containing most of the arsR gene were still inducible by arsenicals. Fusions containing less than 102 residues of the 117-residue ArsR protein were constitutive. When a wild-type arsR gene was placed in trans, the constitutive constructs were again inducible. The results demonstrate that the ArsR protein is a trans-acting regulatory protein which controls its own expression.
Adenosine Triphosphatases↗
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.
Adenosine Triphosphatases↗
arrA is a reliable marker for As(V) respiration.
Arsenate [As(V)]-respiring bacteria affect the speciation and mobilization of arsenic in the environment. This can lead to arsenic contamination of drinking water supplies and deleterious consequences for human health. Using molecular genetics, we show that the functional gene for As(V) respiration, arrA, is highly conserved; that it is required for As(V) reduction to arsenite when arsenic is sorbed onto iron minerals; and that it can be used to identify the presence and activity of As(V)-respiring bacteria in arsenic-contaminated iron-rich sediments. The expression of arrA thus can be used to monitor sites in which As(V)-respiring bacteria may be controlling arsenic geochemistry.
Archaea↗
Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267.
The arsenate, arsenite, and antimonite resistance region of the Staphylococcus xylosus plasmid pSX267 was subcloned in Staphylococcus carnosus. The sequenced DNA region revealed three consecutive open reading frames, named arsR, arsB, and arsC. Expression studies in Escherichia coli with the bacteriophage T7 RNA polymerase-promoter system yielded three polypeptides with apparent molecular weights of 8,000, 35,000, and 15,000, which very likely correspond to ArsR, ArsB, and ArsC, respectively. ArsB was distinguished by its overall hydrophobic character, suggesting a membrane association. The arsenate, arsenite, and antimonite resistance was shown to be inducible by all three heavy metal ions. Inactivation of the first gene, arsR, resulted in constitutive expression of resistance. Similar results were obtained with transcriptional fusions of various portions of the ars genes with a lipase reporter gene, indicating a function of ArsR as a negative regulator of a putative promoter in front of arsR. The inactivation of arsR also resulted in reduction of resistance to arsenite and antimonite, while arsenate resistance was unaffected. The three ars genes conferred arsenite resistance in E. coli and arsenite as well as arsenate resistance in Bacillus subtilis.
Adenosine Triphosphatases↗
Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258.
The arsenic resistance operon from Staphylococcus aureus plasmid pI258 was cloned and sequenced. The DNA sequence contains three genes in the order arsR, arsB, and arsC. The predicted amino acid sequences of the gene products are homologous with those of the products of the ars operons of plasmids pSX267 from Staphylococcus xylosus and R773 from Escherichia coli. The cloned staphylococcal ars operon confers resistances to arsenate, arsenite, and antimonite in S. aureus and Bacillus subtilis. The same operon was also expressed in E. coli and conferred resistance to arsenite but less resistance to arsenate and antimonite. Regulation of the pI258 ars operon was studied by using a translational arsB-blaZ fusion in S. aureus and a transcriptional arsB-luxAB fusion in E. coli. The ars operon was induced by arsenate [As(V)], arsenite [As(III)], and antimonite [Sb(III)], to which the strains were resistant, plus Bi(III) in S. aureus. Only arsenate and arsenite induced the operon in E. coli. Northern (RNA) blot DNA-RNA hybridization analysis showed inducible synthesis of a full-length ars mRNA, about 2.1 kb in size, both in S. aureus and in E. coli. S. aureus ars proteins were expressed in E. coli from the T7 phage promoter under the control of the T7 RNA polymerase. Primer extension (reverse transcriptase) analysis showed that the ars mRNA started at the same position (nucleotides 17 and 18 upstream from the arsR ATG) both in S. aureus and in E. coli. An internal deletion mutation in arsB resulted in decreased resistance to arsenate and total loss of arsenite and antimonite resistances. Partial deletion of 56 bp from the 3' end of the arsC gene resulted in loss of resistance to arsenate; the determinant retained arsenite and antimonite resistances.
Adenosine Triphosphatases↗
The ars operon in the skin element of Bacillus subtilis confers resistance to arsenate and arsenite.
The Bacillus subtilis skin element confers resistance to arsenate and arsenite. The ars operon in the skin element contains four genes in the order arsR, ORF2, arsB, and arsC. Three of these genes are homologous to the arsR, arsB, and arsC genes from the staphylococcal plasmid pI258, while no homologs of ORF2 have been found. Inactivation of arsR, arsB, or arsC results in either constitutive expression of ars, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, respectively. These results suggest that ArsR, ArsB, and ArsC function as a negative regulator, a membrane-associated protein need for extrusion of arsenite, and arsenate reductase, respectively. Expression of the ars operon was induced by arsenate, arsenite, and antimonite. Northern hybridization and primer extension analysis showed that synthesis of a full-length ars transcript of about 2.4 kb was induced by arsenate and that the ars promoter contains sequences that resemble the -10 and -35 regions of promoters that are recognized by E sigmaA.
Adenosine Triphosphatases↗
ATP-dependent arsenite transport in everted membrane vesicles of Escherichia coli.
Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli results from active efflux of these anions out of the cell. Extrusion is an active process mediated by an ATP-dependent pump composed of two types of subunits, the integral membrane ArsB protein and the catalytic ArsA subunit. An in vitro assay for transport in everted membrane vesicles of E. coli was developed. Uptake of 73AsO2- by everted vesicles was time- and temperature-dependent and required both pump subunits. Transport required ATP; no other nucleotide, including GTP, CTP, UTP, or the nonhydrolyzable analog adenosine 5'-O-(thiotriphosphate), could substitute for ATP. Protonophores, ionophores, or inhibitors of other types of ion-motive ATPases did not inhibit arsenite uptake. The sulfhydryl reagent N-ethylmaleimide was a potent inhibitor of ATP-dependent arsenite accumulation in vesicles. The apparent Km values for ATP and arsenite were approximately 2 and 0.1 mM, respectively. Antimonite, the most potent activator of the ArsA ATPase, inhibited arsenite uptake with an apparent Ki of 10 microM.
Adenosine Triphosphatases↗