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ABSTRACT: Accumulation of arsenic has potential health risks through consumption in food. Here we inserted the gene As(III) S-adenosylmethionine methyltransferase (ArsM) into the chromosome of Pseudomonas putida KT2440. Recombinant bacteria methylate inorganic arsenic into less toxic organoarsenicals. This has potential for bioremediation of environmental arsenic and reducing arsenic contamination in food.
Applied and environmental microbiology 05/2013; · 3.69 Impact Factor
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ABSTRACT: Organoarsenicals used as herbicides and growth promoters for farm animals are degraded to inorganic arsenic. Available bacterial whole-cell biosensors detect only inorganic arsenic. We report a biosensor selective for the trivalent organoarsenicals methylarsenite and phenylarsenite over inorganic arsenite. This sensor may be useful for detecting degradation of arsenic-containing herbicides and growth promoters.
Applied and environmental microbiology 07/2012; 78(19):7145-7. · 3.69 Impact Factor
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ABSTRACT: Enzymatic methylation of arsenic is a detoxification process in microorganisms but in humans may activate the metalloid to more carcinogenic species. We describe the first structure of an As(III) S-adenosylmethionine methyltransferase by X-ray crystallography that reveals a novel As(III) binding domain. The structure of the methyltransferase from the thermophilic eukaryotic alga Cyanidioschyzon merolae reveals the relationship between the arsenic and S-adenosylmethionine binding sites to a final resolution of ∼1.6 Å. As(III) binding causes little change in conformation, but binding of SAM reorients helix α4 and a loop (residues 49-80) toward the As(III) binding domain, positioning the methyl group for transfer to the metalloid. There is no evidence of a reductase domain. These results are consistent with previous suggestions that arsenic remains trivalent during the catalytic cycle. A homology model of human As(III) S-adenosylmethionine methyltransferase with the location of known polymorphisms was constructed. The structure provides insights into the mechanism of substrate binding and catalysis.
Biochemistry 06/2012; 51(27):5476-85. · 3.42 Impact Factor
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ABSTRACT: The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508. Substitution of any of the three led to loss of As(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As(III) or MAs(III). As(GS)(3) and MAs(GS)(2) bound significantly faster than As(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb(III), and the purified enzyme methylated and volatilized Sb(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs(III) to dimethylarsenite [DMAs(III)]. The rate-limiting step was identified as the conversion of DMAs(III) to trimethylarsine, and DMAs(III) accumulates as the principal product.
Biochemistry 02/2012; 51(5):944-51. · 3.42 Impact Factor
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ABSTRACT: Arsenic (As) is a ubiquitous element that is widespread in the environment and causes numerous health problems. Biomethylation of As has implications for its mobility and toxicity. Photosynthetic organisms may play a significant role in As geochemical cycling by methylating it to different As species, but little is known about the mechanisms of methylation. Methylated As species have been found in many photosynthetic organisms, and several arsenite S-adenosylmethionine (SAM) methyltransferases have been characterized in cyanobacteria and algae. However, higher plants may not have the ability to methylate As. Instead, methylated arsenicals in plants probably originate from microorganisms in soils and the rhizosphere. Here, we propose possible approaches for developing 'smart' photosynthetic organisms with an enhanced and sensitive biomethylation capacity for bioremediation and safer food.
Trends in Plant Science 01/2012; 17(3):155-62. · 11.05 Impact Factor
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ABSTRACT: Arsenic is the most prevalent environmental toxic substance and ranks first on the U.S. Environmental Protection Agency's Superfund List. Arsenic is a carcinogen and a causative agent of numerous human diseases. Paradoxically arsenic is used as a chemotherapeutic agent for treatment of acute promyelocytic leukemia. Inorganic arsenic has two biological important oxidation states: As(V) (arsenate) and As(III) (arsenite). Arsenic uptake is adventitious because the arsenate and arsenite are chemically similar to required nutrients. Arsenate resembles phosphate and is a competitive inhibitor of many phosphate-utilizing enzymes. Arsenate is taken up by phosphate transport systems. In contrast, at physiological pH, the form of arsenite is As(OH)(3), which resembles organic molecules such as glycerol. Consequently, arsenite is taken into cells by aquaglyceroporin channels. Arsenic efflux systems are found in nearly every organism and evolved to rid cells of this toxic metalloid. These efflux systems include members of the multidrug resistance protein family and the bacterial exchangers Acr3 and ArsB. ArsB can also be a subunit of the ArsAB As(III)-translocating ATPase, an ATP-driven efflux pump. The ArsD metallochaperone binds cytosolic As(III) and transfers it to the ArsA subunit of the efflux pump. Knowledge of the pathways and transporters for arsenic uptake and efflux is essential for understanding its toxicity and carcinogenicity and for rational design of cancer chemotherapeutic drugs.
Current Topics in Membranes 01/2012; 69:325-58. · 0.59 Impact Factor
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ABSTRACT: ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2011; 67(Pt 12):1616-8. · 0.51 Impact Factor
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ABSTRACT: Resistance to arsenite (As(III)) by cells is generally accomplished by arsenite efflux permeases from Acr3 or ArsB unrelated families. We analyzed the function of three Acr3 proteins from Corynebacterium glutamicum, CgAcr3-1, CgAcr3-2, and CgAcr3-3. CgAcr3-1 conferred the highest level of As(III) resistance and accumulation in vivo. CgAcr3-1 was also the most active when everted membranes vesicles from Escherichia coli or C. glutamicum mutants were assayed for efflux with different energy sources. As(III) and antimonite (Sb(III)) resistance and accumulation studies using E. coli or C. glutamicum arsenite permease mutants clearly show that CgAcr3-1 is specific for As(III). In everted membrane vesicles expressing CgAcr3-1, dissipation of either the membrane potential or the pH gradient of the proton motive force did not prevent As(III) uptake, whereas dissipation of both components eliminated uptake. Further, a mutagenesis study of CgAcr3-1 suggested that a conserved cysteine and glutamate are involved in active transport. Therefore, we propose that CgAcr3-1 is an antiporter that catalyzes arsenite-proton exchange with residues Cys129 and Glu305 involved in efflux.
Journal of Biological Chemistry 11/2011; 287(1):723-35. · 4.77 Impact Factor
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ABSTRACT: • Biotransformation of arsenic includes oxidation, reduction, methylation, and conversion to more complex organic arsenicals. Members of the class of arsenite (As(III)) S-adenosylmethyltransferase enzymes catalyze As(III) methylation to a variety of mono-, di-, and trimethylated species, some of which are less toxic than As(III) itself. However, no methyltransferase gene has been identified in plants. • Here, an arsM gene from the soil bacterium Rhodopseudomonas palustris was expressed in Japonica rice (Oryza sativa) cv Nipponbare, and the transgenic rice produced methylated arsenic species, which were measured by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). • Both monomethylarsenate (MAs(V)) and dimethylarsenate (DMAs(V)) were detected in the roots and shoots of transgenic rice. After 12 d exposure to As(III), the transgenic rice gave off 10-fold greater volatile arsenicals. • The present study demonstrates that expression of an arsM gene in rice induces arsenic methylation and volatilization, theoretically providing a potential stratagem for phytoremediation.
New Phytologist 07/2011; 191(1):49-56. · 6.64 Impact Factor
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BioEssays 03/2011; 33(5):350 - 357. · 4.95 Impact Factor
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BioEssays 03/2011; 33(5):350-7. · 4.95 Impact Factor
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ABSTRACT: The ArsD metallochaperone delivers trivalent metalloids, As(III) or Sb(III), to the ArsA ATPase, the catalytic subunit of the ArsAB As(III) efflux pump. Transfer of As(III) increases the affinity of ArsA for As(III), allowing resistance to environmental arsenic concentrations. As(III) transfer is channelled from chaperone to ATPase, implying that ArsD and ArsA form an interface at their metal binding sites. A genetic approach was used to test this hypothesis. Thirteen ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected by either repressed transactivator yeast two-hybrid or reverse yeast two-hybrid assays. Additionally, Lys-37 and Lys-62 were identified as being involved in ArsD function by site-directed mutagenesis and chemical modification. Substitution at either position with arginine was tolerated, suggesting participation of a positive charge. By yeast two-hybrid analysis K37A and K62A mutants lost interaction with ArsA. All 15 mutations were mapped on the surface of the ArsD structure, and their locations are consistent with a structural model generated by in silico docking. Four are close to metalloid binding site residues Cys-12, Cys-13 and Cys-18, and seven are on the surface of helix 1. These results suggest that the interface involves one surface of helix 1 and the metalloid binding site.
Molecular Microbiology 02/2011; 79(4):872-81. · 5.01 Impact Factor
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ABSTRACT: Arsenic is one of the most widespread environmental carcinogens and has created devastating human health problems worldwide, yet little is known about mechanisms of biotransformation in contaminated regions. Methylarsonic acid [MAs(V)], extensively utilized as an herbicide, is largely demethylated to more toxic inorganic arsenite, which causes environmental problems. To understand the process of demethylation of methylarsenicals, soil samples commonly used on Florida golf courses were studied. Several soil extracts were found to demethylate MAs(V) to inorganic arsenite [As(III)]. From these extracts, a bacterial isolate was capable of reducing MAs(V) to MAs(III) but not of demethylating to As(III). A second bacterial isolate was capable of demethylating MAs(III) to As(III) but not of reducing MAs(V). A mixed culture could carry out the complete process of reduction and demethylation, demonstrating that demethylation of MAs(V) to As(III) is a two-step process. Analysis of the 16S ribosomal DNA sequences of the two organisms identified the MAs(V)-reducing and the MAs(III)-demethylating isolates as belong to Burkholderia and Streptomyces species respectively. This is the first report of a novel pathway of degradation of a methylarsenical herbicide by sequential reduction and demethylation in a microbial soil community, which we propose plays a significant role in the arsenic biogeocycle.
Environmental Microbiology 01/2011; 13(5):1205-15. · 5.84 Impact Factor
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ABSTRACT: Arsenic, a toxic metalloid widely existing in the environment, causes a variety of health problems. The ars operon encoded by Escherichia coli plasmid R773 has arsD and arsA genes, where ArsA is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump, and ArsD is an arsenic chaperone for ArsA. ArsD transfers As(III) to ArsA and increases the affinity of ArsA for As(III), allowing resistance to environmental concentrations of arsenic. Cys12, Cys13 and Cys18 in ArsD form a three sulfur-coordinated As(III) binding site that is essential for metallochaperone activity. ATP hydrolysis by ArsA is required for transfer of As(III) from ArsD to ArsA, suggesting that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. The 1.4 Å x-ray crystal structure of ArsD shows a core of four β-strands flanked by four α-helices in a thioredoxin fold. Docking of ArsD with ArsA was modeled in silico. Independently ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected. The locations of the mutations mapped on the surface of ArsD are consistent with the docking model. The results suggest that the interface with ArsA involves one surface of α1 helix and metalloid binding site of ArsD.
Biology of Metals 12/2010; 24(3):391-9. · 3.17 Impact Factor
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ABSTRACT: Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.
Molecular biology of the cell 11/2010; 21(22):3934-41. · 5.98 Impact Factor
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ABSTRACT: ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Conserved ArsD cysteine residues (Cys(12), Cys(13) and Cys(18)) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear. We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA. The predicted solution structure of ArsD is in agreement with recently published crystallographic structural results.
Biomolecular NMR Assignments 11/2010; 5(1):109-12. · 0.72 Impact Factor
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ABSTRACT: Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III) S-adenosylmethionine methyltransferase, an enzyme (EC 2.1.1.137) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic alga Cyanidioschyzon sp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=84.85, b=46.89, c=100.35 A, beta=114.25 degrees and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76 A.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2010; 66(Pt 9):1050-2. · 0.51 Impact Factor
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ABSTRACT: The two putative ars operons in Alkaliphilus metalliredigens QYMF are distinctive in that the arsA gene is split in halves, amarsA1 and amarsA2, and, acr3 but not an arsB gene coexists with arsA. Heterologous expression of one of the A. metalliredigensars operons (ars1) conferred arsenite but not antimonite resistance to DeltaarsEscherichia coli. Only the co-expressed AmArsA1 and AmArsA2 displayed arsenite or antimonite stimulated ATPase activity. The results show that AmArsA1-AmArsA2 interaction is needed to form the functional ArsA ATPase. This novel AmArsA1-AmArsA2 complex may provide insight in how it participates with Acr3 in arsenite detoxification.
FEBS letters 07/2010; 584(14):3089-94. · 3.54 Impact Factor
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ABSTRACT: Arsenic is a carcinogen that tops the Superfund list of hazardous chemicals. Bacterial resistance to arsenic is facilitated by ArsD, which delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump. Here we report the structure of the arsenic metallochaperone ArsD at 1.4 A and a model for its binding of metalloid. There are two ArsD molecules in the asymmetric unit. The overall structure of the ArsD monomer has a thioredoxin fold, with a core of four beta-strands flanked by four alpha-helices. Based on data from structural homologues, ArsD was modeled with and without bound As(III). ArsD binds one arsenic per monomer coordinated with the three sulfur atoms of Cys12, Cys13, and Cys18. Using this structural model, an algorithm was used to dock ArsD and ArsA. The resulting docking model provides testable predictions of the contact points of the two proteins and forms the basis for future experiments.
Biochemistry 06/2010; 49(25):5206-12. · 3.42 Impact Factor
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ABSTRACT: ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 degrees C but not at 4 degrees C. Neither MgADP nor MgATP-gamma-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle.
Biochemistry 04/2010; 49(17):3658-66. · 3.42 Impact Factor
