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Joris Messens - One of the best experts on this subject based on the ideXlab platform.
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Arsenate Reductase mycothiol and mycoredoxin concert thiol disulfide exchange
Journal of Biological Chemistry, 2009Co-Authors: Efren Ordonez, Lode Wyns, Karolien Van Belle, Goedele Roos, Sandra De Galan, Michal Letek, Jose A Gil, Luis M Mateos, Joris MessensAbstract:We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two Arsenate Reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against Arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer Arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of Arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between Arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione Reductase. Cg_ArsCs show a low specificity constant of ∼5 m-1 s-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.
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Arsenate reduction thiol cascade chemistry with convergent evolution
Journal of Molecular Biology, 2006Co-Authors: Joris Messens, Simon SilverAbstract:The frequent abundance of arsenic in the environment has guided the evolution of enzymes for the reduction of Arsenate. The Arsenate Reductases (ArsC) from different sources have unrelated sequences and structural folds, and can be divided into different classes on the basis of their structures, reduction mechanisms and the locations of catalytic cysteine residues. The thioredoxin-coupled Arsenate Reductase class is represented by Staphylococcus aureus pI258 ArsC and Bacillus subtilis ArsC. The ArsC from Escherichia coli plasmid R773 and the eukaryotic ACR2p Reductase from Saccharomyces cerevisiae represent two distinct glutaredoxin-linked ArsC classes. All are small cytoplasmic redox enzymes that reduce Arsenate to arsenite by the sequential involvement of three different thiolate nucleophiles that function as a redox cascade. In contrast, the ArrAB complex is a bacterial heterodimeric periplasmic or a surface-anchored Arsenate Reductase that functions as a terminal electron acceptor and transfers electrons from the membrane respiratory chain to Arsenate. Finally, the less well documented Arsenate Reductase activity of the monomeric arsenic(III) methylase, which is an S-adenosylmethionine (AdoMet)-dependent methyltransferase. After each oxidative methylation cycle and before the next methylation step, As(V) is reduced to As(III). Methylation by this enzyme is also considered an arsenic-resistance mechanism for bacteria, fungi and mammals.
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interplay between ion binding and catalysis in the thioredoxin coupled Arsenate Reductase family
Journal of Molecular Biology, 2006Co-Authors: Goedele Roos, Lode Wyns, Remy Loris, Karolien Van Belle, Lieven Buts, Elke Brosens, Paul Geerlings, Joris MessensAbstract:Abstract In the thioredoxin (Trx)-coupled Arsenate Reductase family, Arsenate Reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of Arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 A from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled Arsenate Reductases. In the P-loop of the Trx-coupled Arsenate Reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in β conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the p K a value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k cat value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled Arsenate Reductases.
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the activation of electrophile nucleophile and leaving group during the reaction catalysed by pi258 Arsenate Reductase
ChemBioChem, 2006Co-Authors: Goedele Roos, Lode Wyns, Karolien Van Belle, Elke Brosens, Paul Geerlings, Stefan Loverix, Joris MessensAbstract:The reduction of Arsenate to arsenite by pI258 Arsenate Reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of Arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic Arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.
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a computational and conceptual dft study on the michaelis complex of pi258 Arsenate Reductase structural aspects and activation of the electrophile and nucleophile
Journal of Physical Chemistry B, 2004Co-Authors: Goedele Roos, Joris Messens, Lode Wyns, Stefan Loverix, Paul GeerlingsAbstract:The first step in the reduction of Arsenate to arsenite catalyzed by the enzyme Arsenate Reductase (ArsC) from Staphylococcus aureus plasmid pI258 involves the nucleophilic attack of a cysteine thiolate (Cys10) on the arsenic atom, leading to a covalent sulfur−arseno intermediate. We present a quantum chemical study on the onset of the nucleophilic displacement reaction. To optimize the reactant state geometry, a density functional study was performed on Cys10, on dianionic Arsenate, and on the catalytic site sequence motif: X-X-Asn13-X-X-Arg16-Ser17. Both the hydrogen bond from Arg16 to the leaving hydroxyl group of Arsenate and the hydrogen bonds from various backbone amide nitrogens of the catalytic site to the other oxygen atoms of Arsenate are responsible for the increased electrophilicity of the central arsenic atom. In particular, Arg16 is identified as a residue that destabilizes the groundstate of the complex. Furthermore, the binding of dianionic Arsenate to the enzyme induces negative charge t...
Lode Wyns - One of the best experts on this subject based on the ideXlab platform.
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Arsenate Reductase mycothiol and mycoredoxin concert thiol disulfide exchange
Journal of Biological Chemistry, 2009Co-Authors: Efren Ordonez, Lode Wyns, Karolien Van Belle, Goedele Roos, Sandra De Galan, Michal Letek, Jose A Gil, Luis M Mateos, Joris MessensAbstract:We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two Arsenate Reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against Arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer Arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of Arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between Arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione Reductase. Cg_ArsCs show a low specificity constant of ∼5 m-1 s-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.
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interplay between ion binding and catalysis in the thioredoxin coupled Arsenate Reductase family
Journal of Molecular Biology, 2006Co-Authors: Goedele Roos, Lode Wyns, Remy Loris, Karolien Van Belle, Lieven Buts, Elke Brosens, Paul Geerlings, Joris MessensAbstract:Abstract In the thioredoxin (Trx)-coupled Arsenate Reductase family, Arsenate Reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of Arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 A from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled Arsenate Reductases. In the P-loop of the Trx-coupled Arsenate Reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in β conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the p K a value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k cat value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled Arsenate Reductases.
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the activation of electrophile nucleophile and leaving group during the reaction catalysed by pi258 Arsenate Reductase
ChemBioChem, 2006Co-Authors: Goedele Roos, Lode Wyns, Karolien Van Belle, Elke Brosens, Paul Geerlings, Stefan Loverix, Joris MessensAbstract:The reduction of Arsenate to arsenite by pI258 Arsenate Reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of Arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic Arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.
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a computational and conceptual dft study on the michaelis complex of pi258 Arsenate Reductase structural aspects and activation of the electrophile and nucleophile
Journal of Physical Chemistry B, 2004Co-Authors: Goedele Roos, Joris Messens, Lode Wyns, Stefan Loverix, Paul GeerlingsAbstract:The first step in the reduction of Arsenate to arsenite catalyzed by the enzyme Arsenate Reductase (ArsC) from Staphylococcus aureus plasmid pI258 involves the nucleophilic attack of a cysteine thiolate (Cys10) on the arsenic atom, leading to a covalent sulfur−arseno intermediate. We present a quantum chemical study on the onset of the nucleophilic displacement reaction. To optimize the reactant state geometry, a density functional study was performed on Cys10, on dianionic Arsenate, and on the catalytic site sequence motif: X-X-Asn13-X-X-Arg16-Ser17. Both the hydrogen bond from Arg16 to the leaving hydroxyl group of Arsenate and the hydrogen bonds from various backbone amide nitrogens of the catalytic site to the other oxygen atoms of Arsenate are responsible for the increased electrophilicity of the central arsenic atom. In particular, Arg16 is identified as a residue that destabilizes the groundstate of the complex. Furthermore, the binding of dianionic Arsenate to the enzyme induces negative charge t...
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the structure of a triple mutant of pi258 Arsenate Reductase from staphylococcus aureus and its 5 thio 2 nitrobenzoic acid adduct
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Joris Messens, I Van Molle, P Vanhaesebrouck, K Van Belle, Khadija Wahni, Jose Martins, Lode Wyns, Remy LorisAbstract:Structural insights into formation of the complex between the ubiquitous thiol–disulfide oxidoReductase thioredoxin and its oxidized substrate are under-documented owing to its entropical instability. In vitro, it is possible via a reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) to make a stable mixed-disulfide complex between thioredoxin from Staphylococcus aureus and one of its substrates, oxidized pI258 Arsenate Reductase (ArsC) from S. aureus. In the absence of the crystal structure of an ArsC–thioredoxin complex, the structures of two precursors of the complex, the ArsC triple mutant ArsC C10SC15AC82S and its 5-thio-2-nitrobenzoic acid (TNB) adduct, were determined. The ArsC triple mutant has a structure very similar to that of the reduced form of wild-type ArsC, with a folded redox helix and a buried catalytic Cys89. In the adduct form, the TNB molecule is buried in a hydrophobic pocket and the disulfide bridge between TNB and Cys89 is sterically inaccessible to thioredoxin. In order to form a mixed disulfide between ArsC and thioredoxin, a change in the orientation of the TNB–Cys89 disulfide in the structure is necessary.
Barry P. Rosen - One of the best experts on this subject based on the ideXlab platform.
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adventitious Arsenate Reductase activity of the catalytic domain of the human cdc25b and cdc25c phosphatases
Biochemistry, 2010Co-Authors: Hiranmoy Bhattacharjee, Rita Mukhopadhyay, Ju Sheng, Abdul A Ajees, Barry P. RosenAbstract:A number of eukaryotic enzymes that function as Arsenate Reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an Arsenate Reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These Reductases contain an active site C-X5-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious Arsenate Reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenat...
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a cdc25 homologue from rice functions as an Arsenate Reductase
New Phytologist, 2007Co-Authors: Gui-lan Duan, Yiping Tong, Rita Mukhopadhyay, Yao Zhou, Barry P. RosenAbstract:Summary • Enzymatic reduction of Arsenate to arsenite is the first step in Arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. • Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the Arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. • The gene products were purified and demonstrated to reduce Arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher Reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)5R motif led to nearly complete loss of both phosphatase and Arsenate Reductase activities. • In planta expression of OsACR2.1 increased dramatically after exposure to Arsenate. OsACR2.2 was observed only in roots following Arsenate exposure, and its expression was less than OsACR2.1.
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hyperaccumulation of arsenic in the shoots of arabidopsis silenced for Arsenate Reductase acr2
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Om Parkash Dhankher, Elizabeth C Mckinney, Barry P. Rosen, R Ichard B. MeagherAbstract:Endogenous plant Arsenate Reductase (ACR) activity converts Arsenate to arsenite in roots, immobilizing arsenic below ground. By blocking this activity, we hoped to construct plants that would mobilize more Arsenate aboveground. We have identified a single gene in the Arabidopsis thaliana genome, ACR2, with moderate sequence homology to yeast Arsenate Reductase. Expression of ACR2 cDNA in Escherichia coli complemented the Arsenate-resistant and Arsenate-sensitive phenotypes of various bacterial ars operon mutants. RNA interference reduced ACR2 protein expression in Arabidopsis to as low as 2% of wild-type levels. The various knockdown plant lines were more sensitive to high concentrations of Arsenate, but not arsenite, than wild type. The knockdown lines accumulated 10- to 16-fold more arsenic in shoots (350–500 ppm) and retained less arsenic in roots than wild type, when grown on Arsenate medium with <8 ppm arsenic. Reducing expression of ACR2 homologs in tree, shrub, and grass species should play a vital role in the phytoremediation of environmental arsenic contamination.
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arginine 60 in the arsc Arsenate Reductase of e coli plasmid r773 determines the chemical nature of the bound as iii product
Protein Science, 2004Co-Authors: Srini Demel, Barry P. Rosen, Jin Shi, Philip D Martin, Brian F P EdwardsAbstract:Arsenic is a ubiquitous environmental toxic metal. Consequently, organisms detoxify Arsenate by reduction to arsenite, which is then excreted or sequestered. The ArsC Arsenate Reductase from Escherichia coli plasmid R773, the best characterized arsenic-modifying enzyme, has a catalytic cysteine, Cys 12, in the active site, surrounded by an arginine triad composed of Arg 60, Arg 94, and Arg 107. During the reaction cycle, the native enzyme forms a unique monohydroxyl Cys 12-thiol-arsenite adduct that contains a positive charge on the arsenic. We hypothesized previously that this unstable intermediate allows for rapid dissociation of the product arsenite. In this study, the role of Arg 60 in product formation was evaluated by mutagenesis. A total of eight new structures of ArsC were determined at resolutions between 1.3 A and 1.8 A, with Rfree values between 0.18 and 0.25. The crystal structures of R60K and R60A ArsC equilibrated with the product arsenite revealed a covalently bound Cys 12-thiol-dihydroxyarsenite without a charge on the arsenic atom. We propose that this intermediate is more stable than the monohydroxyarsenite intermediate of the native enzyme, resulting in slow release of product and, consequently, loss of activity.
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increased cadmium tolerance and accumulation by plants expressing bacterial Arsenate Reductase
New Phytologist, 2003Co-Authors: Om Parkash Dhankher, Barry P. Rosen, Nupur A Shasti, Mark Fuhrmann, R Ichard B. MeagherAbstract:Summary • Cadmium (Cd) is a major environmental pollutant that poses a serious threat to natural ecosystems. However, most initial attempts to engineer phytoremediation of Cd have not succeeded in developing sufficient Cd tolerance for vigorous plant growth. • We found that the bacterial Arsenate Reductase gene (arsC) provided Cd(II) resistance to Escherichia coli. When ArsC is overexpressed in tobacco (Nicotiana tabacum) and Arabidopsis thaliana, both transgenic plant species showed significantly greater Cd tolerance than wild-type controls. • At 50, 75, and 100 µm concentrations of Cd (II), the ArsC expressing transgenic lines grew bigger with broader leaves and longer roots than wild-type controls, which were stunted, turned yellow, flowered early, and often died. At the various Cd(II) concentrations, ArsC transgenic plants attained f. wt 2–3-fold higher than the wild-type plants and had roots significantly longer than wild-type plants. These transgenic plants also contained 30–50% higher Cd concentrations than wild-type plants. • It is likely that the arsC gene directs Cd tolerance via the electrochemical reduction of Cd(II) to Cd(0).
David E. Salt - One of the best experts on this subject based on the ideXlab platform.
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targeted expression of the Arsenate Reductase hac1 identifies cell type specificity of arsenic metabolism and transport in plant roots
Journal of Experimental Botany, 2021Co-Authors: Sina Fischer, Fangjie Zhao, Eduardo Sanchezbermejo, Paulina Flis, Priya Ramakrishna, Mary Lou Guerinot, David E. SaltAbstract:High Arsenic Concentration 1 (HAC1), an Arabidopsis thaliana Arsenate Reductase, plays a key role in Arsenate [As(V)] tolerance. Through conversion of As(V) to arsenite [As(III)], HAC1 enables As(III) export from roots, and restricts translocation of As(V) to shoots. To probe the ability of different root tissues to detoxify As(III) produced by HAC1, we generated A. thaliana lines expressing HAC1 in different cell types. We investigated the As(V) tolerance phenotypes: root growth, As(III) efflux, As translocation, and As chemical speciation. We showed that HAC1 can function in the outer tissues of the root (epidermis, cortex, and endodermis) to confer As(V) tolerance, As(III) efflux, and limit As accumulation in shoots. HAC1 is less effective in the stele at conferring As(V) tolerance phenotypes. The exception is HAC1 activity in the protoxylem, which we found to be sufficient to restrict As translocation, but not to confer As(V) tolerance. In conclusion, we describe cell type-specific functions of HAC1 that spatially separate the control of As(V) tolerance and As translocation. Further, we identify a key function of protoxylem cells in As(V) translocation, consistent with the model where endodermal passage cells, above protoxylem pericycle cells, form a 'funnel' loading nutrients and potentially toxic elements into the vasculature.
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Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants
2016Co-Authors: Daiyin Chao, Ziru Chen, Jiugeng Chen, John Danku, Chengcheng Wang, Fangjie Zhao, Yi Chen, Shulin Shi, David E. SaltAbstract:Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce Arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the Arsenate Reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional Arsenate Reductase confirmed the Arsenate Reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce Arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to Arsenate toxicity. We also confirmed the previous observation that the ACR2 Arsenate Reductase in A
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genome wide association mapping identifies a new Arsenate Reductase enzyme critical for limiting arsenic accumulation in plants
PLOS Biology, 2014Co-Authors: Daiyin Chao, Ziru Chen, Jiugeng Chen, John Danku, Chengcheng Wang, Fangjie Zhao, Yi Chen, David E. SaltAbstract:Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce Arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the Arsenate Reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional Arsenate Reductase confirmed the Arsenate Reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce Arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to Arsenate toxicity. We also confirmed the previous observation that the ACR2 Arsenate Reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.
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a novel Arsenate Reductase from the arsenic hyperaccumulating fern pteris vittata
Plant Physiology, 2006Co-Authors: Danielle R Ellis, Luke Gumaelius, Jo Ann Banks, Ingrid J Pickering, Emily Indriolo, David E. SaltAbstract:Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce Arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an Arsenate Reductase gene (PvACR2) from gametophytes that can suppress the Arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the Arsenate Reductase gene ScACR2. Recombinant PvACR2 protein has in vitro Arsenate Reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both Arsenate Reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant Arsenate Reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and Arsenate Reductases have a conserved HCX 5 R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and Reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of Arsenate, while total Arsenate Reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by Arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the Arsenate Reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process.
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engineering tolerance and hyperaccumulation of arsenic in plants by combining Arsenate Reductase and gamma glutamylcysteine synthetase expression
Nature Biotechnology, 2002Co-Authors: Om Parkash Dhankher, David E. Salt, Barry P. Rosen, Yujing Li, Julie F Senecoff, Nupur A Sashti, R Ichard B. MeagherAbstract:Engineering tolerance and hyperaccumulation of arsenic in plants by combining Arsenate Reductase and γ-glutamylcysteine synthetase expression
Karolien Van Belle - One of the best experts on this subject based on the ideXlab platform.
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Arsenate Reductase mycothiol and mycoredoxin concert thiol disulfide exchange
Journal of Biological Chemistry, 2009Co-Authors: Efren Ordonez, Lode Wyns, Karolien Van Belle, Goedele Roos, Sandra De Galan, Michal Letek, Jose A Gil, Luis M Mateos, Joris MessensAbstract:We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two Arsenate Reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against Arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer Arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of Arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between Arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione Reductase. Cg_ArsCs show a low specificity constant of ∼5 m-1 s-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.
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interplay between ion binding and catalysis in the thioredoxin coupled Arsenate Reductase family
Journal of Molecular Biology, 2006Co-Authors: Goedele Roos, Lode Wyns, Remy Loris, Karolien Van Belle, Lieven Buts, Elke Brosens, Paul Geerlings, Joris MessensAbstract:Abstract In the thioredoxin (Trx)-coupled Arsenate Reductase family, Arsenate Reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of Arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 A from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled Arsenate Reductases. In the P-loop of the Trx-coupled Arsenate Reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in β conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the p K a value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k cat value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled Arsenate Reductases.
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the activation of electrophile nucleophile and leaving group during the reaction catalysed by pi258 Arsenate Reductase
ChemBioChem, 2006Co-Authors: Goedele Roos, Lode Wyns, Karolien Van Belle, Elke Brosens, Paul Geerlings, Stefan Loverix, Joris MessensAbstract:The reduction of Arsenate to arsenite by pI258 Arsenate Reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of Arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic Arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.
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purification of an oxidation sensitive enzyme pi258 Arsenate Reductase from staphylococcus aureus
Journal of Chromatography B, 2003Co-Authors: Joris Messens, Karolien Van Belle, Elke Brosens, Jose C Martins, Ingrid Zegers, Lode WynsAbstract:Abstract Arsenate Reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (Ka=1.1 mM) and potassium was stabilizing ArsC (ΔTm=+6.2 °C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture.
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p of an oxidation sensitive enzyme pi258 Arsenate Reductase from staphylococcus aureus
2003Co-Authors: Joris Messens, Karolien Van Belle, Elke Brosens, Jose C Martins, Ingrid Zegers, Lode WynsAbstract:Arsenate Reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (K 51.1 mM) and potassium a was stabilizing ArsC (DT 516.2 8C). Also due to the use of these salt ions, the final yield of the purification had improved m