Showing papers on "Arsenate reductase published in 2004"

Bacillus macyae sp. nov., an arsenate-respiring bacterium isolated from an Australian gold mine.

Abstract: 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)).

Arginine 60 in the ArsC arsenate reductase of E. coli plasmid R773 determines the chemical nature of the bound As(III) product.

Abstract: 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.

A Computational and Conceptual DFT Study on the Michaelis Complex of pI258 Arsenate Reductase. Structural Aspects and Activation of the Electrophile and Nucleophile

Abstract: 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...

Arsenical resistance genes in Saccharomyces douglasii and other yeast species undergo rapid evolution involving genomic rearrangements and duplications

Abstract: We have isolated and characterized three adjacent Saccharomyces douglasii genes that share remarkable structural homology (97% amino acid sequence identity) with Saccharomyces cerevisiae ARR1 (ACR1), ARR2 (ACR2) and ARR3 (ACR3) genes involved in arsenical resistance. The ARR2 and ARR3 genes encoding the cytoplasmic arsenate reductase and the plasma membrane arsenite transporter are functionally interchangeable in both yeast species. In contrast, a single copy of S. douglasii ARR1 gene is not sufficient to complement the arsenic hypersensitivity of a S. cerevisiae mutant lacking the transcriptional activator Arr1p. This inability may be related to a deletion of a 35-bp sequence including the putative Yap-binding element in the ARR1 promoter of S. douglasii. Different mechanisms of regulation of ARR1 genes expression may therefore explain the increased tolerance of S. douglasii to arsenic in comparison with S. cerevisiae. The apparent duplication of the ARR gene cluster in the S. douglasii genome may constitute another factor contributing to the observed differences in arsenic sensitivity. Comparison of ARR genes from the genomes of several yeast species indicates that they are located in subtelomeric regions undergoing rapid evolution involving large-scale genomic rearrangements.

The structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adduct

Abstract: 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.

Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function.

Abstract: Background: The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. Results: The structure was determined at 1.0 A resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. Conclusion: Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

1H, 13C and 15N resonance assignments of a Bacillus subtilis arsenate reductase.

Abstract: Arsenic compounds are toxic to nearly all kinds of life forms, while some microorganisms such as bacteria, fungi and algae utilize unique arsenical systems to confer arsenic resistance. In most such microorganisms, arsenic compounds are firstly reduced from pentavalent arsenicals to trivalent derivatives by arsenate reductase (ArsC), and then pumped out of the cells via membrane transport systems, though arsenite (III) is at least about 1000 times toxic than arsenate (Cervants et al., 1994). The X-ray structure of the ArsC protein from Bacillus subtilis, was reported previously (Bennett, et al., 2001). The X-ray structure shows that the ArsC protein packs into four ArsC molecules per asymmetric unit, among which, the crucial functional segment (Cys82-Val96) is missing in two of them, while it is visible in the other two ArsC molecules. This may imply an uncommon flexibility. In order to determine the solution structures and obtain further insights into the structure-function correlations of the arsenate reductase, here we report the nearly complete sequence-specific backbone and side-chain 1H, 13C and 15N resonance assignment of the ArsC protein from Bacillus subtilis in the reduced form.