The ArsD As(III) metallochaperone

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 ??-strands flanked by four ??-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 ??1 helix and metalloid binding site of ArsD.     

The Functions of Crucial Cysteine Residues in the Arsenite Methylation Catalyzed by Recombinant Human Arsenic (III) Methyltransferase

Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs³⁺). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs³⁺ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs³⁺ was methylated to dimethylarsinous acid (DMA³⁺). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs³⁺ methylation catalyzed by hAS3MT.     

Transcriptional activation of metalloid tolerance genes in Saccharomyces cerevisiae requires the AP-1-like proteins Yap1p and Yap8p

All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1-like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H(2)O(2) treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1-like proteins respond to metalloids through distinct mechanisms.     

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

The yeast transporter Acr3p is a low affinity As(III)/H⁺ and Sb(III)/H⁺ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane.     

ArsD residues Cys12, Cys13, and Cys18 form an As(III)-binding site required for arsenic metallochaperone activity

The ArsA ATPase is the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. ArsD is a metallochaperone that delivers As(III) to ArsA, increasing its affinity for As(III), thus conferring resistance to environmental concentrations of arsenic. R773 ArsD is a homodimer with three vicinal cysteine pairs, Cys(12)-Cys(13), Cys(112)-Cys(113), and Cys(119)-Cys(120), in each subunit. Each vicinal pair binds As(III) or Sb(III). Alignment of the primary sequence of homologues of ArsD indicates that only the first vicinal cysteine pair, Cys(12)-Cys(13), and an additional cysteine, Cys(18), are conserved. The effect of cysteine-to-alanine substitutions and truncations were examined. By yeast two-hybrid analysis, nearly all of the ArsD mutants were able to interact with wild type ArsD, indicating that the mutations do not interfere with dimerization. ArsD mutants with alanines substituting for Cys(112), Cys(113), Cys(119), or Cys(120) individually or in pairs or truncations lacking the vicinal pairs retained ability to interact with ArsA and to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys(12), Cys(13), or Cys(18), individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. We propose that ArsD residues Cys(12), Cys(13), and Cys(18), but not Cys(112), Cys(113), Cys(119), or Cys(120), are required for delivery of As(III) to and activation of the ArsAB pump.