Hiranmoy Bhattacharjee

Novel Pathway for Arsenic Detoxification in the Legume Symbiont Sinorhizobium meliloti

We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH)3/H+ antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an aquaglyceroporin with a physiological function in arsenic resistance.

Arsenic Metabolism in Prokaryotic and Eukaryotic Microbes

This chapter will focus on recent progress on the mechanisms of metalloid uptake, metabolism, and detoxification in bacteria, archaea, and eukaryotic microbes. One of the initial challenges of the earliest cells would have been the ability to detoxify heavy metal ions, transition metal ions, and metalloids, including arsenic and antimony. The presence of arsenic resistance (ars) genes in the genome of by far most living organisms sequenced to date illustrates firstly that ars genes must be ancient and secondly that arsenic is still ubiquitous in the environment, providing the selective pressure that maintains these genes in present-day organisms. Some early cells also probably could use arsenite as an electron acceptor, giving selective pressure for the evolution of respiratory arsenate reductase. As atmospheric O2 levels increased, arsenite was oxidized to arsenate abiotically. This provided an advantage for the evolution of arsenate reductases, some for arsenate respiration and energy production, and others for arsenate detoxification. Present-day selective pressure for metalloid resistance also comes from sources such as natural release of arsenic from volcanic activities, mining activities, the burning of coal, and other human activities. In addition is the use of arsenicals and antimonials as chemotherapeutic drugs for the treatment of parasitic diseases and cancer. Resistance to these drugs is becoming a major dilemma. Thus, an understanding of the molecular details of metalloid transport systems and detoxification enzymes is essential for the rational design of new drugs, and for treating drug-resistant microorganisms and tumor cells. Finally, this chapter will summarize recent identification of novel enzymes for arsenic reduction, oxidation, and methylation that expand the possibilities for metalloid metabolism and transformations.

Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis

The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of trivalent metalloids, arsenite and antimonite. We have earlier shown that downregulation of LmAQP1 provides resistance to trivalent antimony compounds whereas increased expression of LmAQP1 in drug‐resistant parasites can reverse the resistance. In this paper we describe the biochemical characterization of LmAQP1. Expression of LmAQP1 in Xenopus oocytes rendered them permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. The transport property of LmAQP1 was severely affected when a critical Arg230, located inside the pore of the channel, was altered to either alanine or lysine. Immunofluorescence and immuno‐electron microscopy revealed LmAQP1 to be localized to the flagellum of Leishmania promastigotes and in the flagellar pocket membrane and contractile vacuole/spongiome complex of amastigotes. This is the first report of an aquaglyceroporin being localized to the flagellum of any microbe. Leishmania promastigotes and amastigotes expressing LmAQP1 could regulate their volume in response to hypoosmotic stress. Additionally, Leishmania promastigotes overexpressing LmAQP1 were found to migrate faster towards an osmotic gradient. These results taken together suggest that Leishmania LmAQP1 has multiple physiological roles, being involved in solute transport, volume regulation and osmotaxis.

Spatial Proximity of Cys113, Cys172, and Cys422 in the Metalloactivation Domain of the ArsA ATPase

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys113, Cys172, and Cys422. To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 Å of each other to form a fluorescent adduct. ArsA proteins in which single cysteine residues were altered by site-directed mutagenesis still formed fluorescent adducts. Proteins in which two of the three cysteine residues were substituted did not form fluorescent adducts. These results demonstrate that Cys113, Cys172, and Cys422 are in close proximity of each other. We propose a model in which As(III) or Sb(III) interacts with these three cysteines in a trigonal pyramidal geometry, forming a novel soft metal-thiol cage.

The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance.

Homologues of the bacterial ArsA ATPase are found in nearly every organism. While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified. This article reports the function of the Saccharomyces cerevisiaehomologue encoded by ARR4 gene (YDL100c ORF). Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO43− salts and temperature. A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses. Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain. Wild type and mutant Arr4p were purified from Escherichia coli. The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive. The purified ATPase eluted as a dimer of 80-kDa species. A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed. The gene fusion was able to complement stress-related phenotype of the ARR4 disruption. Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol. Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes. It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.

Mechanism of the ArsA ATPase.

The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells. This transport ATPase shares structural and perhaps mechanism features with ABC transporters. The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase. As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump. The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level. Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains. In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change. The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step. We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters.

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.

Aquaglyceroporins: ancient channels for metalloids

The identification of aquaglyceroporins as uptake channels for arsenic and antimony shows how these toxic elements can enter the food chain, and suggests that food plants could be genetically modified to exclude arsenic while still accumulating boron and silicon.

Crystal structure of the flavoprotein ArsH from Sinorhizobium meliloti

Purified ArsH from Sinorhizobium meliloti exhibits NADPH:FMN‐dependent reduction of molecular O2 to hydrogen peroxide and catalyzes reduction of azo dyes. The structure of ArsH was determined at 1.8 Å resolution. ArsH crystallizes with eight molecules in the asymmetric unit forming two tetramers. Each monomer has a core domain with a central five‐stranded parallel β‐sheet and two monomers interact to form a classical flavodoxin‐like dimer. The N‐ and C‐terminal extensions of ArsH are involved in interactions between subunits and tetramer formation. The structure may provide insight in how ArsH participates in arsenic detoxification.

Aquaglyceroporins and metalloid transport: Implications in human diseases

Aquaglyceroporin (AQP) channels facilitate the diffusion of a wide range of neutral solutes, including water, glycerol, and other small uncharged solutes. More recently, AQPs have been shown to allow the passage of trivalent arsenic and antimony compounds. Arsenic and antimony are metalloid elements. At physiological pH, the trivalent metalloids behave as molecular mimics of glycerol, and are conducted through AQP channels. Arsenicals and antimonials are extremely toxic to cells. Despite their toxicity, both metalloids are used as chemotherapeutic agents for the treatment of cancer and protozoan parasitic diseases. The metalloid homeostasis property of AQPs can be a mixed blessing. In some cases, AQPs form part of the detoxification pathway, and extrude metalloids from cells. In other instances, AQPs allow the transport of metalloids into cells, thereby conferring sensitivity. Understanding the factors that modulate AQP expression will aid in a better understanding of metalloid toxicity and also provide newer approaches to metalloid based chemotherapy.