Arsenic, a toxic metalloid, is currently and has always been ranked first on the Superfund List of Hazardous Substances (available on the World Wide Web), in part because of its environmental ubiquity. As a consequence, nearly all organisms have genes that confer resistance to arsenic. Environmental arsenic is sensed by members of the ArsR/SmtB family of metalloregulatory proteins (1-3). These winged helix repressor proteins specifically bind to arsenic and other toxic metals. Consequently, they control expression of genes involved in arsenic biotransformation and efflux. For example, the ArsR repressor encoded by Escherichia coli plasmid R773 binds to the promoter region of its respective ars operon in the absence of As(III) or Sb(III) (4). This homodimeric repressor has the sequence Cys 32 -Val-Cys 34 -Asp-Leu-Cys 37 in the DNA binding domain of each monomer (5, 6). The three sulfur thiolates of the cysteine residues form a very specific three-coordinate binding site for the trivalent metalloids As(III) and Sb(III). Binding of metalloids to R773 ArsR is presumed to induce a conformational change, leading to dissociation from the DNA and hence derepression. The Staphylococcus aureus CadC is a Cd(II)/Pb(II)/Zn(II)-responsive member of the ArsR/SmtB family that has four cysteine residues in the inducer binding domain (7). Of these four cysteine residues, two come from one subunit, whereas the other two come from the other subunit of the homodimer (8, 9). The position of this metal binding site in CadC is congruent to that of the R773 ArsR but is formed between two monomers. CadC also has a second type of metal binding site (DXHX 10 HX 2 E) for Zn(II) at the dimer interface that is not a regulatory site. This site, however, is identical to the regulatory Zn(II) site of SmtB from Synechococcus PCC 7942 (10). Another member of the ArsR/SmtB family is the ArsR from Acidithiobacillus ferrooxidans (AfArsR), 3 which has three cysteine residues (Cys 95 , Cys 96 , and Cys 102 ) at the dimer interface rather than in or near the DNA binding domain (11). These three cysteine residues form a three-coordinate or S 3 binding site for trivalent metalloids (12). Although both the As(III) binding site of AfArsR and the Zn(II) binding site of SmtB are at the C-terminal dimerization domain, the former is formed of three cysteine residues within a single subunit (two sites per dimer), whereas the latter is formed by four residues, two residues from one monomer and two from the other. Thus, metal(loid) binding sites appear to arise by convergent evolution, even in homologous proteins.

No abstract

A Aq qu ua ag gl ly yc ce er ro op po or ri in ns s: : a an nc ci ie en nt t c ch ha an nn ne el ls s f fo or r m me et ta al ll lo oi id ds s Hiranmoy Bhattacharjee, Rita Mukhopadhyay, Saravanamuthu Thiyagarajan and Barry P Rosen Address: Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.Correspondence: Barry P Rosen. Email: brosen@med.wayne.edu Arsenic, a metalloid, is widely distributed in the Earth's crust and is toxic to all forms of life. Humans can be exposed to arsenic from drinking water that has flowed through arsenicrich rocks or from crops that have been irrigated with arseniccontaminated water. Arsenic occurs predominantly in the environment as the pentavalent arsenate (As(V)) and trivalent arsenite (As(III)) forms. Arsenite is more toxic than arsenate and is primarily responsible for the biological effects of arsenic. The toxicity of arsenite is due to its affinity for closely spaced cysteine thiolates; it inactivates enzymes and receptors by binding to active site cysteine residues or by preventing formation of disulfide bonds. Arsenite also leads to the production of reactive oxygen species by binding to reduced glutathione. In addition to the severe health effects of arsenic in drinking water, its accumulation in crops such as rice jeopardizes the safety of our food supply [1]. An understanding of the pathways of arsenic uptake, metabolism, and elimination will help in developing strategies to produce plants that take up essential metalloids while excluding toxic ones.A Aq qu ua ag gl ly yc ce er ro op po or ri in ns s a an nd d m me et ta al ll lo oi id d t tr ra an ns sp po or rt t A little over a decade ago, Sanders et al.[2] isolated a mutant of GlpF, the glycerol facilitator of Escherichia coli, that was resistant to antimonite (Sb(III)). Later, Meng et al. [3] determined that this mutant also exhibits a 90% reduction in arsenite uptake. Antimony is another metalloid in the same group of the periodic table as arsenic (Figure 1). GlpF is a member of the major intrinsic protein (MIP) or aquaporin superfamily of channels for water and small solutes that is widely expressed in nearly every organism. Aquaporins fall into two broad groups, aquaporins or water-specific channels, and aquaglyceroporins, which conduct water, glycerol and other small, uncharged solutes.It might seem surprising that a transporter for water and small organic compounds could transport a metalloid. However, analysis of the state of trivalent arsenic in solution shows how this is possible. Although trivalent inorganic arsenic is often referred to as an anion, arsenite, in solution, has a pK a of 9.2, and it is therefore protonated at physiological pH. Extended X-ray absorption fine structure (EXAFS) analysis has shown that in aqueous solution there are three oxygen ligands 1.78 Å from the arsenic atom; the major species in solution is therefore the neutral hydroxide As(OH) 3 , which is an inorganic molecular mimic of glycerol [4]. A Ab bs st tr ra ac ct tTh...

ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCEGiven the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor. In reaction to arsenic in their environment, microorganisms orchestrate an organized response that may involve arsenite [As(III)] oxidation, arsenate [As(V)] reduction, or both. These redox reactions serve to detoxify or protect the organism or to generate energy, depending on the organism and the genes involved. Current models depict As(III) being taken up into the cell via aquaglyceroporins (e.g., reviewed in references 1, 2, and 3), where it then interacts with a DNA-binding repressor protein, ArsR, resulting in a conformational change in ArsR and causing it...

Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.