Characterization of a novel ArsR regulates divergent ars operon in Ensifer adhaerens strain ST2.

Microbes evolved resistance determinates for coping with arsenic toxicity are commonly regulated by a variety of transcriptional repressors (ArsRs). Ensifer adhaerens strain ST2 was previously shown tolerance to environmental organoarsenical methylarsenite (MAs(III)), which has been proposed to be a primordial antibiotic. In E. adhaerens strain ST2 chromosomal ars operon, two MAs(III) resistance genes, arsZ, encoding MAs(III) oxidase, and arsK, encoding MAs(III) efflux transporter, are controlled by a novel ArsR transcriptional repressor, EaArsR. It has two conserved cysteine pairs, Cys91-92 and Cys108-109. Electrophoretic mobility shift assays (EMSAs) demonstrate that EaArsR binds to two inverted-repeat sequences within the ars promoter between arsR and arsZ to repress ars operon transcription and that DNA binding is relieved upon binding of As(III) and MAs(III). Mutation of either Cys91 or Cys92 to serine (or both) abolished these mutants binding to the ars promoter. In contrast, both C108S and C109S mutants kept responsiveness to As(III) and MAs(III). These results suggest that cysteine pair Cys91-Cys92 and either Cys108 or Cys109 contribute to form arsenic binding site. Homology modeling of EaArsR indicates the binding site consisted of Cys91-Cys92 pair from one monomer and Cys108-Cys109 pair from the other monomer, which displays the diverse evolution of arsenic binding site in the ArsR metalloregulators.

The ArsI C-As lyase: Elucidating the catalytic mechanism of degradation of organoarsenicals.

Organoarsenicals such as monosodium methylarsenate (MSMA or MAs(V)) and roxarsone (4-hydroxyl-3-nitrophenylarsenate or Rox(V)) have been extensively used as herbicides and growth enhancers for poultry, respectively. Degradation of organoarsenicals to inorganic arsenite (As(III)) contaminates crops and drinking water. One such process is catalyzed by the bacterial enzyme ArsI, whose gene is found in many soil bacteria. ArsI is a non-heme ferrous iron (Fe(II))-dependent dioxygenase that catalyzes oxygen-dependent cleavage of the carbon­arsenic (C-As) bond in trivalent organoarsenicals, degrading them to inorganic As(III). From previous crystal structures of ArsI, we predicted that a loop-gating mechanism controls the catalytic reaction. Understanding the catalytic mechanism of ArsI requires knowledge of the mechanisms of substrate binding and activation of dioxygen. Here we report new ArsI structures with bound Rox(III) and mutant enzymes with alteration of active site residues. Our results elucidate steps in the catalytic cycle of this novel dioxygenase and enhance understanding of the recycling of environmental organoarsenicals.

An ArsRC fusion protein enhances arsenate sensing and detoxification.

Arsenical resistance (ars) operons encode genes for arsenic resistance and biotransformation. The majority are composed of individual genes, but fusion of ars genes is not uncommon, although it is not clear if the fused gene products are functional. Here we report identification of a four-gene ars operon from Paracoccus sp. SY that has two arsR-arsC gene fusions. ArsRC1 and ArsRC2 are related proteins that consist of an N-terminal ArsR arsenite (As(III))-responsive repressor with a C-terminal ArsC arsenate reductase. The other two genes in the operon are gapdh and arsJ. GAPDH, glyceraldehyde 3-phosphate dehydrogenase, forms 1-arseno-3-phosphoglycerate (1As3PGA) from 3-phosphoglyceraldehyde and arsenate (As(V)), ArsJ is an efflux permease for 1As3PGA that dissociates into extracellular As(V) and 3-phosphoglycerate. The net effect is As(V) extrusion and resistance. ArsRs are usually selective for As(III) and do not respond to As(V). However, the substrates and products of this operon are pentavalent, which would not be inducers of the operon. We propose that ArsRC fusions overcome this limitation by channelling the ArsC product into the ArsR binding site without diffusion through the cytosol, a de facto mechanism for As(V) induction. This novel mechanism for arsenate sensing can confer an evolutionary advantage for detoxification of inorganic arsenate.

Aquaglyceroporin AqpS from Sinorhizobium meliloti conducts both trivalent and pentavalent methylarsenicals.

Arsenic is a toxic metalloid that enters cells adventitiously via uptake systems for phosphate transporters, aquaglyceroporins (AQPs) or sugar permeases. However, transport of highly toxic methylarsenite (MAs(III)) and relatively nontoxic methylarsenate (MAs(V)) by bacterial AQPs has not been characterized. MAs(V) has a history of use as an herbicide. Here we used whole genome sequence analysis of AQPs in arsenic resistance (ars) operons. The aqp genes are frequently located next to MAs(III) resistance genes such as arsH, which suggests that they could be involved in MAs(III) uptake. Bacterial AQPs encoded by ars operons can be classified into two subgroups. One subgroup includes AqpS from the plant symbiont Sinorhizobium meliloti 1021. Our data suggests that AqpS has a substrate selectivity filter different from that of other bacterial AQPs. Both Escherichia coli GlpF and AqpS conduct MAs(III) efficiently, but GlpF conducts the MAs(V) anion poorly, so E. coli takes up MAs(V) inefficiently. In contrast, AqpS conducts MAs(V) under physiological conditions. A homology model of AqpS indicates that it has a substrate channel with a selectivity filter containing the nonpolar residue Val177 instead of the charged arginine residue found in other AQPs. While the selectivity filter in most AQPs prevents movement of anions, Val177 is predicted to allow movement of the MAs(V) anion through the channel. We propose that AqpS is a component of an MAs(III) resistance pathway in which MAs(III) enters cells of S. meliloti via AqpS, is oxidized by ArsH to MAs(V), which exits the cells via AqpS.

Chemical synthesis of the organoarsenical antibiotic arsinothricin.

We report two routes of chemical synthesis of arsinothricin (AST), the novel organoarsenical antibiotic. One is by condensation of the 2-chloroethyl(methyl)arsinic acid with acetamidomalonate, and the second involves reduction of the N-acetyl protected derivative of hydroxyarsinothricin (AST-OH) and subsequent methylation of a trivalent arsenic intermediate with methyl iodide. The enzyme AST N-acetyltransferase (ArsN1) was utilized to purify l-AST from racemic AST. This chemical synthesis provides a source of this novel antibiotic for future drug development.

Arsinothricin, an arsenic-containing non-proteinogenic amino acid analog of glutamate, is a broad-spectrum antibiotic.

The emergence and spread of antimicrobial resistance highlights the urgent need for new antibiotics. Organoarsenicals have been used as antimicrobials since Paul Ehrlich's salvarsan. Recently a soil bacterium was shown to produce the organoarsenical arsinothricin. We demonstrate that arsinothricin, a non-proteinogenic analog of glutamate that inhibits glutamine synthetase, is an effective broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, suggesting that bacteria have evolved the ability to utilize the pervasive environmental toxic metalloid arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. The arsN1 gene, widely distributed in bacterial arsenic resistance (ars) operons, selectively confers resistance to arsinothricin by acetylation of the α-amino group. Crystal structures of ArsN1 N-acetyltransferase, with or without arsinothricin, shed light on the mechanism of its substrate selectivity. These findings have the potential for development of a new class of organoarsenical antimicrobials and ArsN1 inhibitors.

A novel MAs(III)-selective ArsR transcriptional repressor.

Microbial expression of genes for resistance to heavy metals and metalloids is usually transcriptionally regulated by the toxic ions themselves. Arsenic is a ubiquitous, naturally occurring toxic metalloid widely distributed in soil and groundwater. Microbes biotransform both arsenate (As(V)) and arsenite (As(III)) into more toxic methylated metabolites methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)). Environmental arsenic is sensed by members of the ArsR/SmtB family. The arsR gene is autoregulated and is typically part of an operon that contains other ars genes involved in arsenic detoxification. To date every identified ArsR is regulated by inorganic As(III). Here we described a novel ArsR from Shewanella putrefaciens selective for MAs(III). SpArsR orthologs control expression of two MAs(III) resistance genes, arsP that encodes the ArsP MAs(III) efflux permease, and arsH encoding the ArsH MAs(III) oxidase. SpArsR has two conserved cysteine residues, Cys101 and Cys102. Mutation of either resulted in loss of MAs(III) binding, indicating that they form an MAs(III) binding site. SpArsR can be converted into an As(III)-responsive repressor by introduction of an additional cysteine that allows for three-coordinate As(III) binding. Our results indicate that SpArsR evolved selectivity for MAs(III) over As(III) in order to control expression of genes for MAs(III) detoxification.

Structure of the ArsI C-As Lyase: Insights into the Mechanism of Degradation of Organoarsenical Herbicides and Growth Promoters.

Arsenic is a ubiquitous and carcinogenic environmental element that enters the biosphere primarily from geochemical sources, but also through anthropogenic activities. Microorganisms play an important role in the arsenic biogeochemical cycle by biotransformation of inorganic arsenic into organic arsenicals and vice versa. ArsI is a microbial non-heme, ferrous-dependent dioxygenase that transforms toxic methylarsenite [MAs(III)] to less toxic and carcinogenic inorganic arsenite [As(III)] by C-As bond cleavage. An ArsI ortholog, TcArsI, from the thermophilic bacterium Thermomonospora curvata was expressed, purified, and crystallized. The structure was solved in both the apo form and with Ni(II), Co(II), or Fe(III). The MAs(III) binding site is a vicinal cysteine pair in a flexible loop. A structure with the loop occupied with ß-mercaptoethanol mimics binding of MAs(III). The structure of a mutant protein (Y100H/V102F) was solved in two different crystal forms with two other orientations of the flexible loop. These results suggest that a loop-gating mechanism controls the catalytic reaction. In the ligand-free open state, the loop is exposed to solvent, where it can bind MAs(III). The loop moves toward the active site, where it forms a closed state that orients the C-As bond for dioxygen addition and cleavage. Elucidation of the enzymatic mechanism of this unprecedented C-As lyase reaction will enhance our understanding of recycling of environmental organoarsenicals.