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Arsenic (As) poisoning in aquifers is a serious problem worldwide, especially in the middle-Gangetic Plain (MGP) of India. Microbially-mediated As speciation in such aquifers is governed by the arsenate-reductase enzyme, ArsC, encoded by the arsC gene of As-metabolizing bacteria. In this study, ArsC1 (119 aa) and ArsC2 (141 aa) of a highly resistant strain to arsenic, Citrobacter youngae IITK SM2 (CyIITKSM2), isolated from a mixed-oxic MGP groundwater were biochemically characterized. Coupled-arsenate-reductase assay and IC-ICP-MS analysis confirmed that ArsC2 showed higher As(V) reduction than ArsC1 in the dissolved phase, which was consistent with the prominent structural changes in ArsC2 as identified through circular dichroism spectroscopy. Furthermore, the two ArsCs were able to mobilize arsenic from solid-bound arsenate [As(V)-loaded goethite, AsG] predominantly as As(III). However, the total arsenic released in the presence of ArsC2 was ∼38 % and ∼88 % higher, respectively, as compared to the ArsC1-containing and ArsC-free conditions. A process-based model that considered ArsC-mediated As(V) reduction to As(III) in the dissolved phase, and surface complexation of As(V) and As(III) on goethite, suggested that the extent of arsenate binding with ArsC was not affected by whether As(V) was dissolved or was sorbed. However, the catalytic reduction rate was at least an order of magnitude lower in sorbed As(V) than in dissolved As(V). Mutants of ArsC2 exhibited variable but reduced efficiencies compared to the wild-type ArsC2. This reduction may be attributed to the C-terminal loop observed in the AlphaFold predicted structure of ArsC2, which was absent in ArsC1. This comprehensive biochemical and biophysical analysis of the arsenate reductases in Citrobacter youngae could enhance our understanding of the role these microbes play in arsenic mobilization within MGP aquifers., Competing Interests: Declaration of competing interest The authors declare that no competing financial interests exist, and this work has been carried out in compliance with ethical standards., (Copyright © 2024 Elsevier Inc. All rights reserved.)

An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351 T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady-state kinetics of wild-type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin-like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni 2+ . As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface-exposed Cys74-Cys76 disulfide, ready for reduction by thioredoxin., (© 2024 John Wiley & Sons Ltd.)

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO 4 3- ) as the terminal electron acceptor, where it is reduced to arsenite (AsO 3 3- ) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.

The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (K AsIII ) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol -1 for ArsC-Vmh2 and to 1200 (± 300) L mol -1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).

The arsC gene-encoded arsenate reductase is a vital catalytic enzyme for remediation of environmental arsenic (As). Microorganisms containing the arsC gene can convert pentavalent arsenate (As[V]) to trivalent arsenite (As[III]) to be either retained in the bacterial cell or released into the air. The molecular mechanism governing this process is unknown. Here we present an in silico model of the enzyme to describe their probable active site cavities using SCFBio servers. We retrieved the amino acid sequence of bacterial arsenate reductase enzymes in FASTA format from the NCBI database. Enzyme structure was predicted using the I-TASSER server and visualized using PyMOL tools. The ProSA and the PROCHECK servers were used to evaluate the overall significance of the predicted model. Accordingly, arsenate reductase from Streptococcus pyogenes, Oligotropha carboxidovorans OM5, Rhodopirellula baltica SH 1, and Serratia ureilytica had the highest quality scores with statistical significance. The plausible cavities of the active site were identified in our examined arsenate reductase enzymes which were abundant in glutamate and lysine residues with 6 to 16 amino acids. This in silico experiment may contribute greatly to the remediation of arsenic pollution through the utilization of microbial species.

Arsenate reductase (ArsC) is a superfamily of enzymes that reduce arsenate. Due to active site similarities, some ArsC can function as low-molecular weight protein tyrosine phosphatases (LMW-PTPs). Broad superfamily classifications align with redox partners (Trx- or Grx-linked). To understand this superfamily's mechanistic diversity, the ArsC superfamily is classified on the basis of active site features utilizing the tools TuLIP (two-level iterative clustering process) and autoMISST (automated multilevel iterative sequence searching technique). This approach identified nine functionally relevant (perhaps isofunctional) protein groups. Five groups exhibit distinct ArsC mechanisms. Three are Grx-linked: group 4AA (classical ArsC), group 3AAA (YffB-like), and group 5BAA. Two are Trx-linked: groups 6AAAAA and 7AAAAAAAA. One is an Spx-like transcriptional regulatory group, group 5AAA. Three are potential LMW-PTP groups: groups 7BAAAA, and 7AAAABAA, which have not been previously identified, and the well-studied LMW-PTP family group 8AAA. Molecular dynamics simulations were utilized to explore functional site details. In several families, we confirm and add detail to literature-based mechanistic information. Mechanistic roles are hypothesized for conserved active site residues in several families. In three families, simulations of the unliganded structure sample specific conformational ensembles, which are proposed to represent either a more ligand-binding-competent conformation or a pathway toward a more binding-competent state; these active sites may be designed to traverse high-energy barriers to the lower-energy conformations necessary to more readily bind ligands. This more detailed biochemical understanding of ArsC and ArsC-like PTP mechanisms opens possibilities for further understanding of arsenate bioremediation and the LMW-PTP mechanism.

The thioredoxin (Trx)-coupled arsenate reductase (ArsC) is a family of enzymes that catalyzes the reduction of arsenate to arsenite in the arsenic detoxification pathway. The catalytic cycle involves a series of relayed intramolecular and intermolecular thiol-disulfide exchange reactions. Structures at different reaction stages have been determined, suggesting significant conformational fluctuations along the reaction pathway. Herein, we use two state-of-the-art NMR methods, the chemical exchange saturation transfer (CEST) and the CPMG-based relaxation dispersion (CPMG RD) experiments, to probe the conformational dynamics of B. subtilis ArsC in all reaction stages, namely the enzymatic active reduced state, the intra-molecular C10-C82 disulfide-bonded intermediate state, the inactive oxidized state, and the inter-molecular disulfide-bonded protein complex with Trx. Our results reveal highly rugged energy landscapes in the active reduced state, and suggest global collective motions in both the C10-C82 disulfide-bonded intermediate and the mixed-disulfide Trx-ArsC complex.

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit ( K m = 44.6 ± 1.6 μM, k cat = 9,810 ± 220 seconds -1 ), and phosphate is a competitive inhibitor of arsenate reduction ( K i = 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that ( i ) arsenate desorption limits microbiologically induced arsenate reductive mobilization and ( ii ) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level., Competing Interests: The authors declare no conflict of interest.

Water sources pollution by arsenic ions is a serious environmental problem all around the world. Arsenate reductase enzyme (TtArsC) from Thermus thermophilus extremophile bacterium, naturally binds arsenic ions, As(V) and As (III), in aqueous solutions. In this research, TtArsC enzyme adsorption onto hybrid polyethylene glycol-stabilized gold nanoparticles (AuNPs) was studied at different pH values as an innovative nanobiosystem for metal concentration monitoring. Characterizations were performed by UV/Vis and circular dichroism spectroscopies, TEM images and in terms of surface charge changes. The molecular interaction between arsenic ions and the TtArsC-AuNPs nanobiosystem was also monitored at all pH values considered by UV/Vis spectroscopy. Tests performed revealed high sensitivities and limits of detection equal to 10 ± 3 M -12 and 7.7 ± 0.3 M -12 for As(III) and As(V), respectively., (© 2016 The Author(s).)

The thermophilic bacterium Thermus thermophilus HB27 encodes chromosomal arsenate reductase (TtArsC), the enzyme responsible for resistance to the harmful effects of arsenic. We report on adsorption of TtArsC onto gold nanoparticles for naked-eye monitoring of biomolecular interaction between the enzyme and arsenic species. Synthesis of hybrid biological-metallic nanoparticles has been characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis), dynamic light scattering (DLS) and phase modulated infrared reflection absorption (PM-IRRAS) spectroscopies. Molecular interactions have been monitored by UV-vis and Fourier transform-surface plasmon resonance (FT-SPR). Due to the nanoparticles' aggregation on exposure to metal salts, pentavalent and trivalent arsenic solutions can be clearly distinguished by naked-eye assay, even at 85 μM concentration. Moreover, the assay shows partial selectivity against other heavy metals.

Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

This study investigated the mechanism of arsenic resistance in the diazotrophic bacterium Herbaspirillum sp. GW103 isolated from rhizosphere soil of Phragmites austrails. The isolate Herbaspirillum sp. GW103 exhibited maximum tolerance to arsenic (550 mg/L). Four different arsenate reductase (arsC) genes (arsC1, arsC2, arsC3 and arsC4) were located in the genome of the isolate Herbaspirillum sp. GW103. The expression pattern of the arsC1 differed from other genes. All four types of arsC genes had different protein secondary structures and stereochemical properties. Molecular modeling and structural analysis of arsC genes revealed close structural homology with arsC family proteins from Escherichia coli (PDB ID: 1I9D) and Pseudomonas aeruginosa (PDB ID: 1RW1)., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Myxococcus xanthus MXAN_0575, ArsA, exhibited sequence homology to low-molecular-weight protein tyrosine phosphatases (LMWPTPs) and arsenate reductases. ArsA exhibited weak phosphatase activity toward p-nitrophenyl phosphate, and high arsenate reductase activity, suggesting that ArsA may play a role in arsenate reductase, but not LMWPTP., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This study focuses on the phylogenetic analysis of all the ArsC protein sequences, obtained from similarity search against Gammaproteobacteria, and also studies the role of Gammaproteobacterial family in arsenic toxicity. The ars gene provides arsenic tolerance for microbial cell system and encodes for an arsenate reductase (ArsC), which is essential for arsenate resistance that converts arsenate into arsenite. Phylogenetic analysis offers an opportunity to understand the evolutionary relationship between organisms of interest. The phylogenetic experiment was set up for all possible ArsC sequences in class Gammaproteobacteria. The results suggested a wide similarity between ArsC sequences in the species of Enterobacteriaceae family rather than other families in Gammaproteobacteria. The three evolutionary clades revealed a role of Enterobacteriaceae species, which has the capability to code ArsC protein. Further phylogenetic analysis of ArsC crystal structure sequences has also shown the separate cluster of Enterobacter species. The overall phylogeny of the ArsC protein sequences suggests the species of Enterobacteriaceae family express more among all family of Gammaproteobacteria. This study could be advantageous to emphasize the importance of Enterobacteriaceae in arsenic toxicity.

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20mM and 15mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram+bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2×10(4)M(-1)s(-1). It also exhibited weak phosphatase activity with a kcat/KM value of 2.7×10(-4)M(-1)s(-1). The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural-functional characterization of a thermophilic arsenate reductase., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study focuses a bioinformatics-based prediction of arsC gene product arsenate reductase (ArsC) protein in Cronobacter sakazakii BAA-894 strain. A protein structure-based study encloses three-dimensional structural modeling of target ArsC protein, was carried out by homology modeling method. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in protein. The ten probable ligand binding sites were predicted for target protein structure and highlighted the common binding residues between target and template protein. It has been first time identified that modeled ArsC protein structure in C. sakazakii was structurally and functionally similar to well-characterized ArsC protein of Escherichia coli because of having same structural motifs and fold with similar protein topology and function. Investigation revealed that ArsC from C. sakazakii can play significant role during arsenic resistance and potential microorganism for bioremediation of arsenic toxicity.

In silico analysis followed by experimental validation leads us to propose that the predicted protein All0195 of Anabaena sp. PCC7120 showing enhanced expression under sodium arsenate (Na2HAsO4) stress belongs to the thioredoxin superfamily with structural similarity to bacterial arsenate reductase. The All0195 protein demonstrated C-X-TC-X-K, NTSG-X2-YR, and D-X2-L-X-KRP as functional motifs that show similarity to seven known bacterial arsenate reductase family protein homologs with Cys, Arg, and Pro as conserved residues. In view of physicochemical properties, such as aliphatic index, ratio of Glu + Lys to Gln + His, and secondary structure, it was evident that All0195 was also a thermostable protein. The predicted three-dimensional structure on molecular docking with arsenate oxyanion ([Formula: see text]) revealed its interaction with conserved Cys residue as also known for other bacterial arsenate reductase. In silico derived properties were experimentally attested by cloning and heterologous expression of all0195. Furthermore, this protein functionally complemented the arsenate reductase-deficient sodium arsenate-hypersensitive phenotype of Escherichia coli strainWC3110 (ΔarsC) and depicted arsenate reductase activity on purification. In view of the above properties, All0195 appears to be a new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120.

Although at low concentrations, arsenic commonly occurs naturally as a local geological constituent. Whereas both arsenate and arsenite are strongly toxic to life, a number of prokaryotes use these compounds as electron acceptors or donors, respectively, for bioenergetic purposes via respiratory arsenate reductase, arsenite oxidase and alternative arsenite oxidase. The recent burst in discovered arsenite oxidizing and arsenate respiring microbes suggests the arsenic bioenergetic metabolisms to be anything but exotic. The first goal of the present review is to bring to light the widespread distribution and diversity of these metabolizing pathways. The second goal is to present an evolutionary analysis of these diverse energetic pathways. Taking into account not only the available data on the arsenic metabolizing enzymes and their phylogenetical relatives but also the palaeogeochemical records, we propose a crucial role of arsenite oxidation via arsenite oxidase in primordial life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A cDNA encoding a putative arsenate reductase homologue (IbArsR) was cloned from sweet potato (Ib). The deduced protein showed a high level of sequence homology (16-66%) with ArsRs from other organisms. A 3-D homology structure was created based on AtArsR (PDB code 1T3K ) from Arabidopsis thaliana. The putative active site of protein tyrosine phosphatase (HC(X)(5)R) is conserved in all reported ArsRs. IbArsR was overexpressed and purified. The monomeric nature of the enzyme was confirmed by 15% SDS-PAGE and molecular mass determination of the native enzyme via ESI Q-TOF. The IbArsR lacks arsenate reductase activity but possesses phosphatase activity. The Michaelis constant (K(M)) value for p-nitrophenyl phosphate (pNPP) was 11.11 mM. The phosphatase activity was inhibited by 0.5 mM sodium arsenate [As(V)]. The protein's half-life of deactivation at 25 °C was 6.1 min, and its inactivation rate constant K(d) was 1.1 × 10(-1) min(-1). The enzyme was active in a broad pH range from 4.0 to 11.0 with optimum activity at pH 10.0. Phosphatase would remove phosphate group from nucleic acid or dephosphorylation of other enzymes as regulation signaling.

Arsenate reductases (ArsC) are a group of enzymes that play essential roles in biological arsenic detoxification pathways by catalyzing the intracellular reduction of arsenate to arsenite, which is subsequently extruded from the cells by specific transport systems. The ArsC protein from cyanobacterium Synechocystis sp. strain PCC 6803 (SynArsC) is related to the thioredoxin-dependent ArsC family, but uses the glutathione/glutaredoxin system for arsenate reduction. Therefore, it is classified to a novel thioredoxin/glutaredoxin hybrid arsenate reductase family. Herein we report the chemical shift assignments of (1)H, (13)C and (15)N atoms for the reduced form of SynArsC, which provides a starting point for further structural analysis and elucidation of its enzymatic mechanism.

The ars gene system provides arsenic resistance to a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. Therefore, arsC gene from Bacillus cereus strain AG27 isolated from soil was amplified, cloned and sequenced. The strain exhibited a minimum inhibitory concentration of 40 and 35 mM to sodium arsenate and sodium arsenite, respectively. Homology of the sequence, when compared with available database using BLASTn search showed that 300 bp amplicons obtained possess partial arsC gene sequence which codes for arsenate reductase, an enzyme involved in the reduction of arsenate to arsenite which is then effluxed out of the cell, thereby indicating the presence of efflux mechanism of resistance in strain. The efflux mechanism was further confirmed by atomic absorption spectroscopy and scanning electron microscopy studies. Moreover, three dimensional structure of modeled arsC from Bacillus cereus strain shares significant structural similarity with arsenate reductase protein of B.subtilis, consisting of, highly similar overall fold with single α/β domain containing a central four stranded, parallel, open-twisted β-sheet flanked by α-helices on both sides. The structure harbors the arsenic binding motif AB loop or P-loop that is highly conserved in arsenate reductase family.

Experimentalists and quantum chemists are living in a different world. A wealth of theoretical enzymology-related publications is hardly known by experimentalists, and vice versa. Our aim is to bring both worlds together and to show the powerful possibilities of a multidisciplinary approach to study subtle details of complicated enzymatic processes to a broad readership. MD simulations and QM/MM approaches often focus on the calculation of reaction paths based on activation energies, which is a time-consuming task. A valuable alternative is the reactivity descriptors founded in conceptual DFT like softness, electrophilicity, and the Fukui function, which describe the kinetic aspects of a reaction in terms of the response to perturbations in N and/or upsilon(r), typical for a chemical reaction, of the reagents in the ground state. As such, the relative energies at the beginning of the reaction predict a sequence of activation energies only based on the properties of the reactants (Figure 5 ). In 2003, Geerlings et al. published a key review giving a detailed description of the principles and concepts of conceptual DFT and highlighting its success to study generalized acid/base reactions including addition, substitution, and elimination reactions. Since the time that this review appeared, conceptual DFT has proven its strength in literally hundreds of papers with application to organic and inorganic reactions. Its role in unravelling enzymatic reaction mechanisms, in handling experimentally difficult accessible biochemical problems, and in the interpretation of biochemical experimental observations is emerging and very promising.

The dissociation mechanism of the thioredoxin (Trx) mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC), was used. In this structure, a Cys29(Trx)-Cys89(ArsC) intermediate disulfide is formed by the nucleophilic attack of Cys29(Trx) on the exposed Cys82(ArsC)-Cys89(ArsC) in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32(Trx) in contact with Cys29(Trx). Cys32(Trx) is activated for its nucleophilic attack by hydrogen bonds, and Cys32(Trx) is found to be more reactive than Cys82(ArsC). Additionally, Cys32(Trx) directs its nucleophilic attack on the more susceptible Cys29(Trx) and not on Cys89(ArsC). This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.

Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.

The arsenate/antimonate reductase LmACR2 has been recently identified in the genome of Leishmania major. Besides displaying phosphatase activity in vitro, this enzyme is able to reduce both As(V) and Sb(V) to their respective trivalent forms and is involved in the activation of Pentostan, a drug containing Sb(V) used in the treatment of leishmaniasis. LmACR2 displays sequence and functional similarity with the arsenate reductase ScACR2 from Saccharomyces cerevisiae, and both proteins are homologous to the catalytic domain of Cdc25 phosphatases, which, in turn, belong to the rhodanese/Cdc25 phosphatase superfamily. In this work, the three-dimensional structure of LmACR2 has been determined with crystallographic methods and refined at 2.15 A resolution. The protein structure maintains the overall rhodanese fold, but substantial modifications are observed in secondary structure position and length. However, the conformation of the active-site loop and the position of the catalytic residue Cys75 are unchanged with respect to the Cdc25 phosphatases. From an evolutionary viewpoint, LmACR2 and the related arsenate reductases form, together with the known Cdc25 phosphatases, a well-defined subfamily of the rhodanese/Cdc25 phosphatase superfamily, characterized by a 7-amino-acid-long active-site loop that is able to selectively bind substrates containing phosphorous, arsenic, or antinomy. The evolutionary tree obtained for these proteins shows that, besides the active-site motif CE[F/Y]SXXR that characterizes Cdc25 phosphatase, the novel CALSQ[Q/V]R motif is also conserved in sequences from fungi and plants. Similar to Cdc25 phosphatase, these proteins are likely involved in cell cycle control. The active-site composition of LmACR2 (CAQSLVR) does not belong to either group, but gives to the enzyme a bifunctional activity of both phosphatase and As/Sb reductase. The subtle dependence of substrate specificity on the amino acid composition of the active-site loop displays the versatility of the ubiquitous rhodanese domain.

Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary phase, at which point it is released from the cell. In intact cells, the enzyme localizes to the periplasm. To purify ARR, a heterologous expression system was developed in Escherichia coli. ARR requires anaerobic conditions and molybdenum for activity. ARR is a heterodimer of approximately 131 kDa, composed of one ArrA subunit (approximately 95 kDa) and one ArrB subunit (approximately 27 kDa). For ARR to be functional, the two subunits must be expressed together. Elemental analysis of pure protein indicates that one Mo atom, four S atoms associated with a bis-molybdopterin guanine dinucleotide cofactor, and four to five [4Fe-4S] are present per ARR. ARR has an apparent melting temperature of 41 degrees C, a Km of 5 microM, and a Vmax of 11,111 micromol of As(V) reduced min(-1) mg of protein(-1) and shows no activity in the presence of alternative electron acceptors such as antimonite, nitrate, selenate, and sulfate. The development of a heterologous overexpression system for ARR will facilitate future structural and/or functional studies of this protein family.

Arsenic compounds commonly exist in nature and are toxic to nearly all kinds of life forms, which directed the evolution of enzymes in many organisms for arsenic detoxification. In bacteria, the thioredoxin-coupled arsenate reductase catalyzes the reduction of arsenate to arsenite by intramolecular thiol-disulfide cascade. The oxidized arsenate reductase ArsC is subsequently regenerated by thioredoxin through an intermolecular thiol-disulfide exchange process. The solution structure of the Bacillus subtilis thioredoxin-arsenate reductase complex represents the transiently formed intermediate during the intermolecular thiol-disulfide exchange reaction. A comparison of the complex structure with that of thioredoxin and arsenate reductase proteins in redox states showed substantial conformational changes coupled to the reaction process, with arsenate reductase, especially, adopting an "intermediate" conformation in the complex. Our current studies provide novel insights into understanding the reaction mechanisms of the thioredoxin-arsenate reductase pathway.