Arsenic Oxidation and Reduction of Inorganic Genes and Enzymes Involved in Bacterial

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Genes and Enzymes Involved in Bacterial Oxidation and Reduction of Inorganic Arsenic

Simon Silver and Le T. Phung Appl. Environ. Microbiol. 2005, 71(2):599. DOI: 10.1128/AEM.71.2.599-608.2005.

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MINIREVIEW

Genes and Enzymes Involved in Bacterial Oxidation and Reduction of Inorganic Arsenic Simon Silver* and Le T. Phung Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois

The human use of toxic heavy metals is here to stay. In functioning as a terminal electron acceptor for an anaerobic

addition to intentional poisoning with arsenic (40) (arsenic respiratory chain (Fig. 1B), as do other minerals, such as Fe Downloaded from levels in the hair of Napoleon Bonaparte approached 40 ppm, and Mn with some microbes. more than 1,000 times above allowable levels), medical, agri- The enzyme responsible for the respiratory oxidation of ar- cultural, and industrial uses of arsenic present major human senite, As(III), to arsenate, As(V) (Fig. 2A), has been found problems (5, 40, 41). Arsenic catastrophes are occurring today, widely in various groups of Bacteria and Archaea and has been most notably as the high arsenic levels present in drinking studied in detail (see below). Indeed, some microbes gain water in East Bengal and West Bengal, where over 40 million energy from oxidizing arsenite (43, 50, 60), although this ac- people are being exposed to more than 50 ppb of arsenic (8); tivity may be an exception limited to chemolithotrophic bacte-

this level was the limit accepted by the World Health Organi- ria; heterotrophic bacteria have not been shown to derive ma- http://aem.asm.org/ zation before that organization (and later the U.S. govern- jor energy from arsenate in growth experiments (12). ment) set the allowable limit ﬁvefold lower, at 10 ppb. Bang- Anaerobic arsenate respiration was discovered (2) with a ladesh maintains an allowable limit of 50 ppb, and the limit in bacterial isolate that coupled anaerobic heterotrophic growth Canada is 25 ppb. The arsenic found in Bengalese drinking to arsenate as the terminal electron acceptor (replacing oxygen water is present naturally in the sediment but is released only in an anaerobic respiratory process) (Fig. 1B). Since then, by humans, primarily from the digging of shallow wells. Simi- diverse bacterial types with anaerobic respiratory arsenate re- larly unacceptably high arsenic levels are found in shallow-well ductase have been discovered (42, 44, 49, 55). In fact, it might domestic drinking water in the American Midwest (7, 7a, 21). be argued that anaerobic terminal electron acceptors, such as

It seems likely that microbial metabolism is involved in mobi- arsenate, and other minerals, such as nitrate and ferric cations, on April 12, 2013 by guest lizing (18, 62) previously immobile “natural” subsurface ar- occurred in early cellular life, preceding aerobic oxygen-utiliz- senic in these areas. ing respiratory electron transport chains. Oxygen respiration Living cells (microbial or human) are generally exposed to could not occur until cyanobacterial photosynthesis resulted in arsenic as arsenate or arsenite. Arsenate, As(V), is frequently an atmosphere with molecular O2, hundreds of millions of 3Ϫ written as AsO4 , which is similar to phosphate, and has a years after the ﬁrst anaerobic bacteria arose from prebiotic 2Ϫ 1Ϫ pKa of 7.0, with HAsO4 and H2AsO4 being equally abun- forms. While recent reports (12, 17, 27, 42, 58) have described dant at pH 7.0. Although arsenate is thought to be highly the broad diversity of microbes able either to reduce arsenate soluble, in many environments with calcium or insoluble iron or to oxidize arsenite, the emphasis here is on genes that compounds, arsenate is precipitated as phosphate would be. determine these transformations and a new understanding of Ϫ Arsenite, As(III), is frequently erroneously written as AsO2 , enzyme structure. although with a pKa of 9.3, it occurs at a neutral or acidic pH as As(OH)3. Arsenite in water can be thought of as an inorganic equivalent of nonionized glycerol and is transported AEROBIC ARSENITE OXIDASE across cell membranes from bacterial cells to human cells by Bacterial oxidation of arsenite to arsenate has long been glyceroporin membrane channel proteins (36, 47). recognized (reviewed in references 11, 12, and 54), especially Nealson et al. (38) introduced the phrase “eating and with aerobic isolates from arsenic-impacted environments (14, breathing” minerals in the context of seeking evidence of in- 17, 50, 51, 56–58). Similar isolates have also been found in soils organic signatures for life on other planets (39), in contrast to and sewage not known to be exposed to elevated levels of earlier searches for life on Mars that emphasized organic car- arsenic (45, 46). It is not currently clear whether arsenite oxi- bon-containing signatures. For arsenic, “eating” means arsen- dation is limited to a few isolates in each species. For example, ite functioning as an electron donor at the start of a membrane although two Alcaligenes faecalis isolates have this activity (45, respiratory chain (Fig. 1A), and “breathing” means arsenate 46), the activity has not been examined in culture collection isolates of this species. It seems that most environmental isolates lack this potential, although a range of Bacteria with * Corresponding author. Mailing address: Department of Microbi- ology and Immunology, MC790, University of Illinois, 835 S. Wolcott arsenite oxidase enzyme activity have been isolated and genes Ave., Chicago, IL 60612-7344. Phone: (312) 996-9608. Fax: (312) 996- apparently encoding arsenite oxidase are found widely in var- 6415. E-mail: [email protected]. ious groups of Bacteria and Archaea (39a).

599 600 MINIREVIEW APPL.ENVIRON.MICROBIOL. Downloaded from http://aem.asm.org/ FIG. 1. Cellular locations and functions of bacterial respiratory arsenite oxidase, respiratory arsenate reductase, and cytoplasmic arsenate reductase.

The 71-kb DNA region encoding arsenite oxidase and asso- newly named member of the ␤-Proteobacteria isolated from ciated functions in A. faecalis strain NCIB8687 was recently industrial wastewater (60). The four identiﬁed genes were sequenced (GenBank accession number AY297781; http: named aoxABCD (for arsenite oxidase); a ﬁfth, partial gene

//www.uic.edu/depts/mcmi/faculty/silver.html; L. T. Phung et upstream of aoxA was also found (Fig. 3). Unfortunately, the on April 12, 2013 by guest al., unpublished data). This is the strain for which the enzyme current names for the A. faecalis genes and the homologous crystal structure has been obtained (13), and this is the only genes from C. arsenoxidans are different, although the asoA currently available structure for a respiratory arsenite oxidase gene product is 73% identical at the amino acid level to the or arsenate reductase. aoxB gene product (Fig. 3); similarly, the asoB and aoxA gene This review summarizes the Alcaligenes arsenic-related products are 62% identical (Fig. 3). Inactivation of the aoxA or genes in what we are calling the first “arsenic gene island.” The aoxB gene eliminated arsenite oxidase activity (37). Santini and asoA and asoB genes (Fig. 3) encode, respectively, the large vanden Hoven (51) reported the DNA (and deduced protein) molybdopterin-containing and the small Rieske (spectroscopy- sequences encoding yet another set of AsoA and AsoB identified [2Fe-2S] cluster) (52) subunits of arsenite oxidase of polypeptides (with yet a third mnemonic, Aro, instead of Aso) A. faecalis (Fig. 2). Upstream of asoB are 15 genes tentatively from chemilithoautotrophic ␣-proteobacterium strain NT-26; considered to be involved in arsenic resistance and metabolism the sequences were homologous to those of A. faecalis AsoA (2 are shown in Fig. 3, and the others are available at GenBank and AsoB (Fig. 3). The DNA sequence similarities are too accession number AY297781 and at http://www.uic.edu/depts weak to be found by standard DNA Southern blotting or de- /mcmi/faculty/silver.html); downstream of asoA are 6 genes generate primer PCR analysis. Strain NT-26 is thought to ␣ ␤ also tentatively identified as being involved in arsenic resis- produce an 2 2 heterotetramer arsenite oxidase (51) with a ␣ ␤ tance and metabolism (3 are shown in Fig. 3). These putative molecular mass approximately twice that for the 1 1 het- genes encode a total of three presumed periplasmic oxyanion erodimer (3, 13) of A. faecalis; yet another ␤-proteobacterium ␣ ␤ binding proteins that are likely to be components of two ABC strain, NT-14, appears to produce an 3 3 heterohexamer en- oxyanion ATPase membrane transport systems and an ArsAB zyme (58). Chemilithoautotrophic strain NT-26 is thought to arsenite chemiosmotic efflux system (Fig. 4). The details are derive useful energy for growth from arsenite oxidation (51), incomplete, but the overall conclusion is that arsenite oxidase although it is questionable whether obligately heterotrophic is encoded in a “gene island” of over 20 functionally related (that is, dependent on fixed carbon for energy) arsenite-oxi- genes, a major change in the understanding of cellular arsenite dizing strains derive useful energy in this way. Researchers are resistance, from a smaller operon to a larger “island” with clearly at an early stage of understanding arsenite-oxidizing multiple related phenotypes (see below). microbes. A cluster of four contiguous genes including those encoding The evolutionary trees of gene and protein sequence rela- arsenite oxidase was identified (Fig. 3) (GenBank accession tionships (Fig. 5) would not have been possible a year or two number AF509588) (37) from Centibacterium arsenoxidans,a ago. New results will undoubtedly make such trees too complex VOL. 71, 2005 MINIREVIEW 601 Downloaded from http://aem.asm.org/

FIG. 2. Models for heterodimer arsenite oxidase (data are from reference [13]) and arsenate reductase (data are from reference [22]). Funnel-shaped active sites are shown at the top. Also shown are embedded Mo-pterin and [Fe-S] cofactors, proposed two-electron (2 eϪ) transfer pathways, and amino acids (Cys or His) linking the [Fe-S] cofactors to the polypeptides. HIPIP, high-potential iron protein. for presentation within a year or two. Two conclusions relevant of sequences precludes the use of gene-speciﬁc universal to environmental microbiology can be drawn at this time: asoA probes or primers for identifying these genes in new isolates. on April 12, 2013 by guest and asoB genes are being found broadly in a wide range of Upstream from asoAB and divergently oriented is a gene prokaryotes (see text above and Fig. 5); however, the diversity that encodes a presumed periplasmic oxyanion binding protein

FIG. 3. Genes for the arsenite oxidase region in A. faecalis (NCBI accession number AY297781; http://www.uic.edu/depts/mcmi/faculty /silver.html), a Sargasso Sea meta-genome environmental isolate (NCBI AACY01082423) (59), C. arsenoxidans (NCBI AF509588) (37), and chemolithoautotrophic strain NT-26 (NCBI AY345225) (51) and upstream and downstream genes. Presumed gene product lengths (in amino acids [aa]) and functions are indicated, as are percent amino acid identities between homologous products and those of A. faecalis. 602 MINIREVIEW APPL.ENVIRON.MICROBIOL. Downloaded from http://aem.asm.org/

FIG. 4. Proposed inorganic arsenic metabolism in A. faecalis, predicted from linked genes in the sequence of GenBank accession number AY297781 (http://www.uic.edu/depts/mcmi/faculty/silver.html). Heterodimeric arsenite oxidase is coupled to the aerobic respiratory chain by a small protein (perhaps azurin or cytochrome c551), whose gene is not in the gene cluster. Two predicted oxyanion ABC ATPases with periplasmic oxyanion binding proteins are indicated, along with their predicted roles, as are an intracellular glutathione-linked ArsC-class arsenate reductase and the ArsAB arsenite efﬂux complex.

and that is homologous to a partial gene (open reading frame arsenite oxidase (Fig. 5). The genes upstream and downstream on April 12, 2013 by guest 253) that occupies the first 759 bp of the sequence of GenBank of the C. aurantiacus putative arsenite oxidase genes are not accession number AF509588 (Fig. 3). Downstream from related to those in this region of the A. faecalis or C. arsenoxi- asoBA and aoxAB, the A. faecalis and C. arsenoxidans se- dans chromosome. Figure 5 contains current trees for AsoA quences are unrelated, both at the DNA level and the protein (presumed orthologs are shown above the broken line, and level (Fig. 3). The A. faecalis chromosome (GenBank accession paralogs are shown below) and AsoB Rieske subunit se- number AY297781; http://www.uic.edu/depts/mcmi/faculty/silver quences. .html; Phung et al., unpublished) contains an moaA gene ho- In addition to bacterial sequences, reasonable candidate molog thought to be involved in molybdopterin biosynthesis and genes for arsenite oxidase have been identified in the genomes a gene, designated phnD, encoding a second periplasmic oxyanion of two hyperthermophilic Archaea, Aeropyrum pernix (Gen- binding protein; the C. arsenoxidans chromosome contains genes Bank accession number NC_000854) and Sulfolobus tokodaii encoding a putative oxyanion reductase and cytochrome c (aoxC (GenBank accession number NC_003106) (Fig. 5). These gene and aoxD, respectively, in Fig. 3) (37). pairs are contiguous and are in the same order as asoA and A partial gene sequence for the large molybdopterin subunit asoB in Fig. 3. of arsenite oxidase from an additional ␤-proteobacterial iso- The evolutionary relationship to other protein sequences in late, a Thiomonas sp., has been deposited in GenBank (acces- the dimethyl sulfoxide (DMSO) reductase family of microbial sion number AJ510263) (V. Bonnefoy, personal communica- molybdopterin enzymes (29) is shown in Fig. 5A. Sequences tion) and is shown in Fig. 5A. In addition to the four sequences above the broken line are presumed to be for the molybdop- that have been recognized as representing arsenite oxidase, the terin subunit of arsenite oxidase, while those below the broken “meta-genome” of numerous chromosomal fragments from line are for other members of the DMSO reductase family, microbial DNA isolated from the Sargasso Sea (59) contains a including the distantly related respiratory arsenate reductase 6.5-kb sequence (NCBI accession number AACY01082423) ArrA. The tree for candidate small Rieske subunits in Fig. 5B with strong candidate genes encoding products homologous to also includes presumed orthologs for other arsenite oxidases AsoA (NCBI accession number EAI76965) and AsoB (NCBI and other paralogous Rieske subunit sequences. accession number EAI76963) (Fig. 3 and 5). The draft genome A comparison of the AsoB small Rieske subunit amino acid of Chloroflexus aurantiacus, a green filamentous anoxygenic sequences predicted from gene sequences (GenBank accession photosynthetic bacterium (Joint Genome Institute contig number AY297781; http://www.uic.edu/depts/mcmi/faculty NZ_AAAH01000321), includes genes Chlo2048 and Chlo2049, /silver.html; Phung et al., unpublished) and X-ray crystallogra- the products of which appear to be subunits of still another phy electron density maps (Protein Data Base deposits GI VOL. 71, 2005 MINIREVIEW 603 Downloaded from http://aem.asm.org/

FIG. 5. Phylogenetic trees of protein sequences for the large Mo-pterin subunit (A) and the small Rieske subunit (B) of arsenite oxidase (presumed orthologs) and selected parologous homologs. NCBI accession numbers are as follows: (A) C. arsenoxidans AoxB, gi 22758844; A. faecalis AsoA, gi 33469597; Thiomonas sp. strain VB-2002 AoxB, gi 23821270; C. aurantiacus Chlo2048, gi 22972154; Sulfolobus tokodaii ST2391, gi 15922722; A. pernix APE2556, gi 14602144; Pseudomonas syringae pv. syringae B728a Psyr020491, gi 23468844; Methanococcus jannaschii FdhF, gi 15669895; Wolinella succinogenes DSMZ 1740 WS0764, gi 34482881; D. hafniense Desu0744, gi 23112121; B. selenitireducens MLS10 ArrA, gi 33466104; Shewanella sp. strain ANA-3 ArrA, gi 33286384; Rhodobacter capsulatus DorA, gi 2981245; Sargasso Sea environmental sequence, gi 44367188; and arsenite-oxidizing ␣-proteobacterium strain NT-26 AroA, gi 37962697; (B) C. arsenoxidans AoxA, gi 22758843; A. faecalis AsoB, gi 33469598; C. aurantiacus Chlo2049, gi 22972155; S. tokodaii ST2392, gi 15922723; A. pernix APE2563, gi 14602146; Aquifex aeolicus VF5 SoxF, gi 2982941; Thermoplasma acidophilum SoxL, gi 10640539; Sulfolobus solfataricus SoxF, gi 13816354; S. tokodaii ST0108, gi 15920287; Sargasso Sea on April 12, 2013 by guest environmental sequence, gi 44367187; and arsenite-oxidizing ␣-proteobacterium strain NT-26 AroB, gi 37962696.

1208496 to GI 1208506) (4, 13) shows a surprising 9% differ- (data not shown). Amino acid sequences predicted from elec- ence (12 of 133 amino acid positions) (Fig. 6). All differences tron density maps are rarely deposited today, since DNA se- are expected to be mistakes in prediction in Protein Data Base quences are generally available prior to the solution of the deposits, as was the case for the 9% difference (78 of 825 protein structure and primary amino acid sequences predicted positions) between the sequences predicted for the molybdop- from DNA sequences are generally consistent with those pre- terin subunit from the asoA gene and the electron density map dicted from electron density maps and accepted.

FIG. 6. Comparison of the amino acid sequences of the AsoB Rieske ([2Fe-2S]) subunit predicted from the DNA sequence translation and from the crystal electron density map. Asterisks indicate identical amino acids; triangles and diamonds indicate, respectively, the cysteine and histidine residues that anchor the [2Fe-2S] center. The TAT leader sequence, with its conserved twin arginines, is indicated, along with the predicted cleavage site. 604 MINIREVIEW APPL.ENVIRON.MICROBIOL.

The differences are not random, and of the 12 discordant center. The Rieske subunit consists of a single domain with the calls in Fig. 6, 3 are lysines at the protein surface that are listed [2Fe-2S] cluster coordinated by C68, H70, C86, and H89 (in- as alanine or serine in the crystal sequence, presumably be- dicated in Fig. 6 with numbering from the unprocessed se- cause the long lysine side chain is mobile in the crystal, and 6 quence). are secondary amines (asparagine or glutamine) that are listed Two guanosine dinucleotide pterin cofactors coordinate as the corresponding dicarboxylic acid or alanine. This pattern each molybdenum center, with one oriented upward and the of missing lysine side chains and secondary amines also dom- other oriented downward in the structure (13), as expected for inated the discordant calls between the DNA translation and bacterial molybdopterin proteins. Unlike most other members the electron density maps for the large molybdopterin subunit of the DMSO family of molybdopterin iron-sulfur oxidoreduc- (data not shown). tases, for which the Mo is anchored with pterin sulfurs and a The crystal structure for the Rieske subunit of arsenite ox- polypeptide serine hydroxyl or cysteine sulfhydryl, the fifth idase starts with R43 (Fig. 6) and lacks eight N-terminal amino position of arsenite oxidase is occupied by double-bonded ox- acids expected to be present in the processed protein. G. L. ygen, and the corresponding amino acid is A199. Extended Anderson (personal communication) obtained the KAPADA X-ray absorption fine-structure spectra and resonance Raman hexapeptide sequence in Fig. 6 by direct N-terminal sequenc- spectroscopy of the active-site molybdenum of arsenite oxidase ing. Q35, which is predicted to follow the twin arginine trans- (9) show the four Mo-S interactions and a single Mo double Downloaded from location (TAT) protease cleavage site (6), was not found. The bonded to O in reduced arsenite oxidase; an additional MoOO N-terminal methionine is present in the 826-amino-acid-long single bond has been found in the oxidized enzyme. Additional AsoA molybdopterin subunit sequence from the translated electrochemical studies (16) have established several key prop- DNA, but not in the crystal structure (electron density) map. erties of arsenite oxidase. Electron spin resonance measure- Direct experiments are needed to test hypotheses from DNA ments have demonstrated the movement of electrons from the sequence-based protein similarity searches. [3Fe-4S] center of the large subunit to the Rieske [2Fe-2S] The alignments of polypeptide gene product sequences and center of the small subunit (3, 13). the phylogenetic trees showing relationships (23) indicate that The molybdopterin center occurs at the bottom of a shallow http://aem.asm.org/ functionally related arsenite oxidases in both Bacteria and Ar- funnel-shaped cavity formed on the subunit surface by domains chaea start with the determinant for a TAT leader sequence (6, I, II, and III, providing solvent access for entry of the arsenite 15, 23, 29) at the N terminus of each Rieske subunit gene. The substrate and exit of the arsenate product (4, 13). The active- first 34 amino acids of the AsoB Rieske subunit sequence (Fig. site surface in the crystal structure contains highly polar amino 6) form the canonical TAT leader sequence, including the acid side groups, with H195, E203, R419, and H423 of the large RRGFLK hexapeptide sequence, which is highly conserved subunit being considered to form the arsenite binding site (4). and followed by about 20 hydrophobic amino acids before the Chemical modification of one histidine residue, perhaps H195

predicted protease cleavage site. The main characteristic for a or H423, by diethylpyrocarbonate inactivates the enzyme ac- on April 12, 2013 by guest TAT-transported periplasmic enzyme such as arsenite oxidase tivity (30); hydroxylamine restores the activity. The predicted is that the nascent polypeptide is folded into its tertiary struc- binding of arsenite places it adjacent to the molybdenum center, ture in the cytoplasm, rather than transported in an unfolded at a distance suitable for nucleophilic attack of Mo(VI)AOby form, as in the sec-dependent pathway (as happens for perhaps an arsenite electron pair, a two-electron reaction (Fig. 2A) (4, 90% of exported proteins in a bacterium such as Escherichia 13). coli) (6, 15). Cofactors such as the molybdopterin complex and After the reduction of molybdenum from Mo(VI) to the [Fe-S] clusters are incorporated within the relatively an- Mo(IV) (Fig. 2A), the electrons are transferred to the [3Fe-4S] aerobic environment of the cytoplasm. Then the preassembled center of the same subunit, then to the [2Fe-2S] center of the protein is translocated to the periplasm by utilizing the TAT Rieske subunit, and then to the ﬁrst coupling protein of the leader sequence, which can be on either subunit of the het- aerobic respiratory chain, possibly azurin or cytochrome c (3, 4, erodimer, apparently on the AsoB small subunit (Fig. 2A) of 13). Within the crystal structure, the [3Fe-4S] cluster is 12 Å arsenite oxidase or on the ArrA large molybdopterin subunit distant from the molybdenum and requires an electron path- of respiratory arsenate reductase (49) (Fig. 2B). After trans- way involving intermediate amino acid residues and/or posi- port, the TAT leader sequence is cleaved by a signal protease tions in the pterin (13). At present, intraprotein electron path- (6). ways are only proposals and require experimental testing. Determination of the protein crystal structure of arsenite Electron transport from the [3Fe-4S] cluster to the [2Fe-2S] oxidase (13) followed extensive analytical and functional stud- cluster occurs over a comparable distance and must involve H ies (3, 4). The molybdopterin- and [Fe-S] cluster-containing bonds with amino acids and/or water molecules. There is pre- enzyme is thought to reside in the periplasmic space associated cedent for such intra- and intermolecular electron transfer with the outer surface of the cytoplasmic membrane and to be pathways with better-studied enzymes, such as nitrogenase (4, associated with the aerobic respiratory chain via an azurin or a 29). c-type cytochrome (Fig. 4) (3, 54). Both [3Fe-4S] and [2Fe-2S] Rieske-type iron-sulfur centers have been found, on different RESPIRATORY ARSENATE REDUCTASE subunits (Fig. 2) (3, 13). Four domains are recognized in the structure of the large arsenite oxidase subunit (13), with the Starting with the discovery of an anaerobic bacterial isolate ﬁrst domain containing the [3Fe-4S] cluster, coordinated to the able to use arsenate as a terminal electron acceptor for a protein by C21, C24, and C28 (Fig. 2). S99 occupies the posi- heterotrophic respiratory chain (2), many diverse bacteria with tion that might anchor the fourth Fe if this were a [4Fe-4S] this potential have been isolated (26, 27, 27a, 42, 44, 55). VOL. 71, 2005 MINIREVIEW 605

FIG. 7. Genes for respiratory (arrAB) and cytoplasmic (arsC) arsenate reductases of Shewanella strain ANA-3 (from GenBank accession number AY271310) (48, 49). Upstream and divergently oriented from the arrA and arrB genes for respiratory arsenate reductase is a four-gene canonical ars operon with the arsC gene for cytoplasmic arsenate reductase. aa, amino acid. Downloaded from

However, no protein structures are available, and the DNA Shewanella appears (from its sequence) to be a member of the sequences of the genes (arr) involved have only recently ap- cytoplasmic glutathione-glutaredoxin-dependent arsenate re- peared (1, 49). ductase clade (36). The arsB and arsA genes of Shewanella The most detailed report of the puriﬁcation and character- probably determine the membrane carrier and ATPase com- ization of the respiratory arsenate reductase enzyme (22) (Fig. ponents of an arsenite efﬂux pump (48), like that shown in Fig. 2B) showed that anaerobic respiratory arsenate reductase, like 4 for the Alcaligenes strain, that removes arsenite from the arsenite oxidase, is a heterodimer periplasmic or membrane- cytoplasm. The Shewanella ars operon confers arsenite resis- http://aem.asm.org/ associated protein consisting of a larger molybdopterin subunit tance when transferred to a different Shewanella strain or to E. (ArrA) which contains an iron-sulfur center, perhaps a high- coli. A mutation disrupting arsB in Shewanella led to arsenite potential [4Fe-4S] cluster, and a smaller [Fe-S] center protein sensitivity, although the arsB mutant strain could still respire (ArrB) that is not homologous to the Rieske polypeptide of arsenate to arsenite anaerobically (48). arsenite oxidase. The smaller ArrB subunit is approximately Upstream and in an orientation opposite that of the ars twice the size of the AsoB subunit of arsenite oxidase (Fig. 2) operon of Shewanella is a two-gene operon determining the (1, 22, 49) and may contain up to four [4Fe-4S] clusters (27). subunits of the periplasmic heterodimer respiratory arsenate

The respiratory arsenate reductase of gram-positive Bacillus reductase (Fig. 2 and 7) (49). The predicted ArrA sequence for on April 12, 2013 by guest differs from that of gram-negative bacteria in that it is an- the large molybdopterin-containing subunit starts with a 42- chored to the membrane of the gram-positive cell (1), which amino-acid TAT leader sequence. Deletions in either arrA or lacks a periplasmic compartment. There may also be anaerobic arrB result in a loss of the ability to grow on arsenate and to respiratory arsenate reductases that are unrelated in structure reduce arsenate anaerobically (ArsC reduces arsenate aerobi- and based on chemistry other than molybdopterin and [Fe-S] cally). Anaerobic growth on arsenate is restored by comple- centers (27). This scenario remains to be shown. mentation by functional genes. The ArrA sequence includes a

Recently, the genetic determinants of two respiratory arsen- CX2CX3CX27C motif predicted to anchor a [4Fe-4S] cluster. ate reductase systems became available; one is from ␥-pro- The shorter, 234-amino-acid ArrB sequence contains four teobacterium gram-negative Shewanella strain ANA-3 (NCBI four-cysteine motifs as candidates for binding [Fe-S] centers accession number AY271310) (48, 49), and the other is from and no TAT leader sequence. The phylogenetic tree of ArrA gram-positive Bacillus selenitireducens (NCBI accession num- and related sequences (Fig. 5A) indicates that ArrA is distantly ber AAQ19491) (1). About 11 kb of DNA from the respiratory related to AsoA in the DMSO oxyreductase family of molyb- arsenate reductase region of Shewanella strain ANA-3 was dopterin- and [Fe-S] cluster-containing enzymes (27a). The sequenced. The DNA sequence consists of two divergently available genome sequence of Desulfitobacteria hafniense con- transcribed operons (Fig. 7) (48, 49) followed by a predicted tains genes that encode the closest currently available ho- transcriptional termination signal and two unrelated genes. mologs of ArrA and ArrB (AY271310) (49). ArrB appears to Upstream of arrA and arrB, encoding the two subunits of re- be an iron-sulfur protein related to DmsB of DMSO reductase spiratory arsenate reductase, is a standard arsDABC operon and NrfC of nitrite reductase. for arsenate and arsenite resistance (Fig. 7) (48). The She- wanella and Bacillus ArrA sequences for the large molybdop- CYTOPLASMIC ARSENATE REDUCTASE FOR terin subunit are 47% identical (Fig. 5A), consistent with or- INTERCELLULAR DEFENSE thologous proteins from gram-positive and gram-negative ArsC cytoplasmic arsenate reductase (Fig. 1C) is found bacteria (see also reference 27a). The transcriptional regula- widely in microbes, and the arsC gene occurs in ars operons in tory gene, arsR, is absent in the Shewanella sequence and is most bacteria with total genomes measuring 2 Mb or larger as predicted to be present elsewhere on the chromosome; this well as in some archaeal genomes. It can be argued that ars situation is unusual, as arsR generally occurs at the beginning operons for arsenic resistance are found more widely in mi- of the operon, upstream of arsD, the determinant of a minor crobes than, for example, trp operons for tryptophan biosyn- secondary regulatory protein (53). The arsC gene product of thesis. The literature on ArsC enzymes has been repeatedly 606 MINIREVIEW APPL.ENVIRON.MICROBIOL. reviewed (e.g., references 36, 53, and 54); therefore, the avail- flight. The third clade of cytoplasmic arsenate reductases has able and newer understanding will be briefly summarized. arsC been found so far only in fungi (36). is almost always found next to the arsB gene for the arsenite membrane pump (as shown in Fig. 7). Surprisingly, three un- FUTURE AND BIOTECHNOLOGY POTENTIALS FOR related clades (trees) of ArsC sequences are currently recog- PRACTICAL USE nized, and these share a common biochemical function but have no evolutionary relationship (36). These three are (i) a Television and newspaper reports related to microbial inor- glutaredoxin-glutathione-coupled enzyme, like that found as- ganic arsenic transformation occur frequently; for example, a sociated with both the arsenite oxidase of Alcaligenes (NCBI recent advertisement promised “arsenic-free lumber” for re- accession number AY297781) and the respiratory arsenate re- placement of home decks, but the U.S. Environmental Protec- ductase of Shewanella (Fig. 7), as well as many plasmids and tion Agency shortly thereafter decided against a requirement chromosomes of gram-negative bacteria; (ii) a less-well-de- to use such lumber. The problems of drinking water containing fined glutaredoxin-dependent arsenate reductase found in high arsenic levels in Bangladesh are reported regularly in yeasts; and (iii) a group of thioredoxin-coupled arsenate re- newspapers and magazines (8) and have their own mailing list ductases found initially in gram-positive bacteria but more ([email protected]) and sites (see, e.g., http:bicn recently also in many gram-negative proteobacteria (28, 31–35, .com/acid/and http: groups.yahoo.com/group/arsenic-crisis- Downloaded from 53, 54). Thioredoxin-coupled arsenate reductase cannot use news/). glutaredoxin, and glutaredoxin-coupled arsenate reductase Among the many websites related to arsenic in drinking cannot use thioredoxin. water and public health and environmental problems, readers ArsC arsenate reductase is a small monomeric protein of may wish to access http://www.who.int/inf-fs/en/fact210.html about 135 amino acid residues and containing three essential and http://www.nlm.nih.gov/medlineplus/arsenic.html. cysteine residues that are involved in a cascade sequence of Microbial metabolism undoubtedly exacerbates environ- enzyme activity. There are no inorganic or other bound cofac- mental arsenic problems (14, 17, 18, 41), perhaps by releasing tors in the ArsC enzyme. In the glutaredoxin-glutathione-cou- arsenic into drinking water in shallow wells (as in East Bengal, http://aem.asm.org/ pled ArsC reductases, the first cysteine residue is located at West Bengal, the American Midwest, and the Canadian Mar- about position 11 from the N terminus of ArsC, but the other itime Provinces). Understanding the mechanisms may help two catalytic cysteines are provided by glutathione and glutare- minimize the impact. It is proposed that microbial anaerobic doxin rather than the ArsC polypeptide. After arsenate asso- respiratory arsenate reductase releases previously immobilized ciates with C11 (E. coli plasmid R773 numbering), forming an underground As(V) into water in newly drilled wells (18). It is As-S covalent bond, glutathione provides a second cysteine possible that microbial metabolism (arsenite oxidase coupled that forms an oxidized Cys-S-S-Cys pair with the ArsC cys- with precipitation in mineral deposits) (18) can be harnessed for practical bioremediation of drinking water arsenic, al- teine; and that disulfide is reduced by a cysteine on glutare- on April 12, 2013 by guest doxin to form a cascade of reduced cysteines to oxidized cys- though this prospect is just beginning to be recognized and no teines (28, 36). sustained efforts in this direction have been made. Microbial The first recognized arsenate reductase was found on a batch reactors to remove arsenic by oxidation of As(III) to gram-positive Staphylococcus plasmid (19, 20). From protein As(V) (25) and the use of bacterial arsenate reductase genes in crystallography, enzymology, and mutational studies (31–35, transgenic plants for potential phytoremediation by intracellu- 61), it is known that the thioredoxin-coupled ArsC arsenate lar sequestration after reduction from As(V) to As(III) (10) reductases utilize three cysteines, all in the ArsC polypeptide were reported recently. primary sequence, again for a cascade of oxidizing and reduc- A bacterial arsC gene for cytoplasmic arsenate reductase ing cysteine residues, with thioredoxin reducing the final and a gene whose enzyme product leads to the overproduction Cys82-S-S-Cys89 oxidized bond. Arsenate covalently bound to of glutathione in the plant model Arabidopsis were tested for the N-terminal Cys10 residue of Staphylococcus is reduced and potential phytoremediation (10). Synthesized alone in Arabi- released as the first internal Cys10-S-S-Cys82 cysteine is dopsis, the bacterial arsenate reductase led to hypersensitivity formed. The thioredoxin-coupled clade of arsenate reductases to arsenate. However, when arsenate reductase was present is found widely among plasmids and genomes of gram-positive together with an increased level of glutathione, greater arsen- bacteria as well as in some gram-negative bacteria (36). The ate resistance, along with the hyperaccumulation of an arsenite Pseudomonas aeruginosa genome, for example, has separate adduct of glutathione, was found (10). It was suggested that the genes for glutaredoxin- and thioredoxin-coupled ArsC reduc- arsenite adduct of glutathione is transported into the vacuole tases, while that for cyanobacteria appears to be an unusual compartment of the plant cell, as is known to happen in yeast hybrid with strong sequence similarity to thioredoxin-depen- cells. This compartmentalization effectively removes the ar- dent reductase but functioning with glutaredoxin and glutathi- senic from the plant cytoplasm and places it in a harmless one instead (24). The cyanobacterial arsenate reductase also subcellular location. Further work is needed to apply these occurs as a homodimer (24), different from other known bac- genetically modified plants to the removal of arsenic from terial enzymes but similar to the yeast enzyme (36). The glu- polluted soils. taredoxin-glutathione-coupled and thioredoxin-coupled ArsC arsenate reductases represent convergent evolution, in which a similar chemical solution has been “invented” more than once, ACKNOWLEDGMENTS analogous to the wings of birds and insects, which have no This research and preparation of this article were supported by evolutionary relationship but which both allow for animal Department of Energy grant ER20056. VOL. 71, 2005 MINIREVIEW 607

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