The rhodanese/Cdc25 phosphatase superfamily. Sequence-structure-function relations

Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. They are found as tandem repeats, with the C-terminal domain hosting the properly structured active-site Cys residue, as single domain proteins or in combination with distinct protein domains. An increasing number of reports indicate that rhodanese modules are versatile sulfur carriers that have adapted their function to fulfill the need for reactive sulfane sulfur in distinct metabolic and regulatory pathways. Recent investigations have shown that rhodanese domains are also structurally related to the catalytic subunit of Cdc25 phosphatase enzymes and that the two enzyme families are likely to share a common evolutionary origin. In this review, the rhodanese/Cdc25 phosphatase superfamily is analyzed. Although the identification of their biological substrates has thus far proven elusive, the emerging picture points to a role for the amino-acid composition of the active-site loop in substrate recognition/specificity. Furthermore, the frequently observed association of catalytically inactive rhodanese modules with other protein domains suggests a distinct regulatory role for these inactive domains, possibly in connection with signaling.     

Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates

Recent investigations have shown that the rhodanese domains, ubiquitous structural modules which might represent an example of conserved structures with possible functional diversity, are structurally related to the catalytic subunit of Cdc25 phosphatase enzymes. The major difference characterizing the active-site of the Azotobacter vinelandii rhodanese RhdA, with respect to the closely related Cdc25s (A, B, C), is that in Cdc25 phosphatases the active site loop [His-Cys-(X)5-Arg] is one residue longer than in RhdA [His-Cys-(X)4-Arg]. According to the hypothesis that the length of the RhdA active-site loop should play a key role in substrate recognition and catalytic activity, RhdA scaffold was the starting point for producing mutants with single-residue insertion to generate the catalytic loop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analyses of the catalytic performances of the engineered RhdAs revealed that elongation of the catalytic loop definitely compromised the ability to catalyze sulfur transfer reactions, while it generated 'phosphatase' enzymes able to interact productively with the artificial substrate 3-O-methylfluorescein phosphate. Although this study is restricted to an example of rhodanese modules (RhdA), it provided experimental evidence of the hypothesis that a specific mutational event (a single-residue insertion or deletion in the active-site loop) could change the selectivity from sulfur- to phosphate-containing substrates (or vice versa).     

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.     

A novel arsenate reductase from the arsenic hyperaccumulating fern Pteris vittata

Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an arsenate reductase gene (PvACR2) from gametophytes that can suppress the arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and arsenate reductases have a conserved HCX5R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of arsenate, while total arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process.     

Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction

Cdc25 phosphatases belong to the family of protein tyrosine phosphatases (PTPs) that contain an active-site cysteine and form a phosphocysteine intermediate. Recently, oxidation/reduction of active-site cysteines of PTPs, including Cdc25, has been proposed to serve as a form of reversible regulation for this class of enzymes. Here we provide in vitro evidence that supports the chemical and kinetic competence for oxidation/reduction of the active-site cysteines of Cdc25B and Cdc25C as a mechanism of regulation. Using kinetic measurements and mass spectrometry, we have found that the active-site cysteines of the Cdc25's are highly susceptible to oxidation. The rate of thiolate conversion to the sulfenic acid by hydrogen peroxide for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. If not for the presence of an adjacent (back-door) cysteine in proximity to the active-site cysteine in the Cdc25's, the sulfenic acid would rapidly oxidize further to the irreversibly inactivated sulfinic acid, as determined by using kinetic partitioning and mass spectrometry with mutants of these back-door cysteines. Thus, the active-site cysteine is protected by rapid intramolecular disulfide formation with the back-door cysteines in the wild-type enzymes. These intramolecular disulfides can then be rapidly and effectively rereduced by thioredoxin/thioredoxin reductase but not glutathione. Thus, the chemistry and kinetics of the active-site cysteines of the Cdc25's support a physiological role for reversible redox-mediated regulation of the Cdc25's, important regulators of the eukaryotic cell cycle.     

Escherichia coli GlpE is a prototype sulfurtransferase for the single-domain rhodanese homology superfamily.

BACKGROUND: Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. RESULTS: Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. CONCLUSIONS: Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.     

The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights the evolutionary relationship between the rhodanese and phosphatase enzyme families

Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a rhodanese. The crystal structure of the A. vinelandii rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.     

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii Rhodanese

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.     

Directed evolution of a yeast arsenate reductase into a protein-tyrosine phosphatase

Arsenic, which is ubiquitous in the environment and comes from both geochemical and anthropogenic sources, has become a worldwide public health problem. Every organism studied has intrinsic or acquired mechanisms for arsenic detoxification. In Saccharomyces cerevisiae arsenate is detoxified by Acr2p, an arsenate reductase. Acr2p is not a phosphatase but is a homologue of CDC25 phosphatases. It has the HCX5R phosphatase motif but not the glycine-rich phosphate binding motif (GXGXXG) that is found in protein-tyrosine phosphatases. Here we show that creation of a phosphate binding motif through the introduction of glycines at positions 79, 81, and 84 in Acr2p resulted in a gain of phosphotyrosine phosphatase activity and a loss of arsenate reductase activity. Arsenate likely achieved geochemical abundance only after the atmosphere became oxidizing, creating pressure for the evolution of an arsenate reductase from a protein-tyrosine phosphatase. The ease by which an arsenate reductase can be converted into a protein-tyrosine phosphatase supports this hypothesis.     

Response to Arsenate Treatment in Schizosaccharomyces pombe and the Role of Its Arsenate Reductase Activity

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.     

Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases.

The reaction mechanism of protein tyrosine phosphatases (PTPases) and dual-specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site-directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site-directed mutagenesis. The D383N mutant enzyme exhibits a 150-fold reduction in kcat, with Kw only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two-domain structure for Cdc25, with a regulatory N-terminal and a catalytic C-terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three-dimensional model for the core region of Cdc25. By comparing this three-dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.     

Catalytic mechanism of Cdc25

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly homologous in sequence or structure to other dual-specificity phosphatases, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. Like other dual-specificity phosphatases, Cdc25 contains an active site cysteine whose pK(a) of 5.9 can be measured in pH-dependent kinetics using both small molecule and protein substrates such as Cdk2-pTpY/CycA. We have previously shown that the catalytic acid expected in phosphatases of this family and apparent in kinetics with the natural protein substrate does not appear to lie within the known structure of Cdc25 [Chen, W., et al. (2000) Biochemistry 39, 10781]. Here we provide experimental evidence for a novel mechanism wherein Cdc25 uses as its substrate a monoprotonated phosphate in contrast to the more typical bisanionic phosphate. Our pH-dependent studies, including one-turnover kinetics, solvent kinetic isotope effects, equilibrium perturbation, substrate depletion, and viscosity measurements, show that the monoprotonated phosphate of the protein substrate Cdk2-pTpY/CycA provides the critical proton to the leaving group. Additionally, we provide evidence that Glu474 on the Cdc25 enzyme serves an important role as a base in the transfer of the proton from the phosphate to the leaving group. Because of its greater intrinsic reactivity, the use of a monoprotonated phosphate as a phosphatase substrate is a chemically attractive solution and suggests the possibility of designing inhibitors specific for the Cdc25 dual-specificity phosphatase, an important anticancer target.     

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.     

Facile synthesis and biological evaluation of a cell-permeable probe to detect redox-regulated proteins

We have developed an improved synthesis for the cell-permeable, sulfenic acid probe DAz-1. Using DAz-1, we detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. In addition, we show that DAz-1 has superior potency in cells compared to a biotinylated derivative. Collectively, these findings set the stage for the development of activity-based inhibitors of Cdc25 cell-cycle phosphatases, which are sensitive to the redox state of the active-site cysteine and demonstrate the advantage of bioorthogonal conjugation methods to detect protein sulfenic acids in cells.     

A new member of the alkaline phosphatase superfamily with a formylglycine nucleophile: structural and kinetic characterisation of a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum

The alkaline phosphatase superfamily comprises a large number of hydrolytic metalloenzymes such as phosphatases and sulfatases. We have characterised a new member of this superfamily, a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum (RlPMH) both structurally and kinetically. The 1.42 Å crystal structure shows structural homology to arylsulfatases with conservation of the core α/β-fold, the mononuclear active site and most of the active-site residues. Sulfatases use a unique formylglycine nucleophile, formed by posttranslational modification of a cysteine/serine embedded in a signature sequence (C/S)XPXR. We provide mass spectrometric and mutational evidence that RlPMH is the first non-sulfatase enzyme shown to use a formylglycine as the catalytic nucleophile. RlPMH hydrolyses phosphonate monoesters and phosphate diesters with similar efficiency. Burst kinetics suggest that substrate hydrolysis proceeds via a double-displacement mechanism. Kinetic characterisation of active-site mutations establishes the catalytic contributions of individual residues. A mechanism for substrate hydrolysis is proposed on the basis of the kinetic data and structural comparisons with E. coli alkaline phosphatase and Pseudomonas aeruginosa arylsulfatase. RlPMH represents a further example of conservation of the overall structure and mechanism within the alkaline phosphatase superfamily.     

The phosphatase C(X)5R motif is required for catalytic activity of the Saccharomyces cerevisiae Acr2p arsenate reductase.

Acr2p detoxifies arsenate by reduction to arsenite in Saccharomyces cerevisiae. This reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. Acr2p has a HC(X)(5)R motif, the signature sequence of the phosphate binding loop of the dual-specific and protein-tyrosine phosphatase family. In Acr2p these are residues His-75, Cys-76, and Arg-82, respectively. Acr2p has another sequence, (118)HCR, that is absent in phosphatases. Acr2p also has a third cysteine residue at position 106. Each of these cysteine residues was changed individually to serine residues, whereas the histidine and arginine residues were altered to alanines. Cells of Escherichia coli heterologously expressing the majority of the mutant ACR2 genes retained wild type resistance to arsenate, and the purified altered Acr2p proteins exhibited normal enzymatic properties. In contrast, cells expressing either the C76S or R82A mutations lost resistance to arsenate, and the purified proteins were inactive. These results suggest that Acr2p utilizes a phosphatase-like Cys(X)(5)Arg motif as the catalytic center to reduce arsenate to arsenite.     

A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25

The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry.     

Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation

The Cdc14 family of phosphatases specifically reverses proline-directed phosphorylation events. In Saccharomyces cerevisiae, Cdc14p promotes Cdk1p inactivation at mitotic exit by reversing Cdk1p-dependent phosphorylations. Cdk1p is a proline-directed kinase whose activity is required in all eukaryotes for the transit into mitosis. At mitotic commitment, Cdk1p participates in its own regulation by activating the mitotic inducing phosphatase, Cdc25p, and inhibiting the opposing kinase, Wee1p. We have investigated the ability of Schizosaccharomyces pombe Clp1p, a Cdc14p homolog, to disrupt this auto-amplification loop. We show here that Clp1p is required to dephosphorylate, destabilize, and inactivate Cdc25p at the end of mitosis. Clp1p promotes recognition of Cdc25p by the anaphase-promoting complex/cyclosome, an E3 ubiquitin ligase. Failure to inactivate and destabilize Cdc25p in late mitosis delays progression through anaphase, interferes with septation initiation network signaling, and additionally advances the commitment to mitotic entry in the next cycle. This may be a widely conserved mechanism whereby Cdc14 proteins contribute to Cdk1p inactivation.     

Regulation of Cdc25C Activity During the Meiotic G2/M Transition

The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G2 arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.     

An arsenate reductase from Synechocystis sp. strain PCC 6803 exhibits a novel combination of catalytic characteristics

The deduced protein product of open reading frame slr0946 from Synechocystis sp. strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecular-weight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase. The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (V(max) = 3.1 micro mol/min. mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (V(max) = 0.08 micro mol/min. mg) indicative of its phosphohydrolytic ancestry. pI258 ArsC from S. aureus is the prototype of one of three distinct families of detoxifying arsenate reductases. The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate. While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features. rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S. aureus enzyme. As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner. rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E. coli enzymes require only one cysteine for catalysis. As in the S. aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction. SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification.