Molecular & Biochemical Parasitology 148 (2006) 161–168

Bifunctional role of the leishmanial antimonate reductaseLmACR2 as a protein tyrosine phosphatase�

Yao Zhou, Hiranmoy Bhattacharjee, Rita Mukhopadhyay ∗Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 East Canfield Avenue,

Detroit, MI 48201, United States

Received 31 January 2006; received in revised form 20 March 2006; accepted 21 March 2006Available online 18 April 2006

Abstract

LmACR2 is the first identified antimonate reductase responsible for the reduction of pentavalent antimony in pentostam to the active trivalentform of the drug in Leishmania. LmACR2 is a homologue of the yeast arsenate reductase Acr2p and Cdc25 phosphatases and has the HC[X]5Rphosphatase motif. Purified LmACR2 exhibited phosphatase activity in vitro and was able to dephosphorylate a phosphotyrosine residue from asynthetic peptide. This phosphatase activity was inhibited by classical inhibitors such as orthovanadate. LmACR2-catalyzed phosphatase activitywTmp©

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as inhibited by either antimonate or arsenate. Site-directed mutagenesis experiments showed that the H74C[X]5R81 motif was involved in catalysis.his is the first report of a metalloid reductase with a bifunctional role in protein tyrosine phosphatase activity. Leishmania is never exposed toetalloids during its life cycle. It is therefore unlikely that it would evolve an enzyme exclusively for drug activation. We propose that the

hysiological function of LmACR2 is to dephosphorylate phosphotyrosine residues in leishmanial proteins.2006 Elsevier B.V. All rights reserved.

eywords: Antimonate; LmACR2; Leishmania; Phosphatase; Reductase

. Introduction

Leishmaniasis is a protozoan, parasitic disease that isndemic in 88 countries on four continents, and is believed toffect over 2 million people each year. Leishmania parasite goeshrough two developmental stages during its life cycle: the pro-

astigote form of the parasite resides in the intestinal tract of thensect vector while the amastigote form resides in macrophagesnd other mononuclear phagocytes in the mammalian host. Pen-avalent antimony containing drugs pentostam and glucantimere the first line of defense against leishmaniasis. It has beenroposed that activation of the drug involves reduction of pen-avalent antimonials to its trivalent form [1] and this processccurs preferentially in the amastigotes [2,3].

LmACR2, from Leishmania major, is the first identified met-lloid reductase with a physiological role in drug activation [4].

Abbreviations: FDP, fluorescein diphosphate; pNPP, p-nitrophenylphos-hate; PTP, protein tyrosine phosphatase� Note: GenBank accession numbers: NP 001780 and AAS73185.∗ Corresponding author. Tel.: +1 313 577 6722; fax: +1 313 577 2765.

This 127 residue enzyme has the ability to reduce either arse-nate [As(V)] or antimonate [Sb(V)]. It is a better antimonatereductase than an arsenate reductase and functions as a drugactivator in Leishmania. LmACR2 has been shown to comple-ment the arsenate-sensitive phenotype of an arsC deletion strainof Escherichia coli or an ScACR2 deletion strain of Saccha-romyces cerevisiae [4]. Transfection of Leishmania infantumwith LmACR2 increased the pentostam sensitivity in intracellu-lar amastigotes of Leishmania [4].

Thiol-linked reductases that confer resistance to arsenatearose at least three times in prokaryotes [5,6] and eukaryotes[7,8], apparently by convergent evolution. The first family isrepresented by E. coli R773 ArsC that utilizes glutathione andglutaredoxin as electron donors [9] and forms a glutathioneintermediate during the reaction cycle [10]. The second familyrepresented by the Staphylococcus aureus plasmid pI258 ArsCuses thioredoxin as the electron donor [5] and is related to afamily of low molecular weight protein tyrosine phosphatase(PTP1). LmACR2 is a member of the third family and is pro-posed to form a mixed disulfide, but does not share any sequencesimilarity with the members of the first two families. Its clos-

E-mail address: rmukhopa@med.wayne.edu (R. Mukhopadhyay). est homologues are yeast arsenate reductase Acr2p and Cdc25

166-6851/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2006.03.009

162 Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168

family of PTPs, which share the same HC[X]5R active site motif.However, LmACR2 is the only known As(V) reductase to exhibitSb(V) reductase activity [11].

Although LmACR2, E. coli ArsC, and S. cerevisiae Acr2phave similar mechanisms to reduce arsenate to arsenite [4],neither Acr2p nor ArsC are phosphatases. Introduction of aGXGXXG motif at the active site of yeast Acr2p, converted it to aPTP, but the altered protein lost arsenate reductase activity [12].pI258 ArsC has similarity to low molecular weight PTPs andwas reported to be a rudimentary phosphatase with low affin-ity for phosphatase substrates such as p-nitrophenylphosphate(pNPP) [13]. However, this protein has not been shown to havePTP activity.

Arsenate, antimonate, and phosphate are chemically similaroxyanions. It is reasonable to assume that the ancestors of Cdc25phosphatases and metalloid reductases had an oxyanion bindingsite that could accommodate either oxyanion. However, to dateno eukaryotic metalloid reductase has been identified with abifunctional phosphatase activity. In this study, we show for thefirst time that besides being a metalloid reductase, LmACR2 isalso a phosphatase, which is most likely related to its physio-logical function. These findings have a greater relevance to theevolution of metalloid reductases.

2. Materials and methods

2

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2

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2

gcgcC5

CTC-3′; C75A, 5′-GAG CTC GCT GTC TTT CAC GCC GCCCAG TCG CTC-3′ and 5′-GAG CGA CTG GGC GGC GTGAAA GAC AGC GAG CTC-3′; C75S, 5′-GAG CTC GCT GTCTTT CAC TCC GCC CAG TCG CTC-3′ and 5′-GAG CGACTG GGC GGA GTG AAA GAC AGC GAG CTC-3′; R81A,5′-GCC CAG TCG CTC GTA GCG GCC CCC AAG GGAGC-3′ and 5′-GC TCC CTT GGG GGC CGC TAC GAG CGACTG GGC-3′; R81K, 5′-GCC CAG TCG CTC GTA AAG GCCCCC AAG GGA GC-3′ and 5′-GC TCC CTT GGG GGC CTTTAC GAG CGA CTG GGC-3′. Each mutation was confirmedby sequencing the entire gene using a CEQ2000 DNA sequencer(Beckman Coulter).

2.4. Assay of phosphatase activity

LmACR2 and its active site mutants were assayed forphosphatase activity by three different methods [12]. In thefirst method, phosphatase activity was assayed at 37 ◦C with10 �M LmACR2 using the indicated amount of pNPP in 0.1 MMOPS/MES buffer, pH 7.5. The assay was initiated by the addi-tion of pNPP, and the rate of hydrolysis was measured from theincrease in absorbance at 405 nm. Each value was corrected fornon-enzymatic pNPP hydrolysis. The data were analyzed withSigma Plot 9.0 using an extinction coefficient for nitrophenol of18,000 M−1 cm−1. In the second approach, fluorescein diphos-pwilramtwwtLttpad

2

idTFl

2

p

.1. Reagents

DNA manipulation reagents were purchased from Qiagen andnvitrogen. Site-directed mutagenesis reagents were purchasedrom Stratagene. Unless otherwise mentioned, all other chemi-als were obtained from Sigma.

.2. Purification of LmACR2

LmACR2 and its active site mutants were purified from cul-ures of E. coli strain TOP10 harboring pBAD/Myc-HisA con-tructs with wild type and mutant LmACR2 genes. Cells wererown at 37 ◦C in Luria-Bertani medium to an A600 of 0.5, athich point 0.02% arabinose was added to induce LmACR2

xpression. The cells were grown for another 3 h before beingarvested by centrifugation. LmACR2 was purified accordingo the protocol described previously [4]. The protein concentra-ion in purified preparations was determined from its absorbancet 280 nm. An extinction coefficient of 16,410 M−1 cm−1 wassed for most preparations except that an extinction coefficientf 16,290 M−1 cm−1 was used to quantitate C75S and C75AmACR2.

.3. Oligonucleotide-directed mutagenesis

Mutations in LmACR2 were introduced by site-directed muta-enesis using the QuikChangeTM site-directed mutagenesis pro-edure (Stratagene), as described previously [12]. The muta-enic oligonucleotides used for both strands and the respectivehanges introduced (underlined) are as follows: H74A, 5′-GAGTC GCT GTC TTT GCC TGC GCC CAG TCG CTC-3′ and′-GAG CGA CTG GGC GCA GGC AAA GAC AGC GAG

hate (FDP) hydrolysis was measured fluorometrically. Assaysere performed at room temperature with 0.5 �M LmACR2

n an SLM-8000C spectrofluorometer with an excitation wave-ength set at 475 nm and emission at 515 nm. Finally, dephospho-ylation of the peptide LCK505 (TEGQpYQPQP) was measuredt room temperature in 0.1 M MOPS/MES buffer, pH 7.5, byonitoring the increase in fluorescence emission resulting from

he formation of free tyrosyl peptide at 305 nm with an excitationavelength of 275 nm. The peptide and enzyme concentrationsere 0.2 mM and 5 �M, respectively. The incubation time with

he wild type and mutant proteins was no more than 3 min. SincemACR2 has its own tyrosine residues, all the reactions where

he peptide and LmACR2 (wild type or mutants) were presentogether, the final curves were generated after subtracting therotein alone numbers at each spectral point (both absorbancend fluorescence). All graphs were plotted and analyzed for theetermination of kinetic parameters using Sigma Plot 9.0.

.5. Inhibitor studies

LmACR2, 10 �M (for pNPP) or 0.5 �M (for FDP) was pre-ncubated with indicated concentrations of sodium orthovana-ate, sodium arsenate or potassium hexahydroxy antimonate.he assay was initiated with 70/100 mM pNPP or 40/60 �MDP. The graphs were plotted and best fit of the data was calcu-

ated by using Sigma Plot 9.0.

.6. pH profile studies

LmACR2 (10 or 0.5 �M) was used for each assay with 70 mMNPP and 40 �M FDP, respectively. For pH 5.5–6.0, 6.5–9.5 and

Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168 163

Fig. 1. Sequence alignment of the active site of Cdc25a and LmACR2. The protein source and GenBank accession numbers of the aligned sequences are Homosapiens Cdc25a (NP 001780) and Leishmania major LmACR2 (AAS73185). The HC[X]5R active site motif is underlined. Amino acids marked with black or greyboxes indicate sequence identity or similarity, respectively. The dashes indicate the gaps introduced to maximize sequence alignment.

10–11, 0.1 M sodium acetate, bis-Tris–propane and CAPS (N-cyclohexyl-3-propanesulfonic acid) buffers were used, respec-tively. Graphs were plotted using Sigma Plot 9.0.

3. Results

3.1. LmACR2 is a phosphatase

All protein phosphatases share the active site sequence motifHC[X]5R, X being any amino acid. LmACR2 also has this motifand shares considerable sequence similarity with the catalytic

domain of a dual specific phosphatase Cdc25a (Fig. 1). Thisprompted us to study the phosphatase activity of LmACR2.Wild type LmACR2 with a C-terminal his tag was purifiedby Ni-affinity and gel filtration chromatography, as describedbefore [4]. Using pNPP as a substrate, LmACR2 was exam-ined for phosphatase activity [12]. A linear increase at 405 nmwas observed over 10 min (Fig. 2A), signifying hydrolysis ofpNPP by LmACR2. A second substrate FDP was also used todemonstrate the phosphatase activity of LmACR2. An increasein fluorescence of FDP upon incubation with LmACR2 clearlydemonstrates the phosphatase activity of LmACR2 (Fig. 2C).

Fok

ig. 2. Phosphatase activity of LmACR2. Phosphatase activity of LmACR2 was deterf 30 mM pNPP by 10 �M LmACR2; (B) kinetics of pNPP hydrolysis and (C) incrinetics of FDP hydrolysis. Each point in the substrate saturation curves represents m

mined by monitoring the (A) increase in absorbance at 405 nm upon hydrolysisease in fluorescence upon hydrolysis of 1 �M FDP by 0.5 �M LmACR2; (D)ean ± S.D. from three independent experiments.

164 Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168

Using the phosphotyrosine containing peptide LCK505 as asubstrate [12], the ability of LmACR2 to dephosphorylate aphosphotyrosine residue was examined. Dephosphorylation ofthe tyrosine residue in LCK505 has been shown to produce anincrease in either the absorbance or fluorescence intensity of the

Fta(tL

peptide [12,14]. A similar phenomenon was observed duringLmACR2-catalyzed dephosphorylation of LCK505 (Fig. 3A andC). An increase in absorbance with 0.2 mM LCK505 and 5 �MLmACR2 at 282 nm was also observed over a period of twoand half minutes (Fig. 3B) signifying hydrolysis of the peptide.LmACR2 alone absorption at 282 nm was subtracted to generatethe final curve in Fig. 3B. Dephosphorylation of the peptide withthe commercially available antarctic phosphatase also produceda similar fluorescence change (Fig. 3A and C). LmACR2 wasfound to be an alkaline phosphatase as the pH optimum for bothpNPP and FDP was between 7.5 and 8.5 (Fig. 4A and B).

The rate of pNPP hydrolysis by LmACR2 as a functionof pNPP concentration was determined (Fig. 2B). The Kmwas calculated to be 36 mM for pNPP, and the Vmax was230 nmol/min/mg of protein. In comparison, either the mam-malian PTP1B [15] or Yersinia PTP [16] show a Km of 2 mMfor pNPP. The turnover number (kcat) for LmACR2 with pNPP asa substrate is 6 × 10−2 s−1, and the catalytic efficiency (kcat/Km)is 1.7 M−1 s−1, respectively (Table 1).

FDP has been used as an alternate substrate to determinephosphatase activity [17]. Kinetic analysis (Fig. 2D) indicated

ig. 3. Phosphotyrosine dephosphorylation by LmACR2. (A) Absorption spec-ra were acquired with 0.2 mM LCK505 and 5 �M LmACR2; (B) increase inbsorbance at 282 nm upon hydrolysis of 0.2 mM LCK505 by 5 �M LmACR2;C) fluorescence emission spectra were acquired with the same concentra-ion of peptide and enzyme: (�), LCK505 only; (©), LCK505 + LmACR2; (�)CK505 + Antarctic phosphatase.

Fy(4i

ig. 4. pH profile of LmACR2-catalyzed phosphatase activity. (A) pNPP hydrol-sis at different pH was determined with 10 �M LmACR2 and 70 mM pNPP;B) FDP hydrolysis at different pH was determined with 0.1 �M LmACR2 and0 �M FDP. Each point represents mean ± S.D. from three independent exper-ments.

Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168 165

Table 1Kinetic parameters of wild type and H74A LmACR2

LmACR2 Substrate Km (mM) Vmax (�mol/min/mg protein) kcat (s−1) kcat/Km (M−1 s−1)

Wild type pNPP 36.0 ± 2.3a 0.23 ± 0.006 6 × 10−2 ± 1.6 × 10−3 1.7 ± 0.3Wild type FDP 0.019 ± 0.002 30.0 ± 1.0 8.0 ± 0.3 4 × 105 ± 2 × 102

H74A pNPP 17.0 ± 3.2 0.03 ± 0.002 8 × 10−3 ± 4.5 × 10−4 0.5 ± 0.06H74A FDP 0.023 ± 0.004 3.0 ± 0.25 1.0 ± 0.08 4 × 104 ± 3 × 102

a ±S.D. values from three independent experiments.

Fig. 5. Inhibition of LmACR2-catalyzed phosphatase activity. The rates of pNPP (A–C) at (�) 70 mM and (©) 100 mM and FDP (D–F) hydrolysis at (�) 40 �Mand (�) 60 �M were assayed in the presence of the indicated concentrations of inhibitors. The inhibitors were (A and D) sodium orthovanadate, (B and E) sodiumarsenate, and (C and F) potassium hexahydroxy antimonate. Solid lines represent the best fit of the data. Each point is an average of two or three separate assays withindependently purified enzyme.

166 Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168

that LmACR2 has a Km of 19 �M for FDP, which is ∼1900-foldgreater affinity than for pNPP. The Vmax was 30 �mol/min/mgof protein. The kcat and kcat/Km values were 8.0 s−1 and4 × 105 M−1 s−1, respectively (Table 1). Thus FDP is a muchbetter substrate for LmACR2 than pNPP. PTP1B also exhibitssimilar affinity for FDP with a Km of 10 �M [18].

3.2. Inhibitors of LmACR2 phosphatase activity

Sodium orthovanadate is a competitive inhibitor of PTPs[19,20]. Sodium orthovanadate competitively inhibited thephosphatase activity of LmACR2 with a Ki of 10 �M (Fig. 5A)with pNPP and 0.5 �M (Fig. 5D) with FDP as in comparisonwith a Ki of ∼1 �M for PTP1B [15]. Arsenate has also beenshown to be a competitive inhibitor of PTPs [16,21]. Arsenatecompetitively inhibited the phosphatase activity of LmACR2with a Ki of 4 mM (Fig. 5B) with pNPP and 1 mM (Fig. 5E)with FDP. Pathak and Yi [20] have shown that Sb(V) in the formof sodium stibogluconate is a potent inhibitor of PTPs such asSHP-1, SHP-2 and PTP1B [20]. Sb(V) competitively inhibitedLmACR2 phosphatase activity with a Ki of 80 �M (Fig. 5C)with pNPP and 50 �M (Fig. 5F) with FDP, indicating Sb(V) isa better inhibitor of LmACR2 phosphatase activity than As(V).In contrast, Sb[III], As[III], sulphate, phosphate or nitrate didnot inhibit LmACR2 phosphatase activity (data not shown).

3.3. Active site residues of LmACR2 phosphatase

LmACR2 shares the same active site motif H74C[X]5R81 asother members of the PTPs (Fig. 1). To examine the role of theconserved His74, Cys75, and Arg81 in catalysis, each of theseresidues were modified individually by site-directed mutagene-sis to generate H74A, C75S, C75A, R81A, and R81K LmACR2.Wild type LmACR2 and each of its mutants were purified bynickel affinity and gel filtration chromatography and examinedfor phosphatase activity in vitro using either pNPP, FDP (Fig. 6Aand C) or LCK505 (Fig. 7A and B) as substrates. The wildtype and mutant proteins were examined by CD spectroscopyto show that the overall structures in the mutant proteins werenot significantly different from the wild type LmACR2 (datanot shown). C75S, C75A, R81A and R81K were completelyinactive, whereas H74A exhibited a low level of phosphataseactivity (Figs. 6A and C and 7A and B]. Addition of 5–7-foldmutant enzymes (C75S, C75A, R81A and R81K) failed to elicitany dephosphorylation of either pNPP or FDP (data not shown).This suggests that Cys75 and Arg81 residues at the active sitemotif are involved in phosphatase activity. There are three addi-tional cysteines located at position 30, 37, and 51 in the primarystructure of LmACR2. From mutagenesis results, none werefound to be involved in LmACR2-catalyzed phosphatase activ-ity (data not shown). The H74A mutant was 10-fold less active

F4hp

ig. 6. Phosphatase activity of LmACR2 mutants. Phosphatase activity of LmACR05 nm upon hydrolysis of 30 mM pNPP; (B) kinetics of pNPP hydrolysis by H74A; (ydrolysis by H74A. The protein concentration in the former was 10 �M and in therotein. Each point in the substrate saturation curves represents mean + S.D. from thr

2 mutants were determined by monitoring the (A) increase in absorbance atC) increase in fluorescence upon hydrolysis of 1 �M FDP; (D) kinetics of FDPlater 1 �M: (�) wild type LmACR2; (♦) H74A; (�) C75S; (�) R81K; (�) noee independent experiments.

Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168 167

Fig. 7. Phosphotyrosine dephosphorylation by LmACR2 mutants. (A) Absorp-tion spectra were acquired with 0.2 mM LCK505 and 5 �M LmACR2; (B)fluorescence emission spectra were acquired with the same concentration ofpeptide and enzyme: (©) wild type LmACR2; (�) H74A; (�) C75S; (�) R81K;(�) LCK505 only.

than the wild type enzyme. The turnover number and catalyticefficiency for either pNPP or FDP were significantly lower forH74A compared to wild type LmACR2 (Table 1 and Fig. 6Band D). However, the affinity for both pNPP and FDP remainedunchanged for the H74A mutant (Table 1). This is in contrastwith other PTPs, where alteration of the histidine residue inthe active site motif, decreased the substrate affinity by 20-foldwithout affecting the rate of catalysis [22].

4. Discussion

LmACR2 belongs to the family of eukaryotic As(V) reduc-tases, such as Acr2p, which has been predicted to have a three-dimensional structure related to rhodaneses and CDC25 dualspecific phophatases [23,24]. Although Acr2p has an HC[X]5Ractive site similar to that of CDC25 [25], it does not exhibitmeasurable phosphatase activity [26].

We had earlier shown that LmACR2 is the only known As(V)reductase to exhibit Sb(V) reductase activity [4]. In this study,we demonstrate that LmACR2 is also a protein tyrosine phos-phatase, as shown by its ability to dephosphorylate the tyrosine

residue in the synthetic peptide LCK505. The H74C[X]5R81

motif was also shown to be involved in catalysis. The histidineresidue at the start of this signature motif of PTPs does not appearto have an essential role in catalysis, but is suggested to performa structural role [22,27,28]. However, the turnover number ofH74A LmACR2 was decreased significantly, while the affinityfor either pNPP or FDP remained nearly the same as the wildtype protein. The inhibitor studies showed that Sb(V) inhibitsthe phosphatase activity 500-fold more efficiently than As(V),consistent with its property as being a much more efficient anti-monate reductase than arsenate reductase. LmACR2 is the onlyenzyme thus so far shown to retain both the metalloid reductaseand PTP activities. Identification of such a bifunctional enzymealso has a significant impact on our views about the evolutionof metalloid reductases. These findings strengthen our earlierhypothesis that eukaryotic metalloid reductases evolved fromphosphatases [12].

Leishmania is not exposed to metalloids during its natu-ral life cycle, since it thrives either in the sand fly gut as apromastigote, or as amastigote inside the macrophage of a ver-tebrate host. The critical question is why the parasite wouldevolve an enzyme such as LmACR2 for drug activation, whenthat is detrimental to the pathogen. We propose that the phys-iological function of Leishmania ACR2 is the dephosphory-lation of protein substrates. The identification of physiologi-ccsdoaaiswabtppaererL

A

Gf

R

al substrates is currently under investigation. PTP often playritical roles during host cell–parasite interaction. It has beenhown that overexpression of human PTP1B in Leishmaniaonovani resulted in partial differentiation of the promastig-te to the amastigote form. This ectopic expression of PTP1Blso increased the virulence of this pathogen, both in vitrond in vivo [29]. Protein phosphorylation at a tyrosine residuen L. donovani virulent promastigotes was shown to decreaseignificantly at higher temperature (37 ◦C) during interactionith macrophages. This clearly indicates modulation of par-

site phosphorylation/dephosphorylation events during verte-rate host infection by Leishmania [30]. It is interesting to notehat Leishmania-induced macrophage phosphotyrosine phos-hatase activity (SHP-1) is necessary for its survival withinhagocytes. SH2-domain-containing PTP, SHP-1, also knowns PTP1C, HCP or SH-PTP1, an enzyme that is abundantlyxpressed in macrophages, has been implicated in the negativeegulation of many activation and growth-promoting hemopoi-tic signaling cascades [31–33]. SHP-1 deficient mice also areesistant to Leishmania infection. We therefore speculate thatmACR2 might be involved in host–parasite interaction.

cknowledgements

This work was supported by National Institutes of Healthrant AI58170 and GM52216. We thank Prof. Barry P. Rosen

or his help and critical review of the manuscript.

eferences

[1] Sereno D, Cavaleyra M, Zemzoumi K, Maquaire S, Ouaissi A, LemesreJL. Axenically grown amastigotes of Leishmania infantum used as an

168 Y. Zhou et al. / Molecular & Biochemical Parasitology 148 (2006) 161–168

in vitro model to investigate the pentavalent antimony mode of action.Antimicrob Agents Chemother 1998;42:3097–102.

[2] Ephros M, Bitnun A, Shaked P, Waldman EZD. Stage-specific activity ofpentavalent antimony against Leishmania donovani axenic amastigotes.Antimicrob Agents Chemother 1999;43:278–82.

[3] Shaked-Mishan P, Ulrich N, Ephros M, Zilberstein D. Novel intracel-lular SbV reducing activity correlates with antimony susceptibility inLeishmania donovani. J Biol Chem 2001;276:3971–6.

[4] Zhou Y, Messier N, Ouellette M, Rosen BP, Mukhopadhyay R. Leish-mania major LmACR2 is a pentavalent antimony reductase that conferssensitivity to the drug pentostam. J Biol Chem 2004;279:37445–51.

[5] Ji G, Garber EAE, Armes LG, Chen CM, Fuchs JA, Silver S. Arse-nate reductase of Staphylococcus aureus plasmid pI258. Biochemistry1994;33:7294–9.

[6] Oden KL, Gladysheva TB, Rosen BP. Arsenate reduction mediated bythe plasmid-encoded ArsC protein is coupled to glutathione. Mol Micro-biol 1994;12:301–6.

[7] Bobrowicz P, Wysocki R, Owsianik G, Goffeau A, Ulaszewski S. Iso-lation of three contiguous genes, ACR1, ACR2 and ACR3, involved inresistance to arsenic compounds in the yeast Saccharomyces cerevisiae.Yeast 1997;13:819–28.

[8] Mukhopadhyay R, Rosen BP. The Saccharomyces cerevisiae ACR2 geneencodes an arsenate reductase. FEMS Microbiol Lett 1998;168:127–36.

[9] Gladysheva TB, Oden KL, Rosen BP. Properties of the arsenate reduc-tase of plasmid R773. Biochemistry 1994;33:7288–93.

[10] Liu J, Gladysheva TB, Lee L, Rosen BP. Identification of an essen-tial cysteinyl residue in the ArsC arsenate reductase of plasmid R773.Biochemistry 1995;34:13472–6.

[11] Mukhopadhyay R, Rosen BP. Arsenate reductases in prokaryotes andeukaryotes. Environ Health Perspect 2002;110(Suppl 5):745–8.

[12] Mukhopadhyay R, Zhou Y, Rosen BP. Directed evolution of a yeast

[

[

[

[

[

[

and molecular monitoring in 11 newly diagnosed and 47 relapsed acutepromyelocytic leukemia patients. Blood 1999;94:3315–24.

[19] Swarup G, Cohen S, Garbers DL. Inhibition of membrane phospho-tyrosyl-protein phosphatase activity by vanadate. Biochem Biophys ResCommun 1982;107:1104–9.

[20] Pathak MK, Yi T. Sodium stibogluconate is a potent inhibitor of proteintyrosine phosphatases and augments cytokine responses in hemopoieticcell lines. J Immunol 2001;167:3391–7.

[21] Zhang YL, Zhang ZY. Low-affinity binding determined by titrationcalorimetry using a high-affinity coupling ligand: a thermodynamic studyof ligand binding to protein tyrosine phosphatase 1B. Anal Biochem1998;261:139–48.

[22] Xu X, Burke SP. Roles of active site residues and the NH2-terminaldomain in the catalysis and substrate binding of human Cdc25. J BiolChem 1996;271:5118–24.

[23] Bordo D, Bork P. The rhodanese/Cdc25 phosphatase superfamily.Sequence–structure–function relations. EMBO 2002;741–6 (Rep 3).

[24] Denu JM, Dixon JE. Protein tyrosine phosphatases: mechanisms of catal-ysis and regulation. Curr Opin Chem Biol 1998;2:633–41.

[25] Mukhopadhyay R, Rosen BP. The phosphatase C[X]5R motif is requiredfor catalytic activity of the Saccharomyces cerevisiae Acr2p arsenatereductase. J Biol Chem 2001;276:34738–42.

[26] Mukhopadhyay R, Shi J, Rosen BP. Purification and characterization ofAcr2p, the Saccharomyces cerevisiae arsenate reductase. J Biol Chem2000;275:21149–57.

[27] Barford D, Flint AJ, Tonks NK. Crystal structure of human proteintyrosine phosphatase 1B. Science 1994;263:1397–404.

[28] Stuckey JA, Schubert HL, Fauman EB, Zhang ZY, Dixon JE, Saper MA.Crystal structure of Yersinia protein tyrosine phosphatase at 2.5 A andthe complex with tungstate. Nature 1994;370:571–5.

[29] Nascimento M, Abourjeily N, Ghosh A, Zhang WW, Matlashewski

[

[

[

[

arsenate reductase into a protein–tyrosine phosphatase. J Biol Chem2003;278:24476–80.

13] Zegers I, Martins JC, Willem R, Wyns L, Messens J. Arsenate reductasefrom S. aureus plasmid pI258 is a phosphatase drafted for redox duty.Nat Struct Biol 2001;8:843–7.

14] Zhang ZY, Maclean D, Thieme-Sefler AM, Roeske RW, Dixon JE. Acontinuous spectrophotometric and fluorimetric assay for protein tyrosinephosphatase using phosphotyrosine-containing peptides. Anal Biochem1993;211:7–15.

15] Guo XL, Shen K, Wang F, Lawrence DS, Zhang ZY. Probing themolecular basis for potent and selective protein–tyrosine phosphatase1B inhibition. J Biol Chem 2002;277:41014–22.

16] Keng YF, Wu L, Zhang ZY. Probing the function of the conserved tryp-tophan in the flexible loop of the Yersinia protein–tyrosine phosphatase.Eur J Biochem 1999;259:809–14.

17] Skorey K, Ly HD, Kelly J, et al. How does alendronate inhibitprotein–tyrosine phosphatases? J Biol Chem 1997;272:22472–80.

18] Niu C, Yan H, Yu T, et al. Studies on treatment of acute promye-locytic leukemia with arsenic trioxide: remission induction, follow-up,

G. Heterologous expression of a mammalian protein tyrosine phos-phatase gene in Leishmania: effect on differentiation. Mol Microbiol2003;50:1517–26.

30] Salotra P, Ralhan R, Sreenivas G. Heat-stress induced modulation of pro-tein phosphorylation in virulent promastigotes of Leishmania donovani.Int J Biochem Cell Biol 2000;32:309–16.

31] Blanchette J, Racette N, Faure R, Siminovitch KA, Olivier M.Leishmania-induced increases in activation of macrophage SHP-1 tyro-sine phosphatase are associated with impaired IFN-gamma-triggeredJAK2 activation. Eur J Immunol 1999;29:3737–44.

32] Olivier M, Romero-Gallo BJ, Matte C, et al. Modulation of interferon-gamma-induced macrophage activation by phosphotyrosine phosphatasesinhibition effect on murine leishmaniasis progression. J Biol Chem1998;273:13944–9.

33] Forget G, Siminovitch KA, Brochu S, Rivest S, Radzioch D, OlivierM. Role of host phosphotyrosine phosphatase SHP-1 in the devel-opment of murine leishmaniasis. Eur J Immunol 2001;31:3185–96.