J Biol Inorg Chem (2014) 19:1277–1285 DOI 10.1007/s00775-014-1184-8
ORIGINAL PAPER
ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase Catarina I. P. Nunes • Joana L. A. Bra´s Shabir Najmudin • Jose´ J. G. Moura • Isabel Moura • Marta S. P. Carepo
•
Received: 21 February 2014 / Accepted: 3 August 2014 / Published online: 20 August 2014 Ó SBIC 2014
Abstract Desulfovibrio alaskensis G20, a sulfate-reducing bacterium, contains an arsRBC2C3 operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in E. coli transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance. Kinetic studies revealed that this enzyme uses thioredoxin as electron donor and therefore belongs to Staphylococcus aureus plasmid pI258 and Bacillus subtilis thioredoxin-coupled arsenate reductases family. Both enzymes from this family contain a potassium-binding site, but only in Sa_ArsC does potassium actually binds resulting in a lower Km. Important differences between the S. aureus and B. subtilis enzymes and DaG20_ArsC3 are observed. DaG20_ArsC3 contains only two (Asn10, Ser33) of the four (Asn10, Ser33, Thr63, Asp65) conserved amino acid residues that form the potassium-binding site and the kinetics is not significantly affected by the presence of either potassium or sulfate ions. Isothermal titration calorimetry measurements confirmed nonspecific binding of K? and Na?, corroborating the non-relevance of these
cations for catalysis. Furthermore, the low Km and high kcat values determined for DaG20_ArsC3 revealed that this enzyme is the most catalytically efficient potassium-independent arsenate reductase described so far and, for the first time indicates that potassium binding is not essential to have low Km, for Trx-arsenate reductases. Keywords Arsenate reductase Thioredoxin Kinetics Potassium-binding site Sulfate-reducing bacteria Abbreviations ArsC DTT ESI-microTOF IPTG LB LMW PTPases pNPP SRB Trx TrxR
Arsenate reductase Dithiothreitol Electrospray ionization micro time of flight Isopropyl-b-D-thiogalactopyranoside Luria Broth Low molecular mass tyrosine phosphatases 4-Nitrophenyl phosphate disodium salt hexahydrate Sulfate-reducing bacterium Thioredoxin Thioredoxin reductase
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1184-8) contains supplementary material, which is available to authorized users. C. I. P. Nunes J. J. G. Moura I. Moura M. S. P. Carepo (&) REQUIMTE-CQFB, Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal e-mail: [email protected] J. L. A. Bra´s S. Najmudin CIISA, Faculdade de Medicina Veterina´ria, Universidade Te´cnica de Lisboa, Po´lo Universita´rio do Alto da Ajuda, Avenida da Universidade Te´cnica, 1300-477 Lisbon, Portugal
Introduction Despite the controversial discovery of a bacterium that may be able to replace phosphate by arsenate [1, 2], arsenate and arsenite are generally considered toxic for the majority of living organisms [3]. To minimize the toxic effects of arsenic, microorganisms have developed several resistance
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strategies including respiratory arsenate reduction, arsenite oxidation or methylation, and active extrusion of arsenite from the cell (ars operon) [4–6]. The ars operon is the best-characterized genetic system for arsenic resistance by microorganisms. This detoxification mechanism acts by decreasing the intracellular arsenic concentration [7–10]. Most ars operons consist of three genes encoding for ArsR, ArsB and ArsC proteins. ArsR is an As(III)-inducible transcription regulator, ArsC is the cytoplasmic arsenate reductase, which converts intracellular As(V) to As(III) and ArsB is the membrane protein responsible for As(III) extrusion. Some operons encode additional proteins that can be responsible for cell extrusion (like ArsA), or act as a metallochaperone protein (ArsD) which transfers arsenite to ArsA [6, 10–12]. Bacterial arsenate reductases can be grouped into three families according to the required electron donor. The arsenate reductases from Escherichia coli plasmid R773 family use glutaredoxin and glutathione as reductants, whereas the arsenate reductases from Staphylococcus aureus plasmid pI258 and Bacillus subtilis family use thioredoxin (Trx) [12–14]. Some arsenate reductases from Corynebacterium glutamicum are able to use the mycothiol/mycoredoxin pathway [15, 16]. The arsenate reductases from S. aureus pI258 (Sa_ArsC) and B. subtilis (Bs_ArsC) have a PTPase-I fold typical for low molecular mass tyrosine phosphatases (LMW PTPases). Both arsenate reductases require Trx and thioredoxin reductase (TrxR) as cofactors to be enzymatically active through a disulfide cascade involving residues Cys10, Cys15, Cys82 and Cys89 [8, 11, 13, 17–20] (Fig. 1). The reduction of arsenate to arsenite also requires the P-loop structural motif (Cys10, Thr11, Gly12, Asn13, Ser14, Cys15, Arg16) and Asp105 [8, 13, 17, 21–24]. However, despite possessing some common features, Sa_ArsC and Bs_ArsC also exhibit some important differences. Sa_ArsC is stabilized by tetrahedral oxyanions binding to the active site and also by a potassium-binding site located near the P-loop active site [25]. The K? ion is coordinated to the protein by Asn13, Ser36, Thr63 and Asp65 and by two water molecules [23, 25, 26]. In the presence of KCl, Sa_ArsC shows a significant Km decrease that is not observed in a buffer containing K2SO4, due to the competition of sulfate with arsenate for the active site. Despite the stabilizing effect of sulfate contributing for a higher kcat, the highest catalytic efficiency is observed with KCl for Sa_ArsC. Bs_ArsC kinetics is not influenced by potassium or sulfate, although the amino acid residues that coordinate K? in Sa_ArsC are conserved [25, 26]. The presence of a positively charged residue, Lys33, on the surface of Bs_ArsC, despite the presence of the negatively charged Asp31 and Glu32 in its vicinity, aids in decreasing the binding affinity of K? [25].
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Desulfovibrio alaskensis G20 is a sulfate-reducing bacterium (SRB) that reduces sulfate to sulfide, and is commonly found in freshwater, saltwater, soil and the intestinal tract of animals [27–30]. This organism grows anaerobically and it has a potential interest as a bioremediator, because it is involved in the reduction of toxic metals present in the Earth crust such as uranium (VI), chromium (VI) and iron (III) [31, 32]. It also has a significant economic impact due to its role in anaerobic biocorrosion of metals, an industrial problem that affects the oil industry in particular [28, 30, 31, 33]. The first study characterizing arsenic resistance systems in SRBs was reported by Li and Krumholz [34] for D. desulfuricans G20 (now called D. alaskensis G20 [35]); this organism contains an arsRBC2C3 operon encoding for two putative arsenate reductases. Resistance assays with the complete arsRBC2C3 operon showed that the operon is functional and confers high arsenate resistance in both the bacterium and E. coli cells [34]. In this work, the role of the individual arsenate reductases DaG20_ArsC2 and DaG20_ArsC3 from D. alaskensis G20 in arsenate resistance is addressed. The presence of potassium and sulfate does not affect DaG20_ArsC3 kinetics, and the low Km and high kcat values determined place this enzyme as one of the most efficient in arsenate detoxification.
Materials and methods Genomic and alignment analysis The D. alaskensis G20 genomic sequence was obtained from NCBI website (http://www.ncbi.nlm.nih.gov); alignment and other bioinformatic analysis were carried out with CLUSTAL W and through VIMSS computational genomics (http://www.microbesonline.org). Resistance assays in E. coli expressing ArsC2 or ArsC3 Single colonies of E. coli BL21 (DE3) transformed with either pETarsC2, pETarsC3, or pET-21c plasmid (control) (Table 1) were inoculated into fresh Luria–Bertani (LB) (Nzytech, Portugal) [36] medium containing ampicillin (100 lg/mL), and grown for 16 h at 37 8C in aerobic conditions. Mid-exponential phase cells were inoculated 1:50 into fresh LB medium containing ampicillin (100 lg/mL) and 0.3 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (Nzytech, Portugal), and grown at 37 8C, 210 rpm, under aerobic conditions for 25 h. Sodium arsenate (Sigma-Aldrich, India) was added to growth medium 6 h after inoculation to obtain the following final concentrations of As (V): 0, 5, 10, 20, 30 and 50 mM. Growth was monitored (O.D.600) after 2, 4, 6, 8, 10, 12, and 14 h for each concentration. Three biological replicates were done for each arsenate concentration.
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Table 1 Plasmids used in this study Plasmids
Description
Source or reference
pET-21c
Expression vector (control)
Novagen, USA
pETarsC2
arsC2 gene cloned on expressing vector pET-21c
This study
pETarsC3
arsC3 gene cloned on expressing vector pET-21c
This study
Expression and purification of arsenate reductase The PCR-amplified arsC3 gene from D. alaskensis G20 was cloned into a pET-21c vector (Novagen, USA) using restriction sites for NdeI and XhoI. The E. coli strain BL21 (DE3) (Novagen, USA) was transformed with pETarsC3 plasmid and was grown overnight at 37 8C in LB medium with 100 lg/mL ampicillin. Protein expression was induced at a cell density of O.D.600 = 0.7 with 1 mM IPTG and was carried out overnight at 37 8C. Cells were harvested by centrifugation for 15 min, 8,0009g at 4 8C and suspended in 50 mM Tris–HCl (pH 7.6), 50 mM NaCl, 2 mM bmercaptoethanol, 0.1 mM EDTA and one Complete, EDTA-free protease inhibitor cocktail tablet (RocheÒ). The cells were lysed through three freeze/thaw cycles in liquid nitrogen followed by disruption on a French Press (ThermoÒ). Cell debris was removed by centrifugation for 20 min at 8,0009g, 4 8C and the supernatant was ultracentrifuged for 1 h at 140,0009g, 4 8C, to separate membrane and soluble fractions. The DaG20_ArsC3 was successfully obtained in the soluble fraction and purified using anionic exchange and gel filtration chromatography. All purification steps were performed aerobically at 4 8C and the purity of the protein was followed by 15 % (wt/vol) Tricine SDS-PAGE gel electrophoresis. Throughout the purification, we used a thiol reductant (either dithiothreitol (DTT) or b-mercaptoethanol, (see below) and flushed all buffers with argon to avoid any possible irreversible oxidation and to keep all four cysteines in the thiol state. Briefly, soluble fraction was dialyzed in dialysis membrane (5 kDa cut-off) at 4 8C overnight, against the same buffer used to equilibrate a DEAE Fast Flow resin (Amersham BiosciencesÒ), which consisted of 10 mM Tris–HCl (pH 7.6), plus 1 mM DTT, 0.1 mM EDTA. The soluble fraction was loaded into the resin and the protein was eluted with a linear ionic strength gradient in Tris–HCl buffer 10–500 mM, pH 7.6, for 2 h at a flow rate of 3 mL/min. The fraction containing DaG20_ArsC3 was concentrated on a VivaCell 250 concentrator over a 5,000 MWCO membrane, at 4 8C, and subsequently loaded into a Superdex 75 column (Amersham BiosciencesÒ). The elution buffer consisted of 20 mM Tris–HCl (pH 7.6), plus 150 mM NaCl, 1 mM DTT and 0.1 mM EDTA, at a flow rate of 2.0 mL/min. The
DaG20_ArsC3 was dialyzed in a dialysis membrane (5 kDa cut-off) overnight at 4 8C, against 10 mM Tris–HCl (pH 7.6), with added 1 mM DTT and 0.1 mM EDTA; the same buffer was used to equilibrate a Resource Q pre-packed 6 mL column (Amersham BiosciencesÒ). The protein was eluted with a linear gradient 10–600 mM Tris–HCl, pH 7.6 for 2 h at a flow rate of 2 mL/min. Quantification of protein was carried out using Bicinchoninic Acid Kit (Sigma-Aldrich, USA) following the manufacturer’s instructions, with bovine serum albumin as standard protein. Determination of molecular mass The molecular mass was assessed using an electrospray ionization micro time of flight (ESI-microTOF) mass spectroscopy and the aggregation state was determined by gel filtration chromatography using a pre-packed Superdex 75 10/300 GL column (Pharmacia) equilibrated in 50 mM Tris–HCl (pH 7.6), 150 mM NaCl with a flow rate of 0.4 mL/min. The proteins used as standards from the Gel Filtration Calibration Kit Low Molecular Weight (GE Healthcare) included aprotinin (6.5 kDa), ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), and conalbumin (75 kDa). Kinetic assays Prior to use in the kinetic assays, DaG20_ArsC3 was reduced with 20 mM DTT for 30 min at room temperature, and dialyzed against the assay buffer (see below for more details) to remove DTT. In this assay, arsenate reduction is coupled to NADPH oxidation via the reduction of Trx by TrxR. Trx is therefore the electron donor for arsenate reduction. E. coli Trx (Sigma-Aldrich, Israel) was reduced with 20 mM DTT at room temperature for 30 min and subsequently dialyzed using a Vivaspin 4 concentrator over a 5 kDa cut-off membrane against 50 mM Tris–HCl (pH 7.6), 100 mM NaCl and 0.1 mM EDTA. E. coli TrxR ammonium sulfate stock (Sigma-Aldrich, Israel) was pelleted, dissolved, reduced and dialyzed against the same buffer. Protein concentrations were determined following the procedure described in the Bicinchoninic Acid Kit (Sigma-Aldrich, USA). The NADPH (Sigma-Aldrich, USA) was dissolved in water to a stock concentration of 10 mM and stored at 4 8C. Arsenate (Na2HAsO47H2O, Sigma-Aldrich, India) was freshly dissolved in water to a concentration of 9 mM, to be used in the different dilution series. Different assay buffers were used to analyze the ion-dependent steady-state kinetics of DaG20_ArsC3: 50 mM Tris–HCl (pH 7.6), 50 mM K2SO4, 0.1 mM EDTA; 50 mM Tris–HCl (pH 7.6), 150 mM KCl, 0.1 mM EDTA; and 50 mM Tris–HCl (pH
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7.6), 150 mM NaCl, 0.1 mM EDTA. All solutions were argon flushed for several minutes prior to use. The reaction mixture was prepared by diluting all components—except the substrate—in the assay buffer to obtain 400 nM DaG20_ArsC3, 6 lM Trx, 0.2 lM TrxR and 300 lM NADPH under anaerobic conditions. The concentrations of Trx and TrxR used were high enough so that their action could not be rate limiting. The component mixture was incubated without substrate at 37 8C in a 1 cm quartz spectrophotometer cell in a SHIMADZU UV-2550 for 1 min. The assay was started by adding various amounts of substrate, 2.5–500 mM final concentration in the cell. The arsenate reduction coupled to NADPH oxidation ( De340 = 6,220 M-1 cm-1) was measured by following the decrease in absorption at 340 nm. Initial rates were calculated and kinetic parameters were also obtained with Origin 5.0 (Microsoft) using the Michaelis–Menten expression to determine the Km and kcat values for DaG20_ArsC3. The phosphatase activity of DaG20_ArsC3 was assayed initially using 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma-Aldrich, USA) as substrate. Briefly, 5 lg of DaG20_ArsC3 was incubated for 30 min at 37 8C in 100 mM Tris–HCl (pH 7.6) containing 5 mM DTT. The concentrations of pNPP used ranged from 10 to 500 mM. The reaction volume was 300 lL. The reaction was stopped by the addition of 600 lL of 1 M NaOH, and the absorbance at 410 nm of the resulting solution was measured. The quantity of p-nitrophenol produced was calculated using an extinction coefficient of 17,800 M-1 cm-1. Kinetic constants were extrapolated using the Michaelis–Menten expression. Isothermal titration calorimetry The heat effects accompanying ion binding to DaG20_ArsC3 were measured at both 25 and 37 8C, using a MicroCal isothermal titration calorimeter (MicroCal VPITC, GE Healthcare). The protein was desalted into 10 mM Tris–HCl (pH 7.6) buffer. During titration, the protein (200 lM) was stirred at 300 rev./min in the reaction cell, which was injected with 28 successive 10 lL aliquots of ligand comprising either KCl or NaCl (15 mM) at 180 s intervals. Integrated heat effects were analysed by nonlinear regression using a single site-binding model (Microcal ORIGIN, Version 5.0; Microcal Software) [25, 37].
Results Genomic analysis From an examination of the D. alaskensis G20 genome sequence (NCBI Reference Sequence NC_007519.1), two putative arsenate reductases, the DaG20_ArsC2 and
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DaG20_ArsC3 with a calculated molecular mass of 16,655.24 and 16,010.26 Da, respectively, were found to be encoded in the ars operon. [38]. DaG20_ArsC2 is a protein sharing 51 and 50 % sequence identity with putative LMW PTPs from Desulfovibrio aespoeensis Aspo-2 and Desulfohalobium retbaense DSM 5692, respectively. DaG20_ArsC3 shares 64 and 62 % sequence identity with LMW PTPs from Desulfovibrio salexigens DSM 2638 and Pelobacter carbinolicus DSM 2380. Both arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3, showed 34 and 53 % amino acid sequence identity with arsenate reductase from B. subtilis (Bs_ArsC) and 28 and 44 % identity with arsenate reductase from S. aureus plasmid pI258 (Sa_ArsC), respectively. The DaG20_ArsC3 amino acid sequence alignment revealed the presence of the signature sequences that are characteristic of S. aureus plasmid pI258 and B. subtilis arsenate reductase family, which uses Trx as electron source. DaG20_ArsC3 has four conserved cysteines, Cys 7, Cys 12, Cys80 and Cys87 and a conserved structural P-loop (Cys7, Thr8, Gly9, Asn10, Ser11, Cys12, Arg13) similar to the Cys-Xn-Arg ArsC P-loop of Sa_ArsC and Bs_ArsC (Cys10, Thr11, Gly12, Asn13, Ser14, Cys15, Arg16) [8, 13, 17, 21–24]. Arg13 and Asp103 are also conserved in DaG20_ArsC3 indicating that thioredoxin is the putative cofactor in catalysis (Fig. 1). Only two residues from the potassium-binding site are conserved in DaG20_ArsC3, Asn10 and Ser33. Thr63 and Asp65 are replaced in DaG20_ArsC3 by Glu60 and Lys62. From the residues Asp31, Glu32 and Lys33—important in reducing the binding affinity of potassium in Bs_ArsC [25]—only Asp31 is conserved in DaG20_ArsC3. Another important residue influencing the kinetics parameters of the arsenate reductases is His62 in Sa_ArsC and Gln62 in Bs_ArsC [25]. DaG20_ArsC3 contains in this position a Gln residue like Bs_ArsC. DaG20_ArsC2 lacks one of the three catalytic cysteines of Trx-dependent arsenate reductases and the amino acids that form the characteristic P-loop. This protein also does not exhibit a recognizable sequence similarity to the arsenate reductase from Escherichia coli plasmid R773, representative of glutathione-linked arsenate reductases class, containing a single catalytic cysteine, Cys12, and five essential amino acids participating in the catalysis, His8, Ser15, Arg60, Arg94 and Arg107 [15, 17, 25, 26, 39]. Thus, the role of DaG20_ArsC2 as a putative arsenate reductase just from the amino acid sequence analysis is questionable. A closer look to D. alaskensis G20 genome revealed a sequence encoding for one cysteine followed by a threonine residue, upstream and on a different frame from the start codon of arsC2 gene. To confirm if this is due to an assembling problem in the genome, primers were designed
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Fig. 1 CLUSTAL W amino acid sequence alignment of arsenate reductases from Desulfovibrio alaskensis G20 (DaG20_ArsC2 and DaG20_ArsC3), Bacillus subtilis (Bs_ArsC), Staphylococcus aureus plasmid pI258 (Sa_ArsC) and Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2) (http://www.ebi.ac.uk/Tools/ clustalw2/index.html)
to amplify this region and the PCR product was sequenced. Analysis of the sequence obtained for the arsC2 gene confirmed that these amino acids are not encoded on the same frame as arsC2. Resistance assays in E. coli expressing ArsC2 or ArsC3 To test if the two individual putative arsenate reductases are functional, two expression vectors were constructed encoding these enzymes, the pETarsC2 and pETarsC3. Preliminary expression tests have shown that DaG20_ArsC3 is present in the soluble fraction and DaG20_ArsC2 is associated with the membrane fraction. The plasmids were transformed into E. coli BL21 (DE3) cells and the expression was induced with IPTG. E. coli pETarsC3 transformants were able to grow in LB medium in the presence of arsenate up to 20 mM (Fig. 2). On the other hand, E. coli pETarsC2 transformants could only grow in arsenate concentrations lower than 10 mM. In this case, the resistance is the same as the control and corresponds to the resistance given by the E. coli BL21 (DE3) chromosomal arsRBC operon.
Protein purification of DaG20_ArsC3 To characterize DaG20_ArsC3, that was proven to provide resistance on the resistance assays, this enzyme was expressed in E. coli BL21 (DE3) and purified using anionic exchange and gel filtration chromatography columns to
obtain the pure protein. The recombinant DaG20_ArsC3 was soluble and migrated on SDS-PAGE in a single band indicating a molecular mass of approximately 16 kDa. The purification yielded about 40 mg of pure protein/L of cell culture. The N-Terminal of the purified DaG20_ArsC3 was confirmed by Edman degradation as MNILF LCTGN SCRSQ MAEGW ARHLK. The molar extinction coefficient determined for the pure protein in 10 mM Tris–HCl (pH 7.6) is 10,989 M-1 cm-1. This value is quite similar to the value obtained by bioinformatics analysis, 10,220 M-1 cm-1 [38]. The molecular mass of DaG20_ArsC3 was determined by ESI-microTOF mass spectrometry to be 16,008.76 Da and by gel filtration chromatography to be 20,000 ± 4,000 Da, indicating that DaG20_ArsC3 is a monomeric protein like the arsenate reductase from S. aureus plasmid pI258 and B. subtilis [5, 19, 23]. Kinetic assays and cation binding Genomic analysis pointed out that DaG20_ArsC3 uses Trx as cofactor and therefore the enzymatic activity was followed indirectly by observing the rate of—NADP formation—with E. coli Trx and TrxR as coupling enzymes, delivering reducing equivalents to the oxidized DaG20_ArsC3. The initial rate of arsenate reduction as a function of DaG20_ArsC3 concentration was determined for 250 lM arsenate and it was shown to increase linearly with increasing concentrations of purified DaG20_ArsC3 within
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results obtained. The kcat values are high for the three iondependent conditions tested, although in the presence of NaCl a decrease is observed. This enzyme is extremely efficient among the arsenate reductases described so far with a kcat/Km of 40.9 9 104 M-1 s-1. Since DaG20_ArsC3 belongs to the LMW protein tyrosine phosphatase family, we have tested this enzyme’s activity towards pNPP hydrolysis (Fig. 3d). Kinetic analysis (Km 160.6 mM; Vmax 2.0 9 10-3 mM min-1) indicates that DaG20_ArsC3 has a very low phosphatase activity compared to its arsenate reductase activity, like others arsenate reductases [18, 40].
Discussion
Fig. 2 a Maximum growth after 8 h of incubation for E. coli BL21 (DE3) recombinants in LB broth with increasing levels of arsenate: E. coli BL21 transformed with pET-21c (open circles), E. coli BL21 transformed with pETarsC3 (filled circles), Insert: Growth of E. coli BL21 recombinants in LB broth with 5 mM arsenate, E. coli BL21 with pET-21c (open circles), E. coli BL21 with pETarsC3 (filled circles). b Maximum growth after 8 h of incubation for E. coli BL21 recombinants in LB broth with increasing levels of arsenate, E. coli BL21 transformed with pET-21c (open squares), E. coli BL21 transformed with pETarsC2 (filled squares). Insert: Growth of E. coli BL21 recombinants in LB broth with 5 mM arsenate, E. coli BL21 with pET-21c (open squares), E. coli BL21 with pETarsC2 (filled squares). The error bars indicate standard deviations
the range of concentrations tested—100–800 nM (data not shown). Due to the importance of potassium and sulfate on the kinetics Trx-coupled arsenate reductases family, we studied the ion-dependent steady-state kinetics of DaG20_ArsC3 in the presence of K2SO4, KCl and NaCl (Fig. 3a–c). The rate of arsenate reduction as a function of arsenate concentration was determined. Data were fitted by the standard Michaelis–Menten model (V = Vmax 9 [S]/ (Km ? [S])) and kinetic parameters were determined (Table 2). The Km of DaG20_ArsC3 is not influenced by potassium/sodium or sulfate. ITC titration curves showed nonspecific binding of potassium to DaG20_ArsC3, as well as nonspecific binding of sodium, at both 37 8C (Fig. 4) and 25 8C (Fig. S1), which is in agreement with the kinetics
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The ars operon from D. alaskensis G20, formerly known as D. desulfuricans G20, was studied for the first time in 2007 by Li and Krumholz [34]. The authors observed that E. coli DH5a cells grew in the presence of arsenate up to a concentration of 20 mM when transformed with a plasmid containing the arsC1 gene, encoding the constitutively expressed arsenate reductase from D. desulfuricans G20. E. coli cells could also grow in LB broth containing 20 mM arsenate and tolerated up to 50 mM, when transformed with the complete arsRBC2C3 operon. The results obtained from the resistance assays, testing the two arsenate reductases individually (without the insertion of the complete operon), provide strong evidence that DaG20_ArsC3 confers arsenate resistance in E. coli cells, whereas DaG20_ArsC2 does not. This result shows that recombinant DaG20_ArsC2 is not functional as arsenate reductase, corroborating the sequence analysis observations, probably due to the lack of important conserved amino acid residues characteristics of the P-loop. However, only future studies on native DaG20_ArsC2 can completely rule out a functional nature for this protein in D. alaskensis G20. DaG20_ArsC3 was confirmed as a monomeric arsenate reductase enzyme belonging to the Trx family by kinetic assays. Despite the common characteristics that unify the members of this family, there are important differences between Sa_ArsC and Bs_ArsC, the Trx family members studied in greater detail. The main differences are related to the kinetic properties of each enzyme, such as the influence of binding cations in kinetic parameters and the influence of oxyanions on enzyme stabilization. Both enzymes contain a cation-binding site, although only in Sa_ArsC is the binding effective and influential on the enzyme’s Km by lowering its value. Sa_ArsC is also stabilized by oxyanions like sulfate, although their binding decreases the substrate affinity for the enzyme, stemming from competition for the active site. On the other hand, Bs_ArsC Km is not
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Fig. 3 Michaelis–Menten plots of DaG20_ArsC3. a Initial rate versus arsenate concentration measured in 50 mM Tris–HCl (pH 7.4), 50 mM K2SO4, 0.1 mM EDTA. b Initial rate versus arsenate concentration measured in 50 mM Tris–HCl (pH 7.4), 150 mM KCl, 0.1 mM EDTA. c Initial rate versus arsenate concentration measured in 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1 mM EDTA. A, B, C
assays were performed at 37 8C with increasing arsenate concentrations in the presence of 400 nM DaG20_ArsC3, 6 lM E. coli Trx, 0.2 lM E. coli TR, and 300 lM NADPH. d Tyrosine phosphatase activity of the DaG20_ArsC3 using pNPP as substrate. The quantity of p-nitrophenol produced was measured at 410 nm for increasing concentrations of substrate at 37 8C
dependent on K? binding. DaG20_ArsC3 sequence identity is higher with Bs_ArsC than with Sa_ArsC, and the Km is not influenced neither by potassium or sodium cations nor by sulfate as observed for Bs_ArsC. However, the Km values are 7 times lower, showing a high affinity for arsenate by the enzyme, quite similar to Sa_ArsC in the presence of KCl. ITC titration curves performed using either K? or Na? as cations corroborate the absence of cation specific binding which is in agreement with the protein primary sequence, lacking two conserved amino acid residues, Thr63 and Asp65, which are conserved on the Sa_ArsC and Bs_ArsC cation-binding sites. Although no potassium binding is observed, DaG20_ArsC3 does not contain Lys33 or Glu32, important residues in preventing potassium binding in Bs_ArsC [25]. An interesting feature observed in the amino acid sequence of DaG20_ArsC3 is the presence of Lys62. In C. glutamicum, (Cg_ArsC2, PDB ID: 3RH0), the positive charge of the side chain N–H group in Lys64 (which is the equivalent of DaG20_ArsC3 Lys62) replaces potassium in its important role on the hydrogenbonding network, involving Cys8 and Asn11 [16], suggesting that the same may occur in DaG20_ArsC3.
The kcat values obtained for DaG20_ArsC3, in the presence of either sulfate, K? or Na?, are relatively constant for the three ion-dependent conditions tested, although some decrease is obtained in the presence of Na?, as observed for Bs_ArsC. However, the kcat values are twice as large as the values obtained for Bs_ArsC. The results presented show, for the first time, a Trx-coupled arsenate reductase in which the high efficiency is not dependent on cation binding or sulfate stabilization. The low Km together with the high kcat observed for DaG20_ArsC3 makes this enzyme one of the most efficient arsenate reductases described so far. The catalytic efficiencies are approximately 10 times higher for DaG20_ArsC3 compared with Bs_ArsC and Sa_ArsC for all conditions tested, except for Sa_ArsC in the presence of KCl. However, the catalytic efficiency of DaG20_ArsC3 under this reaction condition is still 5 times higher. The fact that sulfate-reducing bacteria occupy anaerobic and/or corroded environments and play an important role on the biogeochemical cycles of sulfur, carbon and nitrogen makes it more relevant for them to be able to respond to not only arsenic but also other heavy metal stresses [28, 34, 41, 42]. The low Km observed representing a high
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Table 2 Kinetic parameters determined for DaG20_ArsC3 in different buffers (50 mM Tris–HCl,pH 7.6, 0.1 mM EDTA, with either 50 mM K2SO4, 150 mM KCl or 150 mM NaCl) Buffer
Km (lM)
kcat (min-1)
kcat/Km (M-1 s-1) (9104)
References
This paper
and simultaneously avoiding a competition for sulfate between DaG20_ArsC3 and the respiratory processes.
Conclusion
DaG20_ArsC3 K2SO4
7
172
40.9
KCl
6
191
53.1
NaCl
8
123
25.6
K2SO4
47
95
3.3
KCl
54
96
3.0
NaCl
58
80
2.3
Bs_ArsC 26
Sa_ArsC K2SO4
81
219
4.5
KCl
9
54
10.0
NaCl
22
35
2.7
26
affinity for arsenate of DaG20_ArsC3 that is cation and sulfate independent, together with the high kcat acquires a particular relevance in this type of bacteria that uses sulfate as terminal electron acceptor in the anaerobic respiratory process. This efficient arsenate reducing system allows a fast trapping of arsenate inside the cell that can rapidly be extruded, preventing the toxic effects of this heavy metal,
Time (min)
Time (min) 10 20 30 40 50 60 70 80 90
-0.20
-0.40
-0.40
µcal/sec
-0.20
-0.60
-0.80
-1.00
-1.00
0.00
0.00
-0.04
10 20 30 40 50 60 70 80 90
-0.60
-0.80
-0.04
-0.08
-0.08 0
2
4
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8
10
Molar Ratio
KCl
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-10 0 0.00
kcal mol-1 of injectant
µcal/sec
-10 0 0.00
kcal mol-1 of injectant
Fig. 4 Isothermal calorimetric titration curves of DaG20_ArsC3 with KCl and NaCl at 37 8C, respectively. The upper part of each panel show the raw heats of binding, whereas the lower parts show the integrated areas under the respective peaks in the top panel plotted against the molar ratio of KCl and NaCl titrated into DaG20_ArsC3. These data are illustrative of a lack of binding
Desulfovibrio alaskensis G20 has an operon with two genes encoding two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3 that, according to the amino acid sequence alignment, belong to the Trx-coupled arsenate reductase family. However, DaG20_ArsC2 does not contain a conserved Cys-Xn-Arg P-loop signature motif and resistance assays in E. coli have shown that the expression of this protein does not confer any additional resistance to arsenate; therefore, DaG20_ArsC3 must be the enzyme responsible for arsenate reduction in D. alaskensis G20 encoded by the arsRBC2C3 operon. DaG20_ArsC3 steady-state kinetic assays using thioredoxin, thioredoxin reductase, and NADPH confirmed these proteins as co-factors of arsenate reduction. This enzyme is the first arsenate reductase isolated from an anaerobic sulfate-reducing bacterium. Like Bs_ArsC, DaG20_ArsC3 kinetics is cation and sulfate independent although the low Km is similar to Sa_ArsC in the presence of potassium. These results dissociate for the first time low Km from potassium specific binding on Trx-arsenate family. Kinetics analysis indicated that DaG20_ArsC3 acts more
12
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0
2
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6
8
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Molar Ratio
NaCl
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efficiently as arsenate reductase than phosphatase. Furthermore, DaG20_ArsC3 was shown to be highly efficient compared with other arsenate reductases described in the literature and therefore is a unique member of the Trxcoupled arsenate reductase family. Acknowledgments We would like to thank Fundac¸a˜o para a Cieˆncia e Tecnologia for the grants PEst-C/EQB/LA0006/2011 and PTDC/BIA-PRO/103980/2008 and fellowship SFRH/BD/62051/2009 to CIPN.
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ORIGINAL PAPER
ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase Catarina I. P. Nunes • Joana L. A. Bra´s Shabir Najmudin • Jose´ J. G. Moura • Isabel Moura • Marta S. P. Carepo
•
Received: 21 February 2014 / Accepted: 3 August 2014 / Published online: 20 August 2014 Ó SBIC 2014
Abstract Desulfovibrio alaskensis G20, a sulfate-reducing bacterium, contains an arsRBC2C3 operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in E. coli transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance. Kinetic studies revealed that this enzyme uses thioredoxin as electron donor and therefore belongs to Staphylococcus aureus plasmid pI258 and Bacillus subtilis thioredoxin-coupled arsenate reductases family. Both enzymes from this family contain a potassium-binding site, but only in Sa_ArsC does potassium actually binds resulting in a lower Km. Important differences between the S. aureus and B. subtilis enzymes and DaG20_ArsC3 are observed. DaG20_ArsC3 contains only two (Asn10, Ser33) of the four (Asn10, Ser33, Thr63, Asp65) conserved amino acid residues that form the potassium-binding site and the kinetics is not significantly affected by the presence of either potassium or sulfate ions. Isothermal titration calorimetry measurements confirmed nonspecific binding of K? and Na?, corroborating the non-relevance of these
cations for catalysis. Furthermore, the low Km and high kcat values determined for DaG20_ArsC3 revealed that this enzyme is the most catalytically efficient potassium-independent arsenate reductase described so far and, for the first time indicates that potassium binding is not essential to have low Km, for Trx-arsenate reductases. Keywords Arsenate reductase Thioredoxin Kinetics Potassium-binding site Sulfate-reducing bacteria Abbreviations ArsC DTT ESI-microTOF IPTG LB LMW PTPases pNPP SRB Trx TrxR
Arsenate reductase Dithiothreitol Electrospray ionization micro time of flight Isopropyl-b-D-thiogalactopyranoside Luria Broth Low molecular mass tyrosine phosphatases 4-Nitrophenyl phosphate disodium salt hexahydrate Sulfate-reducing bacterium Thioredoxin Thioredoxin reductase
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1184-8) contains supplementary material, which is available to authorized users. C. I. P. Nunes J. J. G. Moura I. Moura M. S. P. Carepo (&) REQUIMTE-CQFB, Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal e-mail: [email protected] J. L. A. Bra´s S. Najmudin CIISA, Faculdade de Medicina Veterina´ria, Universidade Te´cnica de Lisboa, Po´lo Universita´rio do Alto da Ajuda, Avenida da Universidade Te´cnica, 1300-477 Lisbon, Portugal
Introduction Despite the controversial discovery of a bacterium that may be able to replace phosphate by arsenate [1, 2], arsenate and arsenite are generally considered toxic for the majority of living organisms [3]. To minimize the toxic effects of arsenic, microorganisms have developed several resistance
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strategies including respiratory arsenate reduction, arsenite oxidation or methylation, and active extrusion of arsenite from the cell (ars operon) [4–6]. The ars operon is the best-characterized genetic system for arsenic resistance by microorganisms. This detoxification mechanism acts by decreasing the intracellular arsenic concentration [7–10]. Most ars operons consist of three genes encoding for ArsR, ArsB and ArsC proteins. ArsR is an As(III)-inducible transcription regulator, ArsC is the cytoplasmic arsenate reductase, which converts intracellular As(V) to As(III) and ArsB is the membrane protein responsible for As(III) extrusion. Some operons encode additional proteins that can be responsible for cell extrusion (like ArsA), or act as a metallochaperone protein (ArsD) which transfers arsenite to ArsA [6, 10–12]. Bacterial arsenate reductases can be grouped into three families according to the required electron donor. The arsenate reductases from Escherichia coli plasmid R773 family use glutaredoxin and glutathione as reductants, whereas the arsenate reductases from Staphylococcus aureus plasmid pI258 and Bacillus subtilis family use thioredoxin (Trx) [12–14]. Some arsenate reductases from Corynebacterium glutamicum are able to use the mycothiol/mycoredoxin pathway [15, 16]. The arsenate reductases from S. aureus pI258 (Sa_ArsC) and B. subtilis (Bs_ArsC) have a PTPase-I fold typical for low molecular mass tyrosine phosphatases (LMW PTPases). Both arsenate reductases require Trx and thioredoxin reductase (TrxR) as cofactors to be enzymatically active through a disulfide cascade involving residues Cys10, Cys15, Cys82 and Cys89 [8, 11, 13, 17–20] (Fig. 1). The reduction of arsenate to arsenite also requires the P-loop structural motif (Cys10, Thr11, Gly12, Asn13, Ser14, Cys15, Arg16) and Asp105 [8, 13, 17, 21–24]. However, despite possessing some common features, Sa_ArsC and Bs_ArsC also exhibit some important differences. Sa_ArsC is stabilized by tetrahedral oxyanions binding to the active site and also by a potassium-binding site located near the P-loop active site [25]. The K? ion is coordinated to the protein by Asn13, Ser36, Thr63 and Asp65 and by two water molecules [23, 25, 26]. In the presence of KCl, Sa_ArsC shows a significant Km decrease that is not observed in a buffer containing K2SO4, due to the competition of sulfate with arsenate for the active site. Despite the stabilizing effect of sulfate contributing for a higher kcat, the highest catalytic efficiency is observed with KCl for Sa_ArsC. Bs_ArsC kinetics is not influenced by potassium or sulfate, although the amino acid residues that coordinate K? in Sa_ArsC are conserved [25, 26]. The presence of a positively charged residue, Lys33, on the surface of Bs_ArsC, despite the presence of the negatively charged Asp31 and Glu32 in its vicinity, aids in decreasing the binding affinity of K? [25].
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Desulfovibrio alaskensis G20 is a sulfate-reducing bacterium (SRB) that reduces sulfate to sulfide, and is commonly found in freshwater, saltwater, soil and the intestinal tract of animals [27–30]. This organism grows anaerobically and it has a potential interest as a bioremediator, because it is involved in the reduction of toxic metals present in the Earth crust such as uranium (VI), chromium (VI) and iron (III) [31, 32]. It also has a significant economic impact due to its role in anaerobic biocorrosion of metals, an industrial problem that affects the oil industry in particular [28, 30, 31, 33]. The first study characterizing arsenic resistance systems in SRBs was reported by Li and Krumholz [34] for D. desulfuricans G20 (now called D. alaskensis G20 [35]); this organism contains an arsRBC2C3 operon encoding for two putative arsenate reductases. Resistance assays with the complete arsRBC2C3 operon showed that the operon is functional and confers high arsenate resistance in both the bacterium and E. coli cells [34]. In this work, the role of the individual arsenate reductases DaG20_ArsC2 and DaG20_ArsC3 from D. alaskensis G20 in arsenate resistance is addressed. The presence of potassium and sulfate does not affect DaG20_ArsC3 kinetics, and the low Km and high kcat values determined place this enzyme as one of the most efficient in arsenate detoxification.
Materials and methods Genomic and alignment analysis The D. alaskensis G20 genomic sequence was obtained from NCBI website (http://www.ncbi.nlm.nih.gov); alignment and other bioinformatic analysis were carried out with CLUSTAL W and through VIMSS computational genomics (http://www.microbesonline.org). Resistance assays in E. coli expressing ArsC2 or ArsC3 Single colonies of E. coli BL21 (DE3) transformed with either pETarsC2, pETarsC3, or pET-21c plasmid (control) (Table 1) were inoculated into fresh Luria–Bertani (LB) (Nzytech, Portugal) [36] medium containing ampicillin (100 lg/mL), and grown for 16 h at 37 8C in aerobic conditions. Mid-exponential phase cells were inoculated 1:50 into fresh LB medium containing ampicillin (100 lg/mL) and 0.3 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (Nzytech, Portugal), and grown at 37 8C, 210 rpm, under aerobic conditions for 25 h. Sodium arsenate (Sigma-Aldrich, India) was added to growth medium 6 h after inoculation to obtain the following final concentrations of As (V): 0, 5, 10, 20, 30 and 50 mM. Growth was monitored (O.D.600) after 2, 4, 6, 8, 10, 12, and 14 h for each concentration. Three biological replicates were done for each arsenate concentration.
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Table 1 Plasmids used in this study Plasmids
Description
Source or reference
pET-21c
Expression vector (control)
Novagen, USA
pETarsC2
arsC2 gene cloned on expressing vector pET-21c
This study
pETarsC3
arsC3 gene cloned on expressing vector pET-21c
This study
Expression and purification of arsenate reductase The PCR-amplified arsC3 gene from D. alaskensis G20 was cloned into a pET-21c vector (Novagen, USA) using restriction sites for NdeI and XhoI. The E. coli strain BL21 (DE3) (Novagen, USA) was transformed with pETarsC3 plasmid and was grown overnight at 37 8C in LB medium with 100 lg/mL ampicillin. Protein expression was induced at a cell density of O.D.600 = 0.7 with 1 mM IPTG and was carried out overnight at 37 8C. Cells were harvested by centrifugation for 15 min, 8,0009g at 4 8C and suspended in 50 mM Tris–HCl (pH 7.6), 50 mM NaCl, 2 mM bmercaptoethanol, 0.1 mM EDTA and one Complete, EDTA-free protease inhibitor cocktail tablet (RocheÒ). The cells were lysed through three freeze/thaw cycles in liquid nitrogen followed by disruption on a French Press (ThermoÒ). Cell debris was removed by centrifugation for 20 min at 8,0009g, 4 8C and the supernatant was ultracentrifuged for 1 h at 140,0009g, 4 8C, to separate membrane and soluble fractions. The DaG20_ArsC3 was successfully obtained in the soluble fraction and purified using anionic exchange and gel filtration chromatography. All purification steps were performed aerobically at 4 8C and the purity of the protein was followed by 15 % (wt/vol) Tricine SDS-PAGE gel electrophoresis. Throughout the purification, we used a thiol reductant (either dithiothreitol (DTT) or b-mercaptoethanol, (see below) and flushed all buffers with argon to avoid any possible irreversible oxidation and to keep all four cysteines in the thiol state. Briefly, soluble fraction was dialyzed in dialysis membrane (5 kDa cut-off) at 4 8C overnight, against the same buffer used to equilibrate a DEAE Fast Flow resin (Amersham BiosciencesÒ), which consisted of 10 mM Tris–HCl (pH 7.6), plus 1 mM DTT, 0.1 mM EDTA. The soluble fraction was loaded into the resin and the protein was eluted with a linear ionic strength gradient in Tris–HCl buffer 10–500 mM, pH 7.6, for 2 h at a flow rate of 3 mL/min. The fraction containing DaG20_ArsC3 was concentrated on a VivaCell 250 concentrator over a 5,000 MWCO membrane, at 4 8C, and subsequently loaded into a Superdex 75 column (Amersham BiosciencesÒ). The elution buffer consisted of 20 mM Tris–HCl (pH 7.6), plus 150 mM NaCl, 1 mM DTT and 0.1 mM EDTA, at a flow rate of 2.0 mL/min. The
DaG20_ArsC3 was dialyzed in a dialysis membrane (5 kDa cut-off) overnight at 4 8C, against 10 mM Tris–HCl (pH 7.6), with added 1 mM DTT and 0.1 mM EDTA; the same buffer was used to equilibrate a Resource Q pre-packed 6 mL column (Amersham BiosciencesÒ). The protein was eluted with a linear gradient 10–600 mM Tris–HCl, pH 7.6 for 2 h at a flow rate of 2 mL/min. Quantification of protein was carried out using Bicinchoninic Acid Kit (Sigma-Aldrich, USA) following the manufacturer’s instructions, with bovine serum albumin as standard protein. Determination of molecular mass The molecular mass was assessed using an electrospray ionization micro time of flight (ESI-microTOF) mass spectroscopy and the aggregation state was determined by gel filtration chromatography using a pre-packed Superdex 75 10/300 GL column (Pharmacia) equilibrated in 50 mM Tris–HCl (pH 7.6), 150 mM NaCl with a flow rate of 0.4 mL/min. The proteins used as standards from the Gel Filtration Calibration Kit Low Molecular Weight (GE Healthcare) included aprotinin (6.5 kDa), ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), and conalbumin (75 kDa). Kinetic assays Prior to use in the kinetic assays, DaG20_ArsC3 was reduced with 20 mM DTT for 30 min at room temperature, and dialyzed against the assay buffer (see below for more details) to remove DTT. In this assay, arsenate reduction is coupled to NADPH oxidation via the reduction of Trx by TrxR. Trx is therefore the electron donor for arsenate reduction. E. coli Trx (Sigma-Aldrich, Israel) was reduced with 20 mM DTT at room temperature for 30 min and subsequently dialyzed using a Vivaspin 4 concentrator over a 5 kDa cut-off membrane against 50 mM Tris–HCl (pH 7.6), 100 mM NaCl and 0.1 mM EDTA. E. coli TrxR ammonium sulfate stock (Sigma-Aldrich, Israel) was pelleted, dissolved, reduced and dialyzed against the same buffer. Protein concentrations were determined following the procedure described in the Bicinchoninic Acid Kit (Sigma-Aldrich, USA). The NADPH (Sigma-Aldrich, USA) was dissolved in water to a stock concentration of 10 mM and stored at 4 8C. Arsenate (Na2HAsO47H2O, Sigma-Aldrich, India) was freshly dissolved in water to a concentration of 9 mM, to be used in the different dilution series. Different assay buffers were used to analyze the ion-dependent steady-state kinetics of DaG20_ArsC3: 50 mM Tris–HCl (pH 7.6), 50 mM K2SO4, 0.1 mM EDTA; 50 mM Tris–HCl (pH 7.6), 150 mM KCl, 0.1 mM EDTA; and 50 mM Tris–HCl (pH
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7.6), 150 mM NaCl, 0.1 mM EDTA. All solutions were argon flushed for several minutes prior to use. The reaction mixture was prepared by diluting all components—except the substrate—in the assay buffer to obtain 400 nM DaG20_ArsC3, 6 lM Trx, 0.2 lM TrxR and 300 lM NADPH under anaerobic conditions. The concentrations of Trx and TrxR used were high enough so that their action could not be rate limiting. The component mixture was incubated without substrate at 37 8C in a 1 cm quartz spectrophotometer cell in a SHIMADZU UV-2550 for 1 min. The assay was started by adding various amounts of substrate, 2.5–500 mM final concentration in the cell. The arsenate reduction coupled to NADPH oxidation ( De340 = 6,220 M-1 cm-1) was measured by following the decrease in absorption at 340 nm. Initial rates were calculated and kinetic parameters were also obtained with Origin 5.0 (Microsoft) using the Michaelis–Menten expression to determine the Km and kcat values for DaG20_ArsC3. The phosphatase activity of DaG20_ArsC3 was assayed initially using 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma-Aldrich, USA) as substrate. Briefly, 5 lg of DaG20_ArsC3 was incubated for 30 min at 37 8C in 100 mM Tris–HCl (pH 7.6) containing 5 mM DTT. The concentrations of pNPP used ranged from 10 to 500 mM. The reaction volume was 300 lL. The reaction was stopped by the addition of 600 lL of 1 M NaOH, and the absorbance at 410 nm of the resulting solution was measured. The quantity of p-nitrophenol produced was calculated using an extinction coefficient of 17,800 M-1 cm-1. Kinetic constants were extrapolated using the Michaelis–Menten expression. Isothermal titration calorimetry The heat effects accompanying ion binding to DaG20_ArsC3 were measured at both 25 and 37 8C, using a MicroCal isothermal titration calorimeter (MicroCal VPITC, GE Healthcare). The protein was desalted into 10 mM Tris–HCl (pH 7.6) buffer. During titration, the protein (200 lM) was stirred at 300 rev./min in the reaction cell, which was injected with 28 successive 10 lL aliquots of ligand comprising either KCl or NaCl (15 mM) at 180 s intervals. Integrated heat effects were analysed by nonlinear regression using a single site-binding model (Microcal ORIGIN, Version 5.0; Microcal Software) [25, 37].
Results Genomic analysis From an examination of the D. alaskensis G20 genome sequence (NCBI Reference Sequence NC_007519.1), two putative arsenate reductases, the DaG20_ArsC2 and
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DaG20_ArsC3 with a calculated molecular mass of 16,655.24 and 16,010.26 Da, respectively, were found to be encoded in the ars operon. [38]. DaG20_ArsC2 is a protein sharing 51 and 50 % sequence identity with putative LMW PTPs from Desulfovibrio aespoeensis Aspo-2 and Desulfohalobium retbaense DSM 5692, respectively. DaG20_ArsC3 shares 64 and 62 % sequence identity with LMW PTPs from Desulfovibrio salexigens DSM 2638 and Pelobacter carbinolicus DSM 2380. Both arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3, showed 34 and 53 % amino acid sequence identity with arsenate reductase from B. subtilis (Bs_ArsC) and 28 and 44 % identity with arsenate reductase from S. aureus plasmid pI258 (Sa_ArsC), respectively. The DaG20_ArsC3 amino acid sequence alignment revealed the presence of the signature sequences that are characteristic of S. aureus plasmid pI258 and B. subtilis arsenate reductase family, which uses Trx as electron source. DaG20_ArsC3 has four conserved cysteines, Cys 7, Cys 12, Cys80 and Cys87 and a conserved structural P-loop (Cys7, Thr8, Gly9, Asn10, Ser11, Cys12, Arg13) similar to the Cys-Xn-Arg ArsC P-loop of Sa_ArsC and Bs_ArsC (Cys10, Thr11, Gly12, Asn13, Ser14, Cys15, Arg16) [8, 13, 17, 21–24]. Arg13 and Asp103 are also conserved in DaG20_ArsC3 indicating that thioredoxin is the putative cofactor in catalysis (Fig. 1). Only two residues from the potassium-binding site are conserved in DaG20_ArsC3, Asn10 and Ser33. Thr63 and Asp65 are replaced in DaG20_ArsC3 by Glu60 and Lys62. From the residues Asp31, Glu32 and Lys33—important in reducing the binding affinity of potassium in Bs_ArsC [25]—only Asp31 is conserved in DaG20_ArsC3. Another important residue influencing the kinetics parameters of the arsenate reductases is His62 in Sa_ArsC and Gln62 in Bs_ArsC [25]. DaG20_ArsC3 contains in this position a Gln residue like Bs_ArsC. DaG20_ArsC2 lacks one of the three catalytic cysteines of Trx-dependent arsenate reductases and the amino acids that form the characteristic P-loop. This protein also does not exhibit a recognizable sequence similarity to the arsenate reductase from Escherichia coli plasmid R773, representative of glutathione-linked arsenate reductases class, containing a single catalytic cysteine, Cys12, and five essential amino acids participating in the catalysis, His8, Ser15, Arg60, Arg94 and Arg107 [15, 17, 25, 26, 39]. Thus, the role of DaG20_ArsC2 as a putative arsenate reductase just from the amino acid sequence analysis is questionable. A closer look to D. alaskensis G20 genome revealed a sequence encoding for one cysteine followed by a threonine residue, upstream and on a different frame from the start codon of arsC2 gene. To confirm if this is due to an assembling problem in the genome, primers were designed
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Fig. 1 CLUSTAL W amino acid sequence alignment of arsenate reductases from Desulfovibrio alaskensis G20 (DaG20_ArsC2 and DaG20_ArsC3), Bacillus subtilis (Bs_ArsC), Staphylococcus aureus plasmid pI258 (Sa_ArsC) and Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2) (http://www.ebi.ac.uk/Tools/ clustalw2/index.html)
to amplify this region and the PCR product was sequenced. Analysis of the sequence obtained for the arsC2 gene confirmed that these amino acids are not encoded on the same frame as arsC2. Resistance assays in E. coli expressing ArsC2 or ArsC3 To test if the two individual putative arsenate reductases are functional, two expression vectors were constructed encoding these enzymes, the pETarsC2 and pETarsC3. Preliminary expression tests have shown that DaG20_ArsC3 is present in the soluble fraction and DaG20_ArsC2 is associated with the membrane fraction. The plasmids were transformed into E. coli BL21 (DE3) cells and the expression was induced with IPTG. E. coli pETarsC3 transformants were able to grow in LB medium in the presence of arsenate up to 20 mM (Fig. 2). On the other hand, E. coli pETarsC2 transformants could only grow in arsenate concentrations lower than 10 mM. In this case, the resistance is the same as the control and corresponds to the resistance given by the E. coli BL21 (DE3) chromosomal arsRBC operon.
Protein purification of DaG20_ArsC3 To characterize DaG20_ArsC3, that was proven to provide resistance on the resistance assays, this enzyme was expressed in E. coli BL21 (DE3) and purified using anionic exchange and gel filtration chromatography columns to
obtain the pure protein. The recombinant DaG20_ArsC3 was soluble and migrated on SDS-PAGE in a single band indicating a molecular mass of approximately 16 kDa. The purification yielded about 40 mg of pure protein/L of cell culture. The N-Terminal of the purified DaG20_ArsC3 was confirmed by Edman degradation as MNILF LCTGN SCRSQ MAEGW ARHLK. The molar extinction coefficient determined for the pure protein in 10 mM Tris–HCl (pH 7.6) is 10,989 M-1 cm-1. This value is quite similar to the value obtained by bioinformatics analysis, 10,220 M-1 cm-1 [38]. The molecular mass of DaG20_ArsC3 was determined by ESI-microTOF mass spectrometry to be 16,008.76 Da and by gel filtration chromatography to be 20,000 ± 4,000 Da, indicating that DaG20_ArsC3 is a monomeric protein like the arsenate reductase from S. aureus plasmid pI258 and B. subtilis [5, 19, 23]. Kinetic assays and cation binding Genomic analysis pointed out that DaG20_ArsC3 uses Trx as cofactor and therefore the enzymatic activity was followed indirectly by observing the rate of—NADP formation—with E. coli Trx and TrxR as coupling enzymes, delivering reducing equivalents to the oxidized DaG20_ArsC3. The initial rate of arsenate reduction as a function of DaG20_ArsC3 concentration was determined for 250 lM arsenate and it was shown to increase linearly with increasing concentrations of purified DaG20_ArsC3 within
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results obtained. The kcat values are high for the three iondependent conditions tested, although in the presence of NaCl a decrease is observed. This enzyme is extremely efficient among the arsenate reductases described so far with a kcat/Km of 40.9 9 104 M-1 s-1. Since DaG20_ArsC3 belongs to the LMW protein tyrosine phosphatase family, we have tested this enzyme’s activity towards pNPP hydrolysis (Fig. 3d). Kinetic analysis (Km 160.6 mM; Vmax 2.0 9 10-3 mM min-1) indicates that DaG20_ArsC3 has a very low phosphatase activity compared to its arsenate reductase activity, like others arsenate reductases [18, 40].
Discussion
Fig. 2 a Maximum growth after 8 h of incubation for E. coli BL21 (DE3) recombinants in LB broth with increasing levels of arsenate: E. coli BL21 transformed with pET-21c (open circles), E. coli BL21 transformed with pETarsC3 (filled circles), Insert: Growth of E. coli BL21 recombinants in LB broth with 5 mM arsenate, E. coli BL21 with pET-21c (open circles), E. coli BL21 with pETarsC3 (filled circles). b Maximum growth after 8 h of incubation for E. coli BL21 recombinants in LB broth with increasing levels of arsenate, E. coli BL21 transformed with pET-21c (open squares), E. coli BL21 transformed with pETarsC2 (filled squares). Insert: Growth of E. coli BL21 recombinants in LB broth with 5 mM arsenate, E. coli BL21 with pET-21c (open squares), E. coli BL21 with pETarsC2 (filled squares). The error bars indicate standard deviations
the range of concentrations tested—100–800 nM (data not shown). Due to the importance of potassium and sulfate on the kinetics Trx-coupled arsenate reductases family, we studied the ion-dependent steady-state kinetics of DaG20_ArsC3 in the presence of K2SO4, KCl and NaCl (Fig. 3a–c). The rate of arsenate reduction as a function of arsenate concentration was determined. Data were fitted by the standard Michaelis–Menten model (V = Vmax 9 [S]/ (Km ? [S])) and kinetic parameters were determined (Table 2). The Km of DaG20_ArsC3 is not influenced by potassium/sodium or sulfate. ITC titration curves showed nonspecific binding of potassium to DaG20_ArsC3, as well as nonspecific binding of sodium, at both 37 8C (Fig. 4) and 25 8C (Fig. S1), which is in agreement with the kinetics
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The ars operon from D. alaskensis G20, formerly known as D. desulfuricans G20, was studied for the first time in 2007 by Li and Krumholz [34]. The authors observed that E. coli DH5a cells grew in the presence of arsenate up to a concentration of 20 mM when transformed with a plasmid containing the arsC1 gene, encoding the constitutively expressed arsenate reductase from D. desulfuricans G20. E. coli cells could also grow in LB broth containing 20 mM arsenate and tolerated up to 50 mM, when transformed with the complete arsRBC2C3 operon. The results obtained from the resistance assays, testing the two arsenate reductases individually (without the insertion of the complete operon), provide strong evidence that DaG20_ArsC3 confers arsenate resistance in E. coli cells, whereas DaG20_ArsC2 does not. This result shows that recombinant DaG20_ArsC2 is not functional as arsenate reductase, corroborating the sequence analysis observations, probably due to the lack of important conserved amino acid residues characteristics of the P-loop. However, only future studies on native DaG20_ArsC2 can completely rule out a functional nature for this protein in D. alaskensis G20. DaG20_ArsC3 was confirmed as a monomeric arsenate reductase enzyme belonging to the Trx family by kinetic assays. Despite the common characteristics that unify the members of this family, there are important differences between Sa_ArsC and Bs_ArsC, the Trx family members studied in greater detail. The main differences are related to the kinetic properties of each enzyme, such as the influence of binding cations in kinetic parameters and the influence of oxyanions on enzyme stabilization. Both enzymes contain a cation-binding site, although only in Sa_ArsC is the binding effective and influential on the enzyme’s Km by lowering its value. Sa_ArsC is also stabilized by oxyanions like sulfate, although their binding decreases the substrate affinity for the enzyme, stemming from competition for the active site. On the other hand, Bs_ArsC Km is not
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Fig. 3 Michaelis–Menten plots of DaG20_ArsC3. a Initial rate versus arsenate concentration measured in 50 mM Tris–HCl (pH 7.4), 50 mM K2SO4, 0.1 mM EDTA. b Initial rate versus arsenate concentration measured in 50 mM Tris–HCl (pH 7.4), 150 mM KCl, 0.1 mM EDTA. c Initial rate versus arsenate concentration measured in 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1 mM EDTA. A, B, C
assays were performed at 37 8C with increasing arsenate concentrations in the presence of 400 nM DaG20_ArsC3, 6 lM E. coli Trx, 0.2 lM E. coli TR, and 300 lM NADPH. d Tyrosine phosphatase activity of the DaG20_ArsC3 using pNPP as substrate. The quantity of p-nitrophenol produced was measured at 410 nm for increasing concentrations of substrate at 37 8C
dependent on K? binding. DaG20_ArsC3 sequence identity is higher with Bs_ArsC than with Sa_ArsC, and the Km is not influenced neither by potassium or sodium cations nor by sulfate as observed for Bs_ArsC. However, the Km values are 7 times lower, showing a high affinity for arsenate by the enzyme, quite similar to Sa_ArsC in the presence of KCl. ITC titration curves performed using either K? or Na? as cations corroborate the absence of cation specific binding which is in agreement with the protein primary sequence, lacking two conserved amino acid residues, Thr63 and Asp65, which are conserved on the Sa_ArsC and Bs_ArsC cation-binding sites. Although no potassium binding is observed, DaG20_ArsC3 does not contain Lys33 or Glu32, important residues in preventing potassium binding in Bs_ArsC [25]. An interesting feature observed in the amino acid sequence of DaG20_ArsC3 is the presence of Lys62. In C. glutamicum, (Cg_ArsC2, PDB ID: 3RH0), the positive charge of the side chain N–H group in Lys64 (which is the equivalent of DaG20_ArsC3 Lys62) replaces potassium in its important role on the hydrogenbonding network, involving Cys8 and Asn11 [16], suggesting that the same may occur in DaG20_ArsC3.
The kcat values obtained for DaG20_ArsC3, in the presence of either sulfate, K? or Na?, are relatively constant for the three ion-dependent conditions tested, although some decrease is obtained in the presence of Na?, as observed for Bs_ArsC. However, the kcat values are twice as large as the values obtained for Bs_ArsC. The results presented show, for the first time, a Trx-coupled arsenate reductase in which the high efficiency is not dependent on cation binding or sulfate stabilization. The low Km together with the high kcat observed for DaG20_ArsC3 makes this enzyme one of the most efficient arsenate reductases described so far. The catalytic efficiencies are approximately 10 times higher for DaG20_ArsC3 compared with Bs_ArsC and Sa_ArsC for all conditions tested, except for Sa_ArsC in the presence of KCl. However, the catalytic efficiency of DaG20_ArsC3 under this reaction condition is still 5 times higher. The fact that sulfate-reducing bacteria occupy anaerobic and/or corroded environments and play an important role on the biogeochemical cycles of sulfur, carbon and nitrogen makes it more relevant for them to be able to respond to not only arsenic but also other heavy metal stresses [28, 34, 41, 42]. The low Km observed representing a high
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Table 2 Kinetic parameters determined for DaG20_ArsC3 in different buffers (50 mM Tris–HCl,pH 7.6, 0.1 mM EDTA, with either 50 mM K2SO4, 150 mM KCl or 150 mM NaCl) Buffer
Km (lM)
kcat (min-1)
kcat/Km (M-1 s-1) (9104)
References
This paper
and simultaneously avoiding a competition for sulfate between DaG20_ArsC3 and the respiratory processes.
Conclusion
DaG20_ArsC3 K2SO4
7
172
40.9
KCl
6
191
53.1
NaCl
8
123
25.6
K2SO4
47
95
3.3
KCl
54
96
3.0
NaCl
58
80
2.3
Bs_ArsC 26
Sa_ArsC K2SO4
81
219
4.5
KCl
9
54
10.0
NaCl
22
35
2.7
26
affinity for arsenate of DaG20_ArsC3 that is cation and sulfate independent, together with the high kcat acquires a particular relevance in this type of bacteria that uses sulfate as terminal electron acceptor in the anaerobic respiratory process. This efficient arsenate reducing system allows a fast trapping of arsenate inside the cell that can rapidly be extruded, preventing the toxic effects of this heavy metal,
Time (min)
Time (min) 10 20 30 40 50 60 70 80 90
-0.20
-0.40
-0.40
µcal/sec
-0.20
-0.60
-0.80
-1.00
-1.00
0.00
0.00
-0.04
10 20 30 40 50 60 70 80 90
-0.60
-0.80
-0.04
-0.08
-0.08 0
2
4
6
8
10
Molar Ratio
KCl
123
-10 0 0.00
kcal mol-1 of injectant
µcal/sec
-10 0 0.00
kcal mol-1 of injectant
Fig. 4 Isothermal calorimetric titration curves of DaG20_ArsC3 with KCl and NaCl at 37 8C, respectively. The upper part of each panel show the raw heats of binding, whereas the lower parts show the integrated areas under the respective peaks in the top panel plotted against the molar ratio of KCl and NaCl titrated into DaG20_ArsC3. These data are illustrative of a lack of binding
Desulfovibrio alaskensis G20 has an operon with two genes encoding two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3 that, according to the amino acid sequence alignment, belong to the Trx-coupled arsenate reductase family. However, DaG20_ArsC2 does not contain a conserved Cys-Xn-Arg P-loop signature motif and resistance assays in E. coli have shown that the expression of this protein does not confer any additional resistance to arsenate; therefore, DaG20_ArsC3 must be the enzyme responsible for arsenate reduction in D. alaskensis G20 encoded by the arsRBC2C3 operon. DaG20_ArsC3 steady-state kinetic assays using thioredoxin, thioredoxin reductase, and NADPH confirmed these proteins as co-factors of arsenate reduction. This enzyme is the first arsenate reductase isolated from an anaerobic sulfate-reducing bacterium. Like Bs_ArsC, DaG20_ArsC3 kinetics is cation and sulfate independent although the low Km is similar to Sa_ArsC in the presence of potassium. These results dissociate for the first time low Km from potassium specific binding on Trx-arsenate family. Kinetics analysis indicated that DaG20_ArsC3 acts more
12
14
16
-2
0
2
4
6
8
10
Molar Ratio
NaCl
12
14
16
J Biol Inorg Chem (2014) 19:1277–1285
efficiently as arsenate reductase than phosphatase. Furthermore, DaG20_ArsC3 was shown to be highly efficient compared with other arsenate reductases described in the literature and therefore is a unique member of the Trxcoupled arsenate reductase family. Acknowledgments We would like to thank Fundac¸a˜o para a Cieˆncia e Tecnologia for the grants PEst-C/EQB/LA0006/2011 and PTDC/BIA-PRO/103980/2008 and fellowship SFRH/BD/62051/2009 to CIPN.
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