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Thermostable properties of the periplasmic selenate reductase from Thauera selenatis
Biochimie 92 (2010) 1268e1273
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Research paper
Thermostable properties of the periplasmic selenate reductase from Thauera selenatis Elizabeth J. Dridge, Clive S. Butler* School of Biosciences, Centre for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2010 Accepted 4 June 2010 Available online 12 June 2010
Selenate reductase (SER) from Thauera selenatis is a member of a distinct class of the TAT-translocated type II molybdoenzymes and is closely related to a group of thermostable nitrate reductases (pNAR) found in hyperthermophilic archaea. In the present study the thermostable and thermo-active properties of SER, isolated with either molybdenum (Mo) or tungsten (W) at the active site, are reported. Results show that the purified MoeSER complex is stable and active upon heat-shock incubation for 10 min at temperatures up to 60 C. At temperatures greater than 65 C all three subunits (SerABC) are readily denatured. The optimum temperature for maximum activity recorded was also determined to be 65 C. T. selenatis can grow readily on a tungstate rich medium up to concentrations of 1 mM. SER isolated from periplasmic fractions from cells grown on 1 mM tungstate displayed selenate reductase activities with a 20-fold reduction in Vmax (0.01 mmol [S]/min/mg) and a 23-fold increase in substrate binding affinity (Km 0.7 mM). The thermo-stability and pH dependence of WeSER was shown to be similar to that observed for MoeSER. By contrast, the optimum reaction temperature for WeSER exceeded the maximum temperature tested (>80 C). The combined data from the kinetic analysis and thermal activity profiles provide evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. It is argued that despite the similarity in their catalytic and electron conducting subunits, the presence of a membrane anchor in the archaeal pNAR system appears pivotal to the enhanced hyperthermostability. The fact that MoeSER is thermostable up to 65 C however, could be advantageous when designing selenate contamination remediation strategies. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Selenate reduction Molybdoenzyme Tungstoenzyme thermo-stability
1. Introduction A class of thermostable oxyanion reductases have been identified in a number of hyperthermophilic archaea [1,2]. These reductases are related to the well characterised respiratory nitrate reductase (NAR) family [1], but unlike their mesophilic counterparts, their catalytic NarG-like components, possess an N-terminal signal peptide that facilitates the passage across the cytoplasmic membrane via the twin-arginine translocation (TAT) pathway [1,3,4]. The enzyme from the hyperthermophilic archaeon Pyrobaculum aerophilum has been purified and characterised [2]. The enzyme complex contains molybdenum, non-haem iron and cytochrome b. The protein shows a very high specific activity with
Abbreviations: MGD, molybdopterin guanine dinucleotide; SER, periplasmic selenate reductase; NAR, membrane-bound nitrate reductase; pNAR, membranebound nitrate reductase orientated such that the active site faces the positive side of the membrane; NAP, periplasmic nitrate reductase; DMSO, dimethylsulfoxide; TMAO, trimethylamine N-oxide; TAT, twin-arginine translocation. * Corresponding author. Tel.: þ44 1392 264675; fax: þ44 1392 263434. E-mail address: [email protected] (C.S. Butler). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.06.003
the substrate nitrate (Vmax 1162 s1) and shows optimum temperature for activity at >95 C. When incubated at 100 C, the purified enzyme has a half-life of 1.5 h [2]. These nitrate reductases, referred to recently as positive nitrate reductases (pNAR) due to their location on the positive side (DJþ) of the cytoplasmic membrane, are found in a number of archaea (both hyperthermophiles and halophiles) and analysis of all available genomic data suggests that they are part of a distinct clade of the DMSO reductase family of type II (D-group) molybdoenzymes [1]. Interestingly, this clade includes also a number of enzymes from mesophilic bacteria that function as either dehydrogenases, such as ethylbenzene dehydrogenase from Aromatoleum aromaticum [5,6] and dimethylsulphide dehydrogenase from Rhodovulum sulfidophilium [7,8] or as reductases, such as selenate reductase from Thauera selenatis [9] and chlorate reductase from Ideonella dechloratans [10,11]. Sequence alignments demonstrate that these mesophilic enzymes show similarity to both the catalytic components and the electron-transferring ironesulphur protein components of the archaeal pNAR group [1]. A good example is shown by the comparison of selenate reductase (SER) from T. selenatis with the pNAR from P. aerophilum (Fig. 1). SER is a trimeric (abg) complex
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The bacterial reduction of selenate is considered an essential process in the global selenium cycle and might have significant implications for selenium contamination remediation. The amount of selenate that can be extracted from contaminated soil is of considerable importance when designing a bioremediation strategy and can be increased significantly using water at elevated temperature [18]. Under these conditions SeO2 4 is the predominant Se species in hot aqueous extracts. The hydroxyl ions in hot water substitute for non-specifically absorbed SeO2 4 that is weakly bound in outeresphere complexes. This is also consistent with thermodynamic predictions regarding SeO2 4 solubility [18]. Once extracted, selenate could be reduced by SER to selenite, followed by the reduction to selenium by a number of reducing agents. Consequently, understanding the thermostable properties of SER could aid improving enzyme-based extraction procedures. Given the distinct similarity between SerAB and pNarGH, it raises the question as to whether SER also displays the enhanced thermo-stability shown by the pNAR from P. aerophilum. It is also well documented that growth of a number of the hyperthermophilic archaea are strictly dependent upon tungstate in the growth medium and that tungstoenzyme are more thermostable than their molybdoenzyme counterparts [19]. By contrast to E. coli, where NAR is inactivated by the presence of tungstate in the growth medium, pNAR from P. aerophilum was still active when cultured in elevated concentrations of tungstate [20], suggesting that the thermostable pNAR group might be able to function with a tungsten active site. The aim of the present study was to investigate both the thermostable properties of, and the effect of tungsten on the periplasmic selenate reductase as a means to assess its suitability for thermo-activated selenium extraction procedures.
Fig. 1. TAT-translocated type II (D-group) molybdoproteins. A: Schematic representation of the molybdo-oxidoreductases from the mesophile T. selenatis (SerABC) and the hyperthermophilic archaea P. aerophilum and A. fulgidus (pNarGH). B: Sequence alignment of SER and pNAR showing (in bold) conserved twin-arginine motif and the aspartate (D) Mo ligand. Sequences are from T. selenatis (Ts), P. aerophilum (Pa) and A. fulgidus (Af).
with an apparent molecular mass of w180 kDa that catalyses the 2 reduction of selenate (SeO2 4 ) to selenite (SeO3 ) and supports selenate respiration in T. selenatis [9]. The three subunits consist of SerA (96 kDa e a subunit), SerB (40 kDa e b subunit), and SerC (23 kDa e g subunit) [9]. SerA is the catalytic subunit containing the molybdenum active site in the form of the molybdopterin guanine dinucleotide (bis-MGD) cofactor [9,12,13]. SerB contains a number of cysteine rich motifs that co-ordinate a [3Fee4S] and three [4Fee4S] ironesulphur clusters [12,13]. SerA shows 31% identity and 41% similarity to pNarG from P. aerophilum, and is of a similar size (w100 kDa) [1], approximately 40 kDa smaller than NarG from the mesophilic bacteria. Both SerA and pNarG have a conserved motif (H/CX3CX3CX29e34C) towards the N-terminus [14]. The motif is conserved also in Escherichia coli NarG and has been shown to coordinate a [4Fee4S] (FS0) cluster in the three dimensional crystal stuctures [15,16]. The aspartate residue that co-ordinates the Mo in the bis-MGD cofactor in E. coli NarG (D222) is again conserved in both the pNAR group and SerA (Fig. 1B). Component pNarH is a molybdopterin oxidoreductase ironesulphur binding protein and shows 29% identity and 37% similarity to SerB and low identity with E. coli NarH. The final component in the hyperthermophilic archaeal pNAR system is a membrane-bound b-type cytochrome anchor. By contrast, the SerC subunit is a soluble b-type cytochrome that accepts electrons from either a quinol-cytochrome c oxidoreductase (QCR) or a quinol dehydrogenase via a soluble cytochrome c4 [17].
2. Materials and methods 2.1. Growth conditions of T. selenatis T. selenatis was grown anaerobically at 30 C in mineral salts medium [21] containing yeast extract (0.1%), selenate (10 mM) and acetate (10 mM) in 10 L batch cultures. When grown on tungstate rich medium, ammonium molybdate was removed from the trace elements solution and replaced with sodium tungstate at the required concentration. To ensure that tungstate media were molybdate-free, all glassware was acid washed and only ultra-pure H2O was used during medium preparation. Cultures were harvested during late log phase (after 16e18 h growth) at OD600nm 0.6e0.7 and spheroplasts were prepared as described previously [9]. The spheroplasts were removed by centrifugation and the supernatant containing periplasm was retained. 2.2. Purification of SER from T. selenatis and the preparation of samples for thermo-stability profiling The periplasmic selenate reductase from T. selenatis was purified as described previously [9] following a method adapted from Schroder et al. [9,13]. Purified SerABC samples were heated at a range of temperatures (30e80 C) for 10 min and centrifuged (12,000g, 5 min) to remove precipitated proteins. Protein samples were analysed for thermal stability via SDS-PAGE (gel electrophoresis). Samples were loaded onto a 10% BiseTris NuPAGEÒ Gel. The protein bands were separated by electrophoresis at 200 V, 125 mA, for 50 min. Scanned images of the gels were stored and densitometry was used to calculate the proportion of protein denatured at each temperature. The percentage of protein denatured at increasing temperature was determined by comparison to the amount of soluble protein retained following treatment at 30 C. To determine the metal (Mo and W) content of the selenate reductase,
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samples were analysed by inductively coupled plasma mass spectroscopy (ICPMS). Standards of Mo and W were prepared at the following concentrations 1, 5, 10, 25, 50 and 100 ppb. All samples and standards were treated with 6.5% nitric acid and analysed using a Thermo X-series ICPMS spectrometer in accordance with manufacturer’s instructions. 2.3. Enzyme activity assays Selenate reductase activity was measured in a cuvette assay by monitoring the oxidation of reduced methyl viologen (1 mM) spectrophotometrically at 600 nm, coupled to the reduction of selenate [22]. Activities were calculated from the initial rates using an extinction coefficient of 13,700 M1 cm1 for the methyl viologen radical [23]. Values for Km and Vmax cited in the text and tables are the mean values (n ¼ 3) calculated using non-linear regression analysis in Grafit v3.0 (Erithacus software). To determine the activity retained by the heat-shocked SerABC, samples were incubated at a range of temperatures (30e80 C) for 10 min, prior to activity measurements at ambient temperature. 10 min incubation was chosen because the viologen assay for selenate reductase activity is typically measured over a 5 min time period, so it was considered appropriate that pre-incubation at each temperature was for longer than the assay reaction. To determine the optimum temperature for reductase activity, assays were undertaken at a range of fixed temperatures (ambient e 80 C) and monitored for a 5 min period. The protein concentrations were determined using Biorad reagents following the manufacturers’ protocol.
(Fig. 2A). All three subunits can still be seen clearly in samples heated to 70 C, with only the smallest subunit, SerC, being lost upon heating up to 80 C. In order to quantify the amount of SerABC that remains in a soluble state following heat-shock, the protein band intensities were determined using scanning densitometry (Fig. 2B). Results show that upon incubation up to 60 C for 10 min >70% of the soluble protein is retained. Incubation at 80 C results in retention of soluble SerA and SerB at 17% and 25% respectively. SerC is not detectable after incubation at 80 C and indicates that it is fully denatured. It is noteworthy that a number of contaminating proteins appear to be removed upon heating to >40 C. Most notably, the protein at 28 kDa, which we have previously identified as uridine phosphorylase [13], is completely removed by heating the sample to 50 C. Consequently, this short heat-step makes a useful addition to the purification protocol. The purified enzyme was assayed for selenate reductase activity following incubation at the corresponding range of temperatures to analyse the retention of activity (Fig. 2C). Full activity was maintained up to 60 C, with activity falling to 25% at 70 C. Protein that had been incubated at 80 C for 10 min also retained 25% original activity. All activity was lost upon heating the sample to >90 C. In order to investigate the structural integrity of the SerABC complex, samples were also analysed by circular dichroism (Fig. 2D). Proteins retained a folded confirmation up to 70 C. Selenate reductase activity was also measured at increasing temperature to determine the optimum temperature for methyl viologen dependant activity. Maximum activity was determined at 65 C. 3.2. The effect of tungsten on the growth of T. selenatis
2.4. Circular dichroism analysis SerABC was concentrated to w20 mg/ml and buffer exchanged into 50 mM phosphate buffer. Circular dichroism spectroscopy was used to check for folded protein and the effect of increasing temperature on structural integrity. Samples were incubated at 30 C, 60 C and 80 C for 10 min. Samples were analysed using a Jasco J-810 spectropolarimeter over a wavelength range of 185e250 nm at a scanning speed of 50 nm min1 at room temperature (10 scans). 2.5. Determination of pH optimum Activity measurements to determine pH optimum were undertaken using the microtitre plate assay as described previously [24]. 2.6. Activity gels Proteins were separated using non-denaturing PAGE (6% Triseglycine gels), and gels were stained with sodium dithionitereduced methyl viologen (5 mM) under anaerobic conditions, using a small anaerobic chamber and purged with O2-free nitrogen for 30 min. Protein bands with selenate reductase activity were identified by the addition of selenate for 15 min and observed as clear zones. 3. Results 3.1. Determination of the thermo-stability of SerABC To determine the thermal stability of the purified selenate reductase, samples were heated to a range of temperatures (30 C, 40 C, 50 C, 60 C, 70 C and 80 C) for 10 min and centrifuged to remove precipitated proteins. The supernatant was then analysed by SDS-PAGE and resolution of the retained SerA, SerB and SerC components was taken as evidence of thermostable protein
T. selenatis was grown anaerobically as described, and growth monitored for 26 h (Fig. 3A). Cells grown on Hungates medium or Hungates medium supplemented with 1 mM molybdenum displayed typical growth curves, with stationary phase being reached at OD600nm ¼ 0.7. Cultures supplemented with tungstate at a range of concentrations (10e100 mM) had little effect to both growth rate and yield. With tungstate added to a final concentration of 1 mM, growth was inhibited slightly, giving a final optical density of OD600nm ¼ 0.6. The presence of 15 mM tungstate severely slowed the growth of T. selenatis, with no exponential growth being observed, reaching a final OD600nm ¼ 0.1 after 26 h. The ability to tolerate 1 mM tungsten without significantly inhibiting selenate respiration was interesting and suggested that perhaps selenate reductase can function with tungsten at the active site. To investigate the tungsten substituted selenate reductase (WeSER) further, periplasmic fractions from T. selenatis, grown on either molybdate (1 mM) or tungstate (1 mM) rich medium were generated. Periplasmic fractions were separated by non-denaturing PAGE and stained for selenate reductase activity (Fig. 3B). The resolution of clear bands of similar mobility demonstrated that a functional selenate reductase was detectable in periplasms from both molybdate and tungstate grown cells. 3.3. Thermo-stability of WeSER The thermo-stability of the WeSerABC complex was investigated using SDS-PAGE analysis. Periplasmic fractions were incubated at 30e80 C for 10 min (heat-shocked) and denatured proteins removed by centrifugation. Samples were analysed by SDS-PAGE as before and the % of soluble protein retained determined by densitometry. Levels of expression of SerA were reduced by 50% when compared to molybdate grown cells. Fig. 4A shows the retention of soluble SER proteins at increasing temperature. WeSerA shows a similar stability profile to MoeSerA with approximately 50% of the protein remaining soluble at incubation
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Fig. 2. Thermo-stability of SerABC. A: SDS-PAGE gel showing the thermal stability of SerABC. Lanes 1 and 8, Invitrogen SeeBlueÒ Ladder; Samples were heated for 10 min at; 30 C (lane 2), 40 C (lane 3), 50 C (lane 4), 60 (lane 5), 70 C (lane 6) and 80 C (lane 7). Total protein loaded was 10 mg per lane. B: Graph showing the decreasing thermal stability of the components of SerABC upon heat treatment. Densitometry values of the protein bands were used to calculate the % of soluble protein retained. Symbols represent; SerA (-), SerB (C) and SerC (:). C: Graph showing the thermal stability of selenate reductase activity. Samples were heated to a range of temperatures for 10 min before being assayed for selenate reductase activity. Assays were performed ambient temperature. Results are means of 3 determinants, SD < 5%. D: Circular dichroism spectra of SerABC. Untreated SerABC (solid line), SerABC heat treated at 60 C for 10 min (dashed line) and heat treated at 80 C for 10 min (dotted line).
at w65 C, however, by contrast to MoeSerA, the WeSerA protein appears less stable at 60 C (WeSerA w66%; MoeSerA w80%). Interestingly, the most notably difference is in the stability of the SerB and SerC components. Both seem to be more labile in the WeSER complex. The SerC component is lost from the complex upon heating to temperatures >70 C, with only 50% being retained at 60 C. Similarly, 50% of SerB remains soluble in samples heated to 60 C and none is retained post incubation at 80%. These results suggest that substituting W for Mo at the active site of SerA decreases the overall stability of the SerABC complex, rendering the components less thermostable. Given the much lower level of activity detected, attempts to purify the WeSER using the established chromatography protocols were unsuccessful, so heating samples to w50 C for 10 min was used to obtain a partially-purified sample for subsequent kinetic studies. Partially-pure samples were judged to be enriched such that SER constituted 50% of the total soluble periplasmic protein. Both MoeSER and WeSER were analysed for metal content using ICPMS to confirm the metal active centre. SER isolated for molybdate grown cells had a Mo content of 0.5 mol of Mo per mol of enzyme. SER partially-purified from periplasmic fractions from cells grown in a tungstate rich medium showed no detectable levels of Mo and had an estimated W content of 0.6 mol of W per mol of enzyme. It has been demonstrated previously [13] that purification of MoeSER to homogeneity results in a loss of activity and a concomitant decrease in Mo cofactor. Consequently, the sub-stoicheiometric ratio of W:SER would also suggest that the incorporation of a tungstopterin cofactor does not improve cofactor retention.
3.4. Kinetic parameters of WeSER The partially-purified WeSER was assayed at a range of selenate concentrations. Using reduced methyl viologen as the artificial electron donor, the Km (0.7 mM) and Vmax (0.01 mmol [S]/min/mg) were determined (Table 1.). These results show that although the enzyme is working at a slower rate than MoeSER (0.2 mmol [S]/min/mg), the affinity for selenate is approximately 23-fold higher (MoeSER; Km ¼ 16 mM). The thermal activity of the WeSER was also investigated by assaying for enzyme activity at increasing temperature. When assaying WeSER samples at high temperature (80 C), the samples were equilibrated rapidly and reductase activity measured immediately. The initial rate of viologen oxidation was recorded and shown to increase with temperature up to our limit of 80 C. However, during the assay (after the first 60 s) the steady state activity showed a significant drop-off, consistent with the prolonged incubation (10 min at 80 C), so called “heat-shocked protein”, that resulted in loss of soluble protein at this temperature. 3.5. pH dependence of WeSerABC WeSER was assayed at a range of different pH to determine the pH dependence of the WeSER enzyme (Fig. 4B). The pH optimum for MoeSER in vitro has been determined previously to be pH 6.0, an observation that is consistent with our data [25]. Analysis of WeSER shows that reductase activity is also optimum at pH 6.0, with the enzyme retaining <20% activity when assayed at pH 8.0.
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Fig. 3. The effect of tungsten on selenate respiration. A: Growth curve of T. selenatis on varying concentrations of tungstate. Hungates medium only (-), Hungates medium supplemented with 1 mM Mo (B), Hungates medium supplemented 1 mM W (:) and Hungates medium supplemented with 15 mM W (7). B: Native PAGE gels of periplasmic fractions isolated from Mo and W grown T. selenatis. 10 mg of protein were loaded per lane. Gels were stained for SER activity by submersion in reduced methyl viologen solution (5 mM) and developed by the addition of selenate (10 mM).
Fig. 4. Properties of WeSER. A: Graph showing the decrease in thermal stability of the separate components of the WeSER complex upon heat treatment. Densitometry values of the protein bands were used to calculate the % of soluble protein retained. Symbols represent; SerA (-), SerB (C) and SerC (:). B: Graph showing the pH dependence of selenate reductase activity in periplasmic fractions from T. selenatis grown in the presence of 1 mM tungsten. Results are the means of 6 determinants, SD < 5%.
4. Discussion The data presented demonstrate that the SER enzyme is stable and active upon incubation at temperatures up to 60 C, with an optimum activity recorded at 65 C. The SerC component appears to be least stable once above 60 C and perhaps the loss of this subunit contributes to the overall instability of the SER complex. The SerC component is rather unusual in that it co-ordinates a b-type cytochrome that is rarely found in soluble periplasmic proteins [12,13]. Unlike the common c-type haems, the b-type haems are not covalently attached via a CXXCH motif and as a consequence might not form such highly stable holoproteins. The soluble SerC-like component is missing in the pNAR enzymes from P. aerophilum and A. fulgidus and is replaced by a membrane anchored subunit. It has been suggested that the association with the membrane might aid the overall thermo-stability of the pNAR complexes from the hyperthermophiles [2] and our results support this hypothesis. The observation that SER is thermostable up to 60 C is consistent with reports of other molybdoenzymes from mesophilic bacteria. The TMAO reductase from E. coli, an enzyme also classified as a member of the DMSO reductase molybdoenzyme family, is thermostable up to 60 C with optimum activity recorded at 65 C [26]. Again, similar to SER, TMAO reductase is located as a soluble protein in the periplasm and not anchored to the cytoplasmic membrane. Tungsto-enzymes are often associated with the hyperthermophilic archaea and have been reported to have enhanced thermo-stability and thermo-activity capabilities [19]. Analogue
reaction systems using bis(dithiolene) complexes of MoIV and WIV have both been shown clearly to reduce selenate to selenite by Moand W-mediated oxo-transfer reactions [27], demonstrating unequivocally that chemically W analogues can function as a catalyst for selenate reduction. However, to date no evidence has been presented regarding the effect of tungsten on selenate reduction in an enzyme system (i.e., selenate reductase). Our results demonstrate that T. selenatis can tolerate tungstate concentrations approaching 1 mM without significantly affecting both growth rates and biomass yields. SER isolated form cells grown in a tungstate rich environment showed a 20-fold reduction in selenate reductase activity, but with an increased affinity for selenate (Km 0.7 mM). These observations (kinetics and thermodynamics) are likely to be linked. The tungsten substitution may have enhanced the bond strengths of the WVIesubstrate complexes, thus
Table 1 Kinetic parameters for both Mo and W derivatives of SER Enzyme derivative
Kinetic constanta Vmax (mmol [S]/min/mg)
Km (mM)
MoeSER WeSER
0.2 0.01
16 0.7
a Kinetic constants were measured using methyl viologen as the electron donor. Variation was within 10% of the mean values shown (n ¼ 3). Protein concentration was 14 mg/ml. Selenate was added at a range of concentrations up to 4 mM.
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stabilising the W/OSeO2 3 bond, leading to the observed higher substrate affinity. This in turn may have led to complexes which were kinetically slower than the equivalent Moesubstrate complexes, when measured at the mesophilic range of temperatures [19]. Finally, little is known about selenate reduction/respiration in thermophiles, but emerging data suggests that it might be a widespread activity. P. aerophilum can grow organotrophically in anaerobic conditions in the presence of selenate and/or selenite as the sole electron acceptors [28]. Pyrobaculum arsenaticum, a strictly hyperthermophilic facultative organotrophic archaeon isolated from hot springs in Naples, Italy, can also grow using selenate as the sole electron acceptor, reducing selenate to elemental selenium [28]. It is likely that selenate reduction is a common activity in the hyperthermophiles, since their hydrothermal vent habitats often display elevated levels of selenium compounds [29]. The selenate reductases within these hyperthermophilic organisms must also possess characteristics enabling them to function at elevated temperatures. The results reported in the present work would indicate that a hypertheromophilic selenate reductase is likely to be associated with the membrane and potentially could function with a Wecofactor. 5. Conclusions The present work has investigated the thermostable properties of SER from T. selenatis. The enzyme is stable and active upon incubation at temperatures up to 60 C, with an optimum activity at 65 C. T. selenatis can grow readily on a tungsten rich medium (up to 1 mM) and SER isolated under such conditions displayed reductase activities with a 20-fold reduction in Vmax and a 23-fold increase in Km. The thermo-stability and pH dependence of WeSER was shown to be similar to that observed for MoeSER. The combined data from the kinetic analysis and thermal activity profiles provide convincing evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. The observations that SER from T. selenatis is stable at w65 C might prove usefully when designing selenate remediation strategies. Acknowledgments This work was funded in part by research grants from the Biotechnology and Biological Sciences Research Council (BBSRC). EJD was a recipient of a BBSRC Plant and Microbial Sciences (PMS) committee studentship. Thauera selenatis was kindly provided by Dr Joanne Santini (University College London). We acknowledge help from Dr Carys Watts (University of Newcastle) with protein purification and we thank Prof. David Richardson (University of East Anglia) for useful discussions. References [1] R.M. Martinez-Espinosa, E.J. Dridge, M.J. Bonete, J.N. Butt, C.S. Butler, F. Sargent, D.J. Richardson, Look on the positive side! The orientation, identification and bioenergetics of "Archaeal" membrane-bound nitrate reductases. FEMS Microbiol. Lett. 276 (2007) 129e139. [2] S. Afshar, E. Johnson, S. De Vries, I. Schröder, Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilium. J. Bacteriol. 183 (2001) 5491e5495. [3] F. Sargent, Constructing the wonders of the bacterial world: biosynthesis of complex enzymes. Microbiology 153 (2007) 633e651. [4] B.C. Berks, F. Sargent, T. Palmer, The Tat protein export pathway. Mol. Microbiol. 35 (2000) 260e274.
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Vance, Soil and plant selenium at a reclaimed uranium mine. J. Environ. Qual. 31 (2002) 1516e1521. [19] M.K. Johnson, D.C. Rees, M.W.W. Adams, Tungstoenzymes. Chem. Rev. 96 (1996) 2817e2839. [20] S. Afshar, C. Kim, H.G. Monbouquette, I. Schröder, Effect of tungstate on nitrate reduction by the hyperthermophilic archaeon Pyrobaculum aerophilium. Appl. Environ. Microbiol. 64 (1998) 3004e3008. [21] J.M. Macy, S. Rech, G. Auling, M. Dorsch, E. Stackenbrandt, L.I. Sly, Thauera selenatis gen-nov, sp-nov, a member of the beta-subclass of proteobacteria with a novel type of anaerobic respiration. Int. J. Syst. Bacteriol. 43 (1993) 135e142. [22] A.L. Craske, S.J. Ferguson, The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterisation and kinetic properties. Eur. J. Biochem. 158 (1986) 429e436. [23] T. Watanabe, K. Honda, Measurement of the extinction coefficient of the methyl viologen cation radical and the efficiency of its formation by semiconductor photocatalysis. J. Phys. Chem. 86 (1982) 2617e2619. [24] H. Ridley, C.A. Watts, D.J. Richardson, C.S. Butler, Development of a viologen based microtitre plate assay for the analysis of oxyanion reductase activity: application to the membrane bound selenate reductase from Enterobacter cloacae SLD1a-1. Anal. Biochem. 358 (2006) 289e294. [25] S.A. Rech, J.M. Macy, The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes. J. Bacteriol. 174 (1992) 7316e7320. [26] J. Buc, C.-L. Santini, R. Giordani, M. Czjzek, L.-F. Wu, G. Giordano, Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli. Mol. Microbiol. 32 (1999) 159e168. [27] J.-J. Wang, C. Tessier, R.H. Holm, Analogue reaction systems of selenate reductase. Inorg. Chem. 45 (2006) 2979e2988. [28] R. Huber, M. Sacher, A. Vollmann, H. Huber, D. Rose, Respiration of arsenate and selenate by hyperthermophilic archaea. Syst. Appl. Microbiol. 23 (2000) 305e314. [29] R. Knott, A.E. Fallick, K.J. Richards, H. Bäcker, Mineralogical and sulphur isotope characteristics of a massive sulfide boulder, Galapagos Rift, 85-550W. in: L.M. Parson, C.L. Walker, D.R. Dixon (Eds.), Hydrothermal Vents and Processes, vol. 87. Geological Society Special Publication, 1995, pp. 207e222.
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Research paper
Thermostable properties of the periplasmic selenate reductase from Thauera selenatis Elizabeth J. Dridge, Clive S. Butler* School of Biosciences, Centre for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2010 Accepted 4 June 2010 Available online 12 June 2010
Selenate reductase (SER) from Thauera selenatis is a member of a distinct class of the TAT-translocated type II molybdoenzymes and is closely related to a group of thermostable nitrate reductases (pNAR) found in hyperthermophilic archaea. In the present study the thermostable and thermo-active properties of SER, isolated with either molybdenum (Mo) or tungsten (W) at the active site, are reported. Results show that the purified MoeSER complex is stable and active upon heat-shock incubation for 10 min at temperatures up to 60 C. At temperatures greater than 65 C all three subunits (SerABC) are readily denatured. The optimum temperature for maximum activity recorded was also determined to be 65 C. T. selenatis can grow readily on a tungstate rich medium up to concentrations of 1 mM. SER isolated from periplasmic fractions from cells grown on 1 mM tungstate displayed selenate reductase activities with a 20-fold reduction in Vmax (0.01 mmol [S]/min/mg) and a 23-fold increase in substrate binding affinity (Km 0.7 mM). The thermo-stability and pH dependence of WeSER was shown to be similar to that observed for MoeSER. By contrast, the optimum reaction temperature for WeSER exceeded the maximum temperature tested (>80 C). The combined data from the kinetic analysis and thermal activity profiles provide evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. It is argued that despite the similarity in their catalytic and electron conducting subunits, the presence of a membrane anchor in the archaeal pNAR system appears pivotal to the enhanced hyperthermostability. The fact that MoeSER is thermostable up to 65 C however, could be advantageous when designing selenate contamination remediation strategies. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Selenate reduction Molybdoenzyme Tungstoenzyme thermo-stability
1. Introduction A class of thermostable oxyanion reductases have been identified in a number of hyperthermophilic archaea [1,2]. These reductases are related to the well characterised respiratory nitrate reductase (NAR) family [1], but unlike their mesophilic counterparts, their catalytic NarG-like components, possess an N-terminal signal peptide that facilitates the passage across the cytoplasmic membrane via the twin-arginine translocation (TAT) pathway [1,3,4]. The enzyme from the hyperthermophilic archaeon Pyrobaculum aerophilum has been purified and characterised [2]. The enzyme complex contains molybdenum, non-haem iron and cytochrome b. The protein shows a very high specific activity with
Abbreviations: MGD, molybdopterin guanine dinucleotide; SER, periplasmic selenate reductase; NAR, membrane-bound nitrate reductase; pNAR, membranebound nitrate reductase orientated such that the active site faces the positive side of the membrane; NAP, periplasmic nitrate reductase; DMSO, dimethylsulfoxide; TMAO, trimethylamine N-oxide; TAT, twin-arginine translocation. * Corresponding author. Tel.: þ44 1392 264675; fax: þ44 1392 263434. E-mail address: [email protected] (C.S. Butler). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.06.003
the substrate nitrate (Vmax 1162 s1) and shows optimum temperature for activity at >95 C. When incubated at 100 C, the purified enzyme has a half-life of 1.5 h [2]. These nitrate reductases, referred to recently as positive nitrate reductases (pNAR) due to their location on the positive side (DJþ) of the cytoplasmic membrane, are found in a number of archaea (both hyperthermophiles and halophiles) and analysis of all available genomic data suggests that they are part of a distinct clade of the DMSO reductase family of type II (D-group) molybdoenzymes [1]. Interestingly, this clade includes also a number of enzymes from mesophilic bacteria that function as either dehydrogenases, such as ethylbenzene dehydrogenase from Aromatoleum aromaticum [5,6] and dimethylsulphide dehydrogenase from Rhodovulum sulfidophilium [7,8] or as reductases, such as selenate reductase from Thauera selenatis [9] and chlorate reductase from Ideonella dechloratans [10,11]. Sequence alignments demonstrate that these mesophilic enzymes show similarity to both the catalytic components and the electron-transferring ironesulphur protein components of the archaeal pNAR group [1]. A good example is shown by the comparison of selenate reductase (SER) from T. selenatis with the pNAR from P. aerophilum (Fig. 1). SER is a trimeric (abg) complex
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The bacterial reduction of selenate is considered an essential process in the global selenium cycle and might have significant implications for selenium contamination remediation. The amount of selenate that can be extracted from contaminated soil is of considerable importance when designing a bioremediation strategy and can be increased significantly using water at elevated temperature [18]. Under these conditions SeO2 4 is the predominant Se species in hot aqueous extracts. The hydroxyl ions in hot water substitute for non-specifically absorbed SeO2 4 that is weakly bound in outeresphere complexes. This is also consistent with thermodynamic predictions regarding SeO2 4 solubility [18]. Once extracted, selenate could be reduced by SER to selenite, followed by the reduction to selenium by a number of reducing agents. Consequently, understanding the thermostable properties of SER could aid improving enzyme-based extraction procedures. Given the distinct similarity between SerAB and pNarGH, it raises the question as to whether SER also displays the enhanced thermo-stability shown by the pNAR from P. aerophilum. It is also well documented that growth of a number of the hyperthermophilic archaea are strictly dependent upon tungstate in the growth medium and that tungstoenzyme are more thermostable than their molybdoenzyme counterparts [19]. By contrast to E. coli, where NAR is inactivated by the presence of tungstate in the growth medium, pNAR from P. aerophilum was still active when cultured in elevated concentrations of tungstate [20], suggesting that the thermostable pNAR group might be able to function with a tungsten active site. The aim of the present study was to investigate both the thermostable properties of, and the effect of tungsten on the periplasmic selenate reductase as a means to assess its suitability for thermo-activated selenium extraction procedures.
Fig. 1. TAT-translocated type II (D-group) molybdoproteins. A: Schematic representation of the molybdo-oxidoreductases from the mesophile T. selenatis (SerABC) and the hyperthermophilic archaea P. aerophilum and A. fulgidus (pNarGH). B: Sequence alignment of SER and pNAR showing (in bold) conserved twin-arginine motif and the aspartate (D) Mo ligand. Sequences are from T. selenatis (Ts), P. aerophilum (Pa) and A. fulgidus (Af).
with an apparent molecular mass of w180 kDa that catalyses the 2 reduction of selenate (SeO2 4 ) to selenite (SeO3 ) and supports selenate respiration in T. selenatis [9]. The three subunits consist of SerA (96 kDa e a subunit), SerB (40 kDa e b subunit), and SerC (23 kDa e g subunit) [9]. SerA is the catalytic subunit containing the molybdenum active site in the form of the molybdopterin guanine dinucleotide (bis-MGD) cofactor [9,12,13]. SerB contains a number of cysteine rich motifs that co-ordinate a [3Fee4S] and three [4Fee4S] ironesulphur clusters [12,13]. SerA shows 31% identity and 41% similarity to pNarG from P. aerophilum, and is of a similar size (w100 kDa) [1], approximately 40 kDa smaller than NarG from the mesophilic bacteria. Both SerA and pNarG have a conserved motif (H/CX3CX3CX29e34C) towards the N-terminus [14]. The motif is conserved also in Escherichia coli NarG and has been shown to coordinate a [4Fee4S] (FS0) cluster in the three dimensional crystal stuctures [15,16]. The aspartate residue that co-ordinates the Mo in the bis-MGD cofactor in E. coli NarG (D222) is again conserved in both the pNAR group and SerA (Fig. 1B). Component pNarH is a molybdopterin oxidoreductase ironesulphur binding protein and shows 29% identity and 37% similarity to SerB and low identity with E. coli NarH. The final component in the hyperthermophilic archaeal pNAR system is a membrane-bound b-type cytochrome anchor. By contrast, the SerC subunit is a soluble b-type cytochrome that accepts electrons from either a quinol-cytochrome c oxidoreductase (QCR) or a quinol dehydrogenase via a soluble cytochrome c4 [17].
2. Materials and methods 2.1. Growth conditions of T. selenatis T. selenatis was grown anaerobically at 30 C in mineral salts medium [21] containing yeast extract (0.1%), selenate (10 mM) and acetate (10 mM) in 10 L batch cultures. When grown on tungstate rich medium, ammonium molybdate was removed from the trace elements solution and replaced with sodium tungstate at the required concentration. To ensure that tungstate media were molybdate-free, all glassware was acid washed and only ultra-pure H2O was used during medium preparation. Cultures were harvested during late log phase (after 16e18 h growth) at OD600nm 0.6e0.7 and spheroplasts were prepared as described previously [9]. The spheroplasts were removed by centrifugation and the supernatant containing periplasm was retained. 2.2. Purification of SER from T. selenatis and the preparation of samples for thermo-stability profiling The periplasmic selenate reductase from T. selenatis was purified as described previously [9] following a method adapted from Schroder et al. [9,13]. Purified SerABC samples were heated at a range of temperatures (30e80 C) for 10 min and centrifuged (12,000g, 5 min) to remove precipitated proteins. Protein samples were analysed for thermal stability via SDS-PAGE (gel electrophoresis). Samples were loaded onto a 10% BiseTris NuPAGEÒ Gel. The protein bands were separated by electrophoresis at 200 V, 125 mA, for 50 min. Scanned images of the gels were stored and densitometry was used to calculate the proportion of protein denatured at each temperature. The percentage of protein denatured at increasing temperature was determined by comparison to the amount of soluble protein retained following treatment at 30 C. To determine the metal (Mo and W) content of the selenate reductase,
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samples were analysed by inductively coupled plasma mass spectroscopy (ICPMS). Standards of Mo and W were prepared at the following concentrations 1, 5, 10, 25, 50 and 100 ppb. All samples and standards were treated with 6.5% nitric acid and analysed using a Thermo X-series ICPMS spectrometer in accordance with manufacturer’s instructions. 2.3. Enzyme activity assays Selenate reductase activity was measured in a cuvette assay by monitoring the oxidation of reduced methyl viologen (1 mM) spectrophotometrically at 600 nm, coupled to the reduction of selenate [22]. Activities were calculated from the initial rates using an extinction coefficient of 13,700 M1 cm1 for the methyl viologen radical [23]. Values for Km and Vmax cited in the text and tables are the mean values (n ¼ 3) calculated using non-linear regression analysis in Grafit v3.0 (Erithacus software). To determine the activity retained by the heat-shocked SerABC, samples were incubated at a range of temperatures (30e80 C) for 10 min, prior to activity measurements at ambient temperature. 10 min incubation was chosen because the viologen assay for selenate reductase activity is typically measured over a 5 min time period, so it was considered appropriate that pre-incubation at each temperature was for longer than the assay reaction. To determine the optimum temperature for reductase activity, assays were undertaken at a range of fixed temperatures (ambient e 80 C) and monitored for a 5 min period. The protein concentrations were determined using Biorad reagents following the manufacturers’ protocol.
(Fig. 2A). All three subunits can still be seen clearly in samples heated to 70 C, with only the smallest subunit, SerC, being lost upon heating up to 80 C. In order to quantify the amount of SerABC that remains in a soluble state following heat-shock, the protein band intensities were determined using scanning densitometry (Fig. 2B). Results show that upon incubation up to 60 C for 10 min >70% of the soluble protein is retained. Incubation at 80 C results in retention of soluble SerA and SerB at 17% and 25% respectively. SerC is not detectable after incubation at 80 C and indicates that it is fully denatured. It is noteworthy that a number of contaminating proteins appear to be removed upon heating to >40 C. Most notably, the protein at 28 kDa, which we have previously identified as uridine phosphorylase [13], is completely removed by heating the sample to 50 C. Consequently, this short heat-step makes a useful addition to the purification protocol. The purified enzyme was assayed for selenate reductase activity following incubation at the corresponding range of temperatures to analyse the retention of activity (Fig. 2C). Full activity was maintained up to 60 C, with activity falling to 25% at 70 C. Protein that had been incubated at 80 C for 10 min also retained 25% original activity. All activity was lost upon heating the sample to >90 C. In order to investigate the structural integrity of the SerABC complex, samples were also analysed by circular dichroism (Fig. 2D). Proteins retained a folded confirmation up to 70 C. Selenate reductase activity was also measured at increasing temperature to determine the optimum temperature for methyl viologen dependant activity. Maximum activity was determined at 65 C. 3.2. The effect of tungsten on the growth of T. selenatis
2.4. Circular dichroism analysis SerABC was concentrated to w20 mg/ml and buffer exchanged into 50 mM phosphate buffer. Circular dichroism spectroscopy was used to check for folded protein and the effect of increasing temperature on structural integrity. Samples were incubated at 30 C, 60 C and 80 C for 10 min. Samples were analysed using a Jasco J-810 spectropolarimeter over a wavelength range of 185e250 nm at a scanning speed of 50 nm min1 at room temperature (10 scans). 2.5. Determination of pH optimum Activity measurements to determine pH optimum were undertaken using the microtitre plate assay as described previously [24]. 2.6. Activity gels Proteins were separated using non-denaturing PAGE (6% Triseglycine gels), and gels were stained with sodium dithionitereduced methyl viologen (5 mM) under anaerobic conditions, using a small anaerobic chamber and purged with O2-free nitrogen for 30 min. Protein bands with selenate reductase activity were identified by the addition of selenate for 15 min and observed as clear zones. 3. Results 3.1. Determination of the thermo-stability of SerABC To determine the thermal stability of the purified selenate reductase, samples were heated to a range of temperatures (30 C, 40 C, 50 C, 60 C, 70 C and 80 C) for 10 min and centrifuged to remove precipitated proteins. The supernatant was then analysed by SDS-PAGE and resolution of the retained SerA, SerB and SerC components was taken as evidence of thermostable protein
T. selenatis was grown anaerobically as described, and growth monitored for 26 h (Fig. 3A). Cells grown on Hungates medium or Hungates medium supplemented with 1 mM molybdenum displayed typical growth curves, with stationary phase being reached at OD600nm ¼ 0.7. Cultures supplemented with tungstate at a range of concentrations (10e100 mM) had little effect to both growth rate and yield. With tungstate added to a final concentration of 1 mM, growth was inhibited slightly, giving a final optical density of OD600nm ¼ 0.6. The presence of 15 mM tungstate severely slowed the growth of T. selenatis, with no exponential growth being observed, reaching a final OD600nm ¼ 0.1 after 26 h. The ability to tolerate 1 mM tungsten without significantly inhibiting selenate respiration was interesting and suggested that perhaps selenate reductase can function with tungsten at the active site. To investigate the tungsten substituted selenate reductase (WeSER) further, periplasmic fractions from T. selenatis, grown on either molybdate (1 mM) or tungstate (1 mM) rich medium were generated. Periplasmic fractions were separated by non-denaturing PAGE and stained for selenate reductase activity (Fig. 3B). The resolution of clear bands of similar mobility demonstrated that a functional selenate reductase was detectable in periplasms from both molybdate and tungstate grown cells. 3.3. Thermo-stability of WeSER The thermo-stability of the WeSerABC complex was investigated using SDS-PAGE analysis. Periplasmic fractions were incubated at 30e80 C for 10 min (heat-shocked) and denatured proteins removed by centrifugation. Samples were analysed by SDS-PAGE as before and the % of soluble protein retained determined by densitometry. Levels of expression of SerA were reduced by 50% when compared to molybdate grown cells. Fig. 4A shows the retention of soluble SER proteins at increasing temperature. WeSerA shows a similar stability profile to MoeSerA with approximately 50% of the protein remaining soluble at incubation
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Fig. 2. Thermo-stability of SerABC. A: SDS-PAGE gel showing the thermal stability of SerABC. Lanes 1 and 8, Invitrogen SeeBlueÒ Ladder; Samples were heated for 10 min at; 30 C (lane 2), 40 C (lane 3), 50 C (lane 4), 60 (lane 5), 70 C (lane 6) and 80 C (lane 7). Total protein loaded was 10 mg per lane. B: Graph showing the decreasing thermal stability of the components of SerABC upon heat treatment. Densitometry values of the protein bands were used to calculate the % of soluble protein retained. Symbols represent; SerA (-), SerB (C) and SerC (:). C: Graph showing the thermal stability of selenate reductase activity. Samples were heated to a range of temperatures for 10 min before being assayed for selenate reductase activity. Assays were performed ambient temperature. Results are means of 3 determinants, SD < 5%. D: Circular dichroism spectra of SerABC. Untreated SerABC (solid line), SerABC heat treated at 60 C for 10 min (dashed line) and heat treated at 80 C for 10 min (dotted line).
at w65 C, however, by contrast to MoeSerA, the WeSerA protein appears less stable at 60 C (WeSerA w66%; MoeSerA w80%). Interestingly, the most notably difference is in the stability of the SerB and SerC components. Both seem to be more labile in the WeSER complex. The SerC component is lost from the complex upon heating to temperatures >70 C, with only 50% being retained at 60 C. Similarly, 50% of SerB remains soluble in samples heated to 60 C and none is retained post incubation at 80%. These results suggest that substituting W for Mo at the active site of SerA decreases the overall stability of the SerABC complex, rendering the components less thermostable. Given the much lower level of activity detected, attempts to purify the WeSER using the established chromatography protocols were unsuccessful, so heating samples to w50 C for 10 min was used to obtain a partially-purified sample for subsequent kinetic studies. Partially-pure samples were judged to be enriched such that SER constituted 50% of the total soluble periplasmic protein. Both MoeSER and WeSER were analysed for metal content using ICPMS to confirm the metal active centre. SER isolated for molybdate grown cells had a Mo content of 0.5 mol of Mo per mol of enzyme. SER partially-purified from periplasmic fractions from cells grown in a tungstate rich medium showed no detectable levels of Mo and had an estimated W content of 0.6 mol of W per mol of enzyme. It has been demonstrated previously [13] that purification of MoeSER to homogeneity results in a loss of activity and a concomitant decrease in Mo cofactor. Consequently, the sub-stoicheiometric ratio of W:SER would also suggest that the incorporation of a tungstopterin cofactor does not improve cofactor retention.
3.4. Kinetic parameters of WeSER The partially-purified WeSER was assayed at a range of selenate concentrations. Using reduced methyl viologen as the artificial electron donor, the Km (0.7 mM) and Vmax (0.01 mmol [S]/min/mg) were determined (Table 1.). These results show that although the enzyme is working at a slower rate than MoeSER (0.2 mmol [S]/min/mg), the affinity for selenate is approximately 23-fold higher (MoeSER; Km ¼ 16 mM). The thermal activity of the WeSER was also investigated by assaying for enzyme activity at increasing temperature. When assaying WeSER samples at high temperature (80 C), the samples were equilibrated rapidly and reductase activity measured immediately. The initial rate of viologen oxidation was recorded and shown to increase with temperature up to our limit of 80 C. However, during the assay (after the first 60 s) the steady state activity showed a significant drop-off, consistent with the prolonged incubation (10 min at 80 C), so called “heat-shocked protein”, that resulted in loss of soluble protein at this temperature. 3.5. pH dependence of WeSerABC WeSER was assayed at a range of different pH to determine the pH dependence of the WeSER enzyme (Fig. 4B). The pH optimum for MoeSER in vitro has been determined previously to be pH 6.0, an observation that is consistent with our data [25]. Analysis of WeSER shows that reductase activity is also optimum at pH 6.0, with the enzyme retaining <20% activity when assayed at pH 8.0.
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Fig. 3. The effect of tungsten on selenate respiration. A: Growth curve of T. selenatis on varying concentrations of tungstate. Hungates medium only (-), Hungates medium supplemented with 1 mM Mo (B), Hungates medium supplemented 1 mM W (:) and Hungates medium supplemented with 15 mM W (7). B: Native PAGE gels of periplasmic fractions isolated from Mo and W grown T. selenatis. 10 mg of protein were loaded per lane. Gels were stained for SER activity by submersion in reduced methyl viologen solution (5 mM) and developed by the addition of selenate (10 mM).
Fig. 4. Properties of WeSER. A: Graph showing the decrease in thermal stability of the separate components of the WeSER complex upon heat treatment. Densitometry values of the protein bands were used to calculate the % of soluble protein retained. Symbols represent; SerA (-), SerB (C) and SerC (:). B: Graph showing the pH dependence of selenate reductase activity in periplasmic fractions from T. selenatis grown in the presence of 1 mM tungsten. Results are the means of 6 determinants, SD < 5%.
4. Discussion The data presented demonstrate that the SER enzyme is stable and active upon incubation at temperatures up to 60 C, with an optimum activity recorded at 65 C. The SerC component appears to be least stable once above 60 C and perhaps the loss of this subunit contributes to the overall instability of the SER complex. The SerC component is rather unusual in that it co-ordinates a b-type cytochrome that is rarely found in soluble periplasmic proteins [12,13]. Unlike the common c-type haems, the b-type haems are not covalently attached via a CXXCH motif and as a consequence might not form such highly stable holoproteins. The soluble SerC-like component is missing in the pNAR enzymes from P. aerophilum and A. fulgidus and is replaced by a membrane anchored subunit. It has been suggested that the association with the membrane might aid the overall thermo-stability of the pNAR complexes from the hyperthermophiles [2] and our results support this hypothesis. The observation that SER is thermostable up to 60 C is consistent with reports of other molybdoenzymes from mesophilic bacteria. The TMAO reductase from E. coli, an enzyme also classified as a member of the DMSO reductase molybdoenzyme family, is thermostable up to 60 C with optimum activity recorded at 65 C [26]. Again, similar to SER, TMAO reductase is located as a soluble protein in the periplasm and not anchored to the cytoplasmic membrane. Tungsto-enzymes are often associated with the hyperthermophilic archaea and have been reported to have enhanced thermo-stability and thermo-activity capabilities [19]. Analogue
reaction systems using bis(dithiolene) complexes of MoIV and WIV have both been shown clearly to reduce selenate to selenite by Moand W-mediated oxo-transfer reactions [27], demonstrating unequivocally that chemically W analogues can function as a catalyst for selenate reduction. However, to date no evidence has been presented regarding the effect of tungsten on selenate reduction in an enzyme system (i.e., selenate reductase). Our results demonstrate that T. selenatis can tolerate tungstate concentrations approaching 1 mM without significantly affecting both growth rates and biomass yields. SER isolated form cells grown in a tungstate rich environment showed a 20-fold reduction in selenate reductase activity, but with an increased affinity for selenate (Km 0.7 mM). These observations (kinetics and thermodynamics) are likely to be linked. The tungsten substitution may have enhanced the bond strengths of the WVIesubstrate complexes, thus
Table 1 Kinetic parameters for both Mo and W derivatives of SER Enzyme derivative
Kinetic constanta Vmax (mmol [S]/min/mg)
Km (mM)
MoeSER WeSER
0.2 0.01
16 0.7
a Kinetic constants were measured using methyl viologen as the electron donor. Variation was within 10% of the mean values shown (n ¼ 3). Protein concentration was 14 mg/ml. Selenate was added at a range of concentrations up to 4 mM.
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stabilising the W/OSeO2 3 bond, leading to the observed higher substrate affinity. This in turn may have led to complexes which were kinetically slower than the equivalent Moesubstrate complexes, when measured at the mesophilic range of temperatures [19]. Finally, little is known about selenate reduction/respiration in thermophiles, but emerging data suggests that it might be a widespread activity. P. aerophilum can grow organotrophically in anaerobic conditions in the presence of selenate and/or selenite as the sole electron acceptors [28]. Pyrobaculum arsenaticum, a strictly hyperthermophilic facultative organotrophic archaeon isolated from hot springs in Naples, Italy, can also grow using selenate as the sole electron acceptor, reducing selenate to elemental selenium [28]. It is likely that selenate reduction is a common activity in the hyperthermophiles, since their hydrothermal vent habitats often display elevated levels of selenium compounds [29]. The selenate reductases within these hyperthermophilic organisms must also possess characteristics enabling them to function at elevated temperatures. The results reported in the present work would indicate that a hypertheromophilic selenate reductase is likely to be associated with the membrane and potentially could function with a Wecofactor. 5. Conclusions The present work has investigated the thermostable properties of SER from T. selenatis. The enzyme is stable and active upon incubation at temperatures up to 60 C, with an optimum activity at 65 C. T. selenatis can grow readily on a tungsten rich medium (up to 1 mM) and SER isolated under such conditions displayed reductase activities with a 20-fold reduction in Vmax and a 23-fold increase in Km. The thermo-stability and pH dependence of WeSER was shown to be similar to that observed for MoeSER. The combined data from the kinetic analysis and thermal activity profiles provide convincing evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. The observations that SER from T. selenatis is stable at w65 C might prove usefully when designing selenate remediation strategies. Acknowledgments This work was funded in part by research grants from the Biotechnology and Biological Sciences Research Council (BBSRC). EJD was a recipient of a BBSRC Plant and Microbial Sciences (PMS) committee studentship. Thauera selenatis was kindly provided by Dr Joanne Santini (University College London). We acknowledge help from Dr Carys Watts (University of Newcastle) with protein purification and we thank Prof. David Richardson (University of East Anglia) for useful discussions. References [1] R.M. Martinez-Espinosa, E.J. Dridge, M.J. Bonete, J.N. Butt, C.S. Butler, F. Sargent, D.J. Richardson, Look on the positive side! The orientation, identification and bioenergetics of "Archaeal" membrane-bound nitrate reductases. FEMS Microbiol. Lett. 276 (2007) 129e139. [2] S. Afshar, E. Johnson, S. De Vries, I. Schröder, Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilium. J. Bacteriol. 183 (2001) 5491e5495. [3] F. Sargent, Constructing the wonders of the bacterial world: biosynthesis of complex enzymes. Microbiology 153 (2007) 633e651. [4] B.C. Berks, F. Sargent, T. Palmer, The Tat protein export pathway. Mol. Microbiol. 35 (2000) 260e274.
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