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Cloning, purification, and characterization of inorganic pyrophosphatase from the hyperthermophilic archaea Pyrococcus horikoshii
Protein Expression and Purification 99 (2014) 94–98
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Cloning, purification, and characterization of inorganic pyrophosphatase from the hyperthermophilic archaea Pyrococcus horikoshii Dongmei Lu a,⇑, Guiqiu Xie b, Renjun Gao b a b
College of Life Science and Technology, Zhanjiang Normal University, Zhanjiang City, Guangdong Province, 524048, PR China Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China
a r t i c l e
i n f o
Article history: Received 18 February 2014 and in revised form 9 April 2014 Available online 20 April 2014 Keywords: ArchaeaPyrococcus horikoshii Inorganic pyrophosphatase Ni-chelating chromatography Thermostability
a b s t r a c t The gene encoding inorganic pyrophosphatase (PPiase) from the hyperthermophilic archaea Pyrococcus horikoshii (Pho PPiase) was cloned in the Escherichia coli strain BL21/pET15b, and the recombinant PPiase was purified by Ni-chelating chromatography in only an one-step procedure. The PPiase showed optimal activity at 88 °C and pH of 10.3. Kinetic analysis revealed Km, kcat, Vm of 14.27 lM, 3436 s 1, and 34.35 lmol/min/mg protein, respectively. Pho PPiase was stable against denaturant chemicals as well as heat. It retained 19.61% of the original activity after incubation at 100 °C for 12 h and 25.96% of the original activity in the presence of 8 M urea after incubation at 50 °C for 120 h. Pho PPiase showed high specificity for inorganic pyrophosphate but low reactivity to sodium tripolyphosphate and sodium tetrapolyphosphate. ADP and ATP could not serve as substrates. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Inorganic pyrophosphatase (PPiase1, EC 3.6.1.1) is widely distributed among living organisms, and the enzyme has been purified from various sources [1–9]. On the basis of subunit structure and cellular localization, PPiases can be discriminated in two types: membrane-bound PPiase and cytoplasmic PPiase [10]. Membrane-bound PPiase is an unique, electrogenic proton pump distributed among most terrestrial plants but is distributed only among some algae, protozoa, bacteria, and archaebacteria. In plant cells, H+-PPiase coexists with H+-ATPase in a single vacuolar membrane and has the ability to transport protons across the membrane [11]. However, PPiases in the plant thylakoid membrane [12] and plant mitochondria [13] have recently been found to not show proton pump activity. In addition to functions in inorganic pyrophosphate (PPi) hydrolysis and energy coupling, it has been suggested that mitochondrial PPiase could be involved in the calcium-mediated response to certain hormones [14]. It specifically catalyzes the hydrolysis of PPi to orthophosphate [15]. This essential function is performed by a soluble cytoplasmic PPiase. The substrate for PPiase, PPi, which is necessary for life, is produced as a by-product of several metabolic processes
⇑ Corresponding author. Tel.: +86 759 3183271. E-mail address: [email protected] (D. Lu). Abbreviations used: PPiase, inorganic pyrophosphatase; Pho PPiase, PPiase from the hyperthermophilic archaea P. horikoshii; Y-PPiase, PPiases from Saccharomyces cerevisiae; PPi, inorganic pyrophosphate; PPPi, sodium tripolyphosphate; PPPPi, sodium tetrapolyphosphate. 1
http://dx.doi.org/10.1016/j.pep.2014.04.006 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
such as deoxyribonucleic and ribonucleic acid (DNA and RNA) polymerization, amino acid activation (aminoacyl-tRNA synthesis), fatty acid b-oxidation (fatty acyl-CoA synthesis), cellulose synthesis (UDPglucose synthesis), and starch synthesis (ADP-glucose synthesis). PPi hydrolysis can provide energy via many biosynthetic reactions. In addition, PPi can be synthesized by photophosphorylation, oxidative phosphorylation, and glycolysis [11,16,17]. We report here on the purification and characterization of PPiase from the hyperthermophilic archaea Pyrococcus horikoshii (Pho PPiase). The results obtained are compared with those for other known PPiases. In addition, the role of PPiase in the organism is discussed. Materials and methods Materials Restriction endonucleases, T4 DNA ligase, Ex-Taq DNA polymerase, DNA Marker DL2000, k-HindIII digested DNA Marker, and isopropyl-b-D-thiogalacto-pyranoside (IPTG) were purchased from TaKaRa Biotechnology (Dalian, PR China). ATP, ADP, sodium tripolyphosphate (PPPi), and sodium tetrapolyphosphate (PPPPi) were obtained from Sigma (St. Louis, MO, USA). Other chemicals were of the highest grade available. Cell growth In total, 500 ll of cells from a stock strain was inoculated into 50 ml 2YT medium and incubated with vigorous shaking at 37 °C
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overnight until OD600 reached 0.4–1.0. IPTG was then added to achieve a final concentration of 1 mM, and the solution was incubated at 25 °C overnight with shaking. The cells were harvested by centrifugation at 6000 rpm for 15 min at 4 °C the following day and then maintained at 20 °C. Amplification and expression The genome of Pyrococcus horikoshii was isolated and then used as a template for the gene encoding PPiase (Accession No. PH1907). The gene was amplified by polymerase chain reaction (PCR) and prime was designed based on an open reading frame (ORF) sequence for a protein of 178 amino acids. The amplified DNA sequence was digested and inserted into pET15b vector with the same restriction sites. The nucleotide sequence of the inserted DNA was determined to verify the sequence. The amplified gene was expressed in the Escherichia coli BL21 (DE3) Codon Plus-RIL (Stratagene, USA). The transformed bacteria were grown in 2YT medium. Protein expression was induced by adding IPTG. Purification of Pho PPiase Cells were resuspended in 50 mM Tris–HCl (pH 8.0) at 25 °C, heated at 85 °C for 30 min, and then cooled. Cell debris was centrifuged at 12,000 rpm for 20 min. The supernatant was removed and subjected to Ni-chelating chromatography. Fractions possessing PPiase activity were pooled, concentrated, and dialyzed against 10 mM Tris–HCl buffer (pH 8.0) overnight. Assay of Pho PPiase activity Pho PPiase activity was determined spectrophotometrically via the change in absorbance at 350 nm. The reaction mixture contained 2 mM MgCl2, 1 mM PPi, 50 mM Tris–HCl (pH 8.0), and the enzyme. The reaction was initiated by the addition of Pho PPiase and was stopped by rapid cooling on ice after being maintained at 90 °C for 10 min. One unit (U) of activity was expressed as the release of 1 lmol phosphate per min under the assay conditions. A calibration curve was obtained by the foregoing procedure using K2HPO4 solutions of known concentrations as standards [18].
Results Identification of Pho PPiase gene Pho PPiase gene was amplified by PCR from a P. horikoshii genomic DNA, cloned and sequenced to confirm the sequences in the database (data not shown). Purification of Pho PPiase Pho PPiase was inserted in a pET15b vector with the His-Tag oligohistidine domain. The cells were collected after culturing; most of the proteins from E. coli were separated by centrifugation after heating at 85 °C. The enzyme obtained by Ni-chelating chromatography was almost 41-fold more pure than the crude extract, and this purification was achieved in a single step (Table 1). The apparent enzymes molecular mass of the purified enzyme was estimated to be about 24.5 kDa by SDS–PAGE using a gradient gel (Fig. 1A). This value is inconsistent with the size (20,833 Da) calculated from the amino acid sequence. This discrepancy might be explained by the fact that Pho PPiase has many positively charged amino acid residues. Western blotting analysis, which was performed to confirm the identity of the major purified protein as Pho PPiase, revealed it was strongly immunoreactive with anti-His6 antibodies. Western blotting was carried out to confirm the presence of Pho PPiase (Fig. 1B). Effects of temperature and pH on Pho PPiase activity The temperature profile of Pho PPiase was determined by assaying its activity from 50 °C to 100 °C. The optimum temperature was 88 °C (Fig. 2). Activity profiles of Pho PPiase were also investigated at different pH values at 90 °C in 100 mM potassium phosphate
Table 1 Purification of Pho PPiase. Step
Protein concentration (mg/ml)
Specific activity (U/mg)
Crude extract Ni-chelating column
4.80 0.25935
2.45 101.61
Protein determination During the purification steps, protein concentration was determined according to the Bradford method using BSA as the standard [19]. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and Western blot analysis The identity of the target protein was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis. SDS–PAGE was performed with a 15% (w/v) polyacrylamide gel. The gel was stained with Coomassie brilliant blue. For Western blotting, protein extracts separated by SDS–PAGE were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h in 5% non-fat dry milk in Tris-buffered saline (TBS), and incubated with primary anti-His6 antibodies over-night at 4 °C. Bound antibodies were detected using horseradish peroxidase conjugated goat anti-mouse IgG antibodies. Finally, the target protein was visualized with 3,3-diaminobenzidine (DAB) as a substrate.
Fig. 1. Detection of the purified Pho PPiase. (A) Detection of Pho PPiase by SDS– PAGE. (B) Detection of Pho PPiase by Western blotting analysis.
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D. Lu et al. / Protein Expression and Purification 99 (2014) 94–98 Table 2 Effects of different metal cations. Metals
Specific activity (U/mg)
EDTA Cd2+ Mn2+ Ca2+ K+ Mg2+
0 0 19.410 0.867 2.426 74.696
Fig. 2. Temperature dependence of Pho PPiase activity. The assay mixture contained 2 mM MgCl2, 1 mM PPi, 50 mM Tris–HCl (pH 8.0), and Pho PPiase.
Fig. 4. Effect of Mg2+ on Pho PPiase activity. The reaction system was the same as that for the standard activity assay conditions.
Fig. 3. pH dependence of Pho PPiase activity. The reactions were carried out at 90 °C for 10 min.
buffer (pH 5.0–7.5), 100 mM Tris–HCl buffer (pH 7.5–9.0), and 100 mM glycine–NaOH buffer (pH 9–11). As observed in Fig. 3, the optimum pH value was 10.3. Effects of various salts and denaturants on Pho PPiase activity As observed in Table 2, Pho PPiase showed maximum activity in the presence of Mg2+. The result was compared with the results for EDTA, Cd2+, Ca2+, Mn2+, K+, and Zn2+, and it was determined that the influence of other metal cations was negligible. Variations in Pho PPiase activity based on the ionic strength of Mg2+ in the 0– 8 mM concentration range were investigated using the standard activity assay conditions. Pho PPiase activity was found to increase along with the Mg2+ concentration (Fig. 4). Stability The effect of temperature on stability was determined by incubating the purified Pho PPiase at temperatures ranging from 25 °C to 100 °C in the 50 mM Tris–HCl buffer (pH 8.0) and measuring residual activity after appropriate incubation periods at the different temperatures. The results in Fig. 5 show that the half-life was
Fig. 5. Thermal stability of Pho PPiase. The system contained 50 mM Tris–HCl (pH 8.0), EDTA, and Pho PPiase. The mixture was incubated at 95 °C (d) for 24 h and 100 °C (N) for 12 h. Aliquots were removed to determine the remaining activity.
4 h at 100 °C and 5 h at 95 °C. In addition, it remained active at 25 °C for 7 days. The recombinant Pho PPiase retained 19.61% of its original activity after incubation at 100 °C for 12 h, indicating it was highly thermostable. PPiase was also incubated with different concentrations of urea, and aliquots were removed to determine the residual activity. The enzyme retained 40.66% of the original activity in the presence of 8 M urea after incubation for 24 h at 50 °C and 39.3% of the original activity in the presence of 6 M urea at 50 °C after incubation for
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D. Lu et al. / Protein Expression and Purification 99 (2014) 94–98 Table 3 Substrate specificity of Pho PPiase. Substrates
Relative activity (%)
PPi ATP ADP PPPi PPPPi
100 2.4 0 40.3 25.2
Discussion
Fig. 6. Effect of denaturants on Pho PPiase. The assay mixture was incubated in 8 M urea (d) and 6 M urea (N) at 50 °C. The reaction was carried out at 90 °C for 10 min.
24 h (Fig. 6). It also retained 25.96% of its original activity in the presence of 8 M urea after incubation for 120 h at 50 °C. Thus, Pho PPiase was relatively stable against denaturants. Kinetic characterizations of Pho PPiase The effect of PPi concentration on the initial rate of the catalyzed reaction was investigated using the Lineweaver–Burk double reciprocal plot (Fig. 7). Km, kcat, and Vm values were calculated from the plot. Km (14.27 lM) was somewhat higher than that of other PPiases, such as those from the thermoacidophilic archaebacterium Thermoplasma acidophilum (7 lM). On the other hand, Vm was 34.35 lmol/min/mg protein and kcat was 3436 s 1. Thus, Pho PPiase showed pronounced specific activity. Substrate specificity Activity against other substrates, such as ADP, ATP, PPPi, and PPPPi, was determined using the same method as above, except PPi was replaced by each substrate. Table 3 shows that Pho PPiase was specific for PPi. It barely showed any specificity to the higher polyphosphates (PPPi and PPPPi) and showed no specificity to ADP and ATP.
Fig. 7. Determination of kinetic parameters. The reaction was carried out at 88 °C and pH 10.3.
PPiases from various sources are divided into two groups on the basis of their molecular mass and oligomeric structure. The first is the eukaryotic group and contains PPiases from Saccharomyces cerevisiae (Y-PPiase) [20], Kluyveromyces lactis [21], Arabidopsis thaliana [22], and the bovine retina [23]. The other is the prokaryotic group and includes PPiases from E. coli [24], Thermus thermophilus (Tth PPiase) [25], Bacillus stearothermophilus (Bst PPiase) [26], Rhodospirillum rubrum [27], thermophilic bacterium PS-3 [20,28], archaeon Methanobacterium thermoautotrophicum (strainDH) [29], Sulfolobus acidocaldarius strain 7 [30], and Sulfolobus sp. strain 7 [31]. Eubacterial and archaeal enzymes are homotetramers or hexamers comprising 19- to 23-kDa subunits [1,2,10], and eukaryotic enzymes are homodimers comprising 32- to 35-kDa subunits [20,32,33]. Thus, Pho PPiase resembles the bacterial and archaeal type [34], based on the molecular mass of a single subunit. All known PPiases, both cytoplasmic and membrane-bound, require a divalent metal ion, with Mg2+ conferring the highest activity. Previous kinetic studies of PPi hydrolysis and Pi/PPi exchange have suggested that Mg2+ has a dual role: free magnesium ion activates the enzyme, whereas magnesium pyrophosphate is the substrate for soluble PPiase [17]. The active site structure of soluble PPiases is remarkably similar to that of membrane-bound PPiases, supporting the notion that they share a common catalytic mechanism [27]. Despite similar requirements for all types of PPiases, the differences in their kinetic properties could be important in their regulation [35]. Pho PPiase was dependent on the presence of divalent cations for catalytic activity, with the highest activity in the presence of Mg2+. In particular, a drastic increase in enzyme activity was observed in the presence of Mg2+ (0–2 mM). The enzyme also retained high levels of activity in the presence of up to 8 mM Mg2+. Ca2+ and K+ had little impact on enzyme activity for catalysis. Other cations (Mn2+) might be able to replace Mg2+, but their impact needs to be studied further. PPiases reported to date are relatively thermostable, especially in the presence of divalent metal cations, as reported for the other PPiases [26]. However, most become inactive at high temperatures (80–100 °C). Pho PPiase was particularly thermostable and did not become inactive at 100 °C even in the absence of Mg2+. In addition, Pho PPiase was stable against various denaturants, retaining some activity in 8 M urea. Urea was found to inhibit the enzyme at very high concentrations. The inhibitory effect of urea on enzyme activity is probably due to some subtle conformational change or direct interaction of urea with the active site of the enzyme. Further studies are needed to determine why there is residual enzyme activity after a 5 h incubation in 8 M urea. Pho PPiase showed optimal activity at alkaline pH and at a higher optimal temperature than PPiases from other archaea such as M. thermoautotrophicum (7.7 and 70 °C, respectively) [29], S. acidocaldarius (6.0 and 75 °C, respectively) [36], and T. acidophilum (6.7 and 85 °C, respectively). Therefore, it is likely to have widespread applications. PPiases are becoming an important model for enzymatic hydrolysis and synthesis of polyphosphates [37]. The present method of
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genetic engineering provides an effective means of producing diverse folding patterns and enzymatic properties. Therefore, desired enzymes have become more accessible. Attention has recently been directed to the mechanism and regulation of active transport systems in PPiases, including proton transport systems in the membrane. One example is the study of the tomoplast PPiase from the mung bean [38]. PPiases may also have an important role in evolution by affecting the accuracy with which DNA molecules are copied during chromosome duplication [39]. Further study of the structure and function of PPiases is therefore extremely important. In conclusion, P. horikoshii is a hyperthermophilic archaea, and Pho PPiase is thus an excellent model for studies of thermostability and folding dynamics. Large-scale production of highly thermostable enzymes for industrial use may become a practical application. Acknowledgments This work was supported by Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University. The authors would like to thank Enago (www.enago.cn) for the English language review. References [1] D.A. Ware, J.R. Postgate, Physiological and chemical properties of a reductantactivated inorganic pyrophosphatase from Desulfovibrio desulfuricans, J. Gen. Microbiol. 67 (1971) 145–160. [2] N. Tomonaga, T. Mori, Purification and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans, J. Biochem. 81 (1977) 477–483. [3] R. Lahti, T. Niemi, Purification and some properties of inorganic pyrophosphatase from Streptococcus faecalis, J. Biochem. 90 (1981) 79–85. [4] A. Hachimori, A. Takeda, M. Kaibuchi, N. Ohkaeara, T. Samejima, Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus, J. Biochem. 77 (1975) 1177–1183. [5] J. Josse, Constitutive inorganic pyrophosphatase of Escherichia coli I. Purification and catalytic properties, J. Biol. Chem. 241 (1966) 1938–1947. [6] M.Y. Liu, J. Le Gall, Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibrio vulgaris: rubrerythrin and a new, highly active, enzyme, Biochem. Biophys. Res. Commun. 171 (1990) 313–318. [7] J.A. Verhoeven, K.M. Schenck, R.R. Meyer, J.M. Trela, Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus, J. Bacteriol. 168 (1986) 318–321. [8] M.S.M. Jetten, T.J. Fluit, A.J.M. Stams, A.J.B. Zehnder, A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii, Arch. Microbiol. 157 (1992) 284–289. [9] O.M.H. Richter, G. Schäfer, Purification and enzymatic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum, Eur. J. Biochem. 209 (1992) 343–349. [10] G.J.W.M. van Alebeek, J.T. Keltjens, C. van der Drift, Purification and characterization of inorganic pyrophosphatase from Methanobacterium thermoautotrophicum (strain delta H), Biochim. Biophys. Acta 1206 (1994) 231–239. [11] M. Maeshima, Vacuolar H+-pyrophosphatase, Biochim. Biophys. Acta 1465 (2000) 37–51. [12] S.S. Jiang, L.L. Fan, S.J. Yang, S.Y. Kuo, R.L. Pan, Purification and characterization of thylakoid membrane-bound inorganic pyrophosphatase from Spinacia oleracia, L, Arch. Biochem. Biophys. 346 (1997) 105–112. [13] M. Zancani, F. Macri, A.D.B. Peruffo, A. Vianello, Isolation of the catalytic subunit of a membrane-bound H+-pyrophosphatase from pea stem mitochondria, Eur. J. Biochem. 228 (1995) 138–143. [14] M. Lundin, H. Baltscheffsky, H. Ronne, Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function, J. Biol. Chem. 266 (1991) 12168–12172. [15] T. Sivula, A. Salminen, A.N. Parfenyev, P. Pohjanjoki, A. Goldman, B.S. Cooperman, A.A. Baykov, R. Lahti, Evolutionary aspects of inorganic pyrophosphatase, FEBS Lett. 454 (1999) 75–80.
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Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Cloning, purification, and characterization of inorganic pyrophosphatase from the hyperthermophilic archaea Pyrococcus horikoshii Dongmei Lu a,⇑, Guiqiu Xie b, Renjun Gao b a b
College of Life Science and Technology, Zhanjiang Normal University, Zhanjiang City, Guangdong Province, 524048, PR China Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China
a r t i c l e
i n f o
Article history: Received 18 February 2014 and in revised form 9 April 2014 Available online 20 April 2014 Keywords: ArchaeaPyrococcus horikoshii Inorganic pyrophosphatase Ni-chelating chromatography Thermostability
a b s t r a c t The gene encoding inorganic pyrophosphatase (PPiase) from the hyperthermophilic archaea Pyrococcus horikoshii (Pho PPiase) was cloned in the Escherichia coli strain BL21/pET15b, and the recombinant PPiase was purified by Ni-chelating chromatography in only an one-step procedure. The PPiase showed optimal activity at 88 °C and pH of 10.3. Kinetic analysis revealed Km, kcat, Vm of 14.27 lM, 3436 s 1, and 34.35 lmol/min/mg protein, respectively. Pho PPiase was stable against denaturant chemicals as well as heat. It retained 19.61% of the original activity after incubation at 100 °C for 12 h and 25.96% of the original activity in the presence of 8 M urea after incubation at 50 °C for 120 h. Pho PPiase showed high specificity for inorganic pyrophosphate but low reactivity to sodium tripolyphosphate and sodium tetrapolyphosphate. ADP and ATP could not serve as substrates. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Inorganic pyrophosphatase (PPiase1, EC 3.6.1.1) is widely distributed among living organisms, and the enzyme has been purified from various sources [1–9]. On the basis of subunit structure and cellular localization, PPiases can be discriminated in two types: membrane-bound PPiase and cytoplasmic PPiase [10]. Membrane-bound PPiase is an unique, electrogenic proton pump distributed among most terrestrial plants but is distributed only among some algae, protozoa, bacteria, and archaebacteria. In plant cells, H+-PPiase coexists with H+-ATPase in a single vacuolar membrane and has the ability to transport protons across the membrane [11]. However, PPiases in the plant thylakoid membrane [12] and plant mitochondria [13] have recently been found to not show proton pump activity. In addition to functions in inorganic pyrophosphate (PPi) hydrolysis and energy coupling, it has been suggested that mitochondrial PPiase could be involved in the calcium-mediated response to certain hormones [14]. It specifically catalyzes the hydrolysis of PPi to orthophosphate [15]. This essential function is performed by a soluble cytoplasmic PPiase. The substrate for PPiase, PPi, which is necessary for life, is produced as a by-product of several metabolic processes
⇑ Corresponding author. Tel.: +86 759 3183271. E-mail address: [email protected] (D. Lu). Abbreviations used: PPiase, inorganic pyrophosphatase; Pho PPiase, PPiase from the hyperthermophilic archaea P. horikoshii; Y-PPiase, PPiases from Saccharomyces cerevisiae; PPi, inorganic pyrophosphate; PPPi, sodium tripolyphosphate; PPPPi, sodium tetrapolyphosphate. 1
http://dx.doi.org/10.1016/j.pep.2014.04.006 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
such as deoxyribonucleic and ribonucleic acid (DNA and RNA) polymerization, amino acid activation (aminoacyl-tRNA synthesis), fatty acid b-oxidation (fatty acyl-CoA synthesis), cellulose synthesis (UDPglucose synthesis), and starch synthesis (ADP-glucose synthesis). PPi hydrolysis can provide energy via many biosynthetic reactions. In addition, PPi can be synthesized by photophosphorylation, oxidative phosphorylation, and glycolysis [11,16,17]. We report here on the purification and characterization of PPiase from the hyperthermophilic archaea Pyrococcus horikoshii (Pho PPiase). The results obtained are compared with those for other known PPiases. In addition, the role of PPiase in the organism is discussed. Materials and methods Materials Restriction endonucleases, T4 DNA ligase, Ex-Taq DNA polymerase, DNA Marker DL2000, k-HindIII digested DNA Marker, and isopropyl-b-D-thiogalacto-pyranoside (IPTG) were purchased from TaKaRa Biotechnology (Dalian, PR China). ATP, ADP, sodium tripolyphosphate (PPPi), and sodium tetrapolyphosphate (PPPPi) were obtained from Sigma (St. Louis, MO, USA). Other chemicals were of the highest grade available. Cell growth In total, 500 ll of cells from a stock strain was inoculated into 50 ml 2YT medium and incubated with vigorous shaking at 37 °C
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overnight until OD600 reached 0.4–1.0. IPTG was then added to achieve a final concentration of 1 mM, and the solution was incubated at 25 °C overnight with shaking. The cells were harvested by centrifugation at 6000 rpm for 15 min at 4 °C the following day and then maintained at 20 °C. Amplification and expression The genome of Pyrococcus horikoshii was isolated and then used as a template for the gene encoding PPiase (Accession No. PH1907). The gene was amplified by polymerase chain reaction (PCR) and prime was designed based on an open reading frame (ORF) sequence for a protein of 178 amino acids. The amplified DNA sequence was digested and inserted into pET15b vector with the same restriction sites. The nucleotide sequence of the inserted DNA was determined to verify the sequence. The amplified gene was expressed in the Escherichia coli BL21 (DE3) Codon Plus-RIL (Stratagene, USA). The transformed bacteria were grown in 2YT medium. Protein expression was induced by adding IPTG. Purification of Pho PPiase Cells were resuspended in 50 mM Tris–HCl (pH 8.0) at 25 °C, heated at 85 °C for 30 min, and then cooled. Cell debris was centrifuged at 12,000 rpm for 20 min. The supernatant was removed and subjected to Ni-chelating chromatography. Fractions possessing PPiase activity were pooled, concentrated, and dialyzed against 10 mM Tris–HCl buffer (pH 8.0) overnight. Assay of Pho PPiase activity Pho PPiase activity was determined spectrophotometrically via the change in absorbance at 350 nm. The reaction mixture contained 2 mM MgCl2, 1 mM PPi, 50 mM Tris–HCl (pH 8.0), and the enzyme. The reaction was initiated by the addition of Pho PPiase and was stopped by rapid cooling on ice after being maintained at 90 °C for 10 min. One unit (U) of activity was expressed as the release of 1 lmol phosphate per min under the assay conditions. A calibration curve was obtained by the foregoing procedure using K2HPO4 solutions of known concentrations as standards [18].
Results Identification of Pho PPiase gene Pho PPiase gene was amplified by PCR from a P. horikoshii genomic DNA, cloned and sequenced to confirm the sequences in the database (data not shown). Purification of Pho PPiase Pho PPiase was inserted in a pET15b vector with the His-Tag oligohistidine domain. The cells were collected after culturing; most of the proteins from E. coli were separated by centrifugation after heating at 85 °C. The enzyme obtained by Ni-chelating chromatography was almost 41-fold more pure than the crude extract, and this purification was achieved in a single step (Table 1). The apparent enzymes molecular mass of the purified enzyme was estimated to be about 24.5 kDa by SDS–PAGE using a gradient gel (Fig. 1A). This value is inconsistent with the size (20,833 Da) calculated from the amino acid sequence. This discrepancy might be explained by the fact that Pho PPiase has many positively charged amino acid residues. Western blotting analysis, which was performed to confirm the identity of the major purified protein as Pho PPiase, revealed it was strongly immunoreactive with anti-His6 antibodies. Western blotting was carried out to confirm the presence of Pho PPiase (Fig. 1B). Effects of temperature and pH on Pho PPiase activity The temperature profile of Pho PPiase was determined by assaying its activity from 50 °C to 100 °C. The optimum temperature was 88 °C (Fig. 2). Activity profiles of Pho PPiase were also investigated at different pH values at 90 °C in 100 mM potassium phosphate
Table 1 Purification of Pho PPiase. Step
Protein concentration (mg/ml)
Specific activity (U/mg)
Crude extract Ni-chelating column
4.80 0.25935
2.45 101.61
Protein determination During the purification steps, protein concentration was determined according to the Bradford method using BSA as the standard [19]. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and Western blot analysis The identity of the target protein was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis. SDS–PAGE was performed with a 15% (w/v) polyacrylamide gel. The gel was stained with Coomassie brilliant blue. For Western blotting, protein extracts separated by SDS–PAGE were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h in 5% non-fat dry milk in Tris-buffered saline (TBS), and incubated with primary anti-His6 antibodies over-night at 4 °C. Bound antibodies were detected using horseradish peroxidase conjugated goat anti-mouse IgG antibodies. Finally, the target protein was visualized with 3,3-diaminobenzidine (DAB) as a substrate.
Fig. 1. Detection of the purified Pho PPiase. (A) Detection of Pho PPiase by SDS– PAGE. (B) Detection of Pho PPiase by Western blotting analysis.
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D. Lu et al. / Protein Expression and Purification 99 (2014) 94–98 Table 2 Effects of different metal cations. Metals
Specific activity (U/mg)
EDTA Cd2+ Mn2+ Ca2+ K+ Mg2+
0 0 19.410 0.867 2.426 74.696
Fig. 2. Temperature dependence of Pho PPiase activity. The assay mixture contained 2 mM MgCl2, 1 mM PPi, 50 mM Tris–HCl (pH 8.0), and Pho PPiase.
Fig. 4. Effect of Mg2+ on Pho PPiase activity. The reaction system was the same as that for the standard activity assay conditions.
Fig. 3. pH dependence of Pho PPiase activity. The reactions were carried out at 90 °C for 10 min.
buffer (pH 5.0–7.5), 100 mM Tris–HCl buffer (pH 7.5–9.0), and 100 mM glycine–NaOH buffer (pH 9–11). As observed in Fig. 3, the optimum pH value was 10.3. Effects of various salts and denaturants on Pho PPiase activity As observed in Table 2, Pho PPiase showed maximum activity in the presence of Mg2+. The result was compared with the results for EDTA, Cd2+, Ca2+, Mn2+, K+, and Zn2+, and it was determined that the influence of other metal cations was negligible. Variations in Pho PPiase activity based on the ionic strength of Mg2+ in the 0– 8 mM concentration range were investigated using the standard activity assay conditions. Pho PPiase activity was found to increase along with the Mg2+ concentration (Fig. 4). Stability The effect of temperature on stability was determined by incubating the purified Pho PPiase at temperatures ranging from 25 °C to 100 °C in the 50 mM Tris–HCl buffer (pH 8.0) and measuring residual activity after appropriate incubation periods at the different temperatures. The results in Fig. 5 show that the half-life was
Fig. 5. Thermal stability of Pho PPiase. The system contained 50 mM Tris–HCl (pH 8.0), EDTA, and Pho PPiase. The mixture was incubated at 95 °C (d) for 24 h and 100 °C (N) for 12 h. Aliquots were removed to determine the remaining activity.
4 h at 100 °C and 5 h at 95 °C. In addition, it remained active at 25 °C for 7 days. The recombinant Pho PPiase retained 19.61% of its original activity after incubation at 100 °C for 12 h, indicating it was highly thermostable. PPiase was also incubated with different concentrations of urea, and aliquots were removed to determine the residual activity. The enzyme retained 40.66% of the original activity in the presence of 8 M urea after incubation for 24 h at 50 °C and 39.3% of the original activity in the presence of 6 M urea at 50 °C after incubation for
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Relative activity (%)
PPi ATP ADP PPPi PPPPi
100 2.4 0 40.3 25.2
Discussion
Fig. 6. Effect of denaturants on Pho PPiase. The assay mixture was incubated in 8 M urea (d) and 6 M urea (N) at 50 °C. The reaction was carried out at 90 °C for 10 min.
24 h (Fig. 6). It also retained 25.96% of its original activity in the presence of 8 M urea after incubation for 120 h at 50 °C. Thus, Pho PPiase was relatively stable against denaturants. Kinetic characterizations of Pho PPiase The effect of PPi concentration on the initial rate of the catalyzed reaction was investigated using the Lineweaver–Burk double reciprocal plot (Fig. 7). Km, kcat, and Vm values were calculated from the plot. Km (14.27 lM) was somewhat higher than that of other PPiases, such as those from the thermoacidophilic archaebacterium Thermoplasma acidophilum (7 lM). On the other hand, Vm was 34.35 lmol/min/mg protein and kcat was 3436 s 1. Thus, Pho PPiase showed pronounced specific activity. Substrate specificity Activity against other substrates, such as ADP, ATP, PPPi, and PPPPi, was determined using the same method as above, except PPi was replaced by each substrate. Table 3 shows that Pho PPiase was specific for PPi. It barely showed any specificity to the higher polyphosphates (PPPi and PPPPi) and showed no specificity to ADP and ATP.
Fig. 7. Determination of kinetic parameters. The reaction was carried out at 88 °C and pH 10.3.
PPiases from various sources are divided into two groups on the basis of their molecular mass and oligomeric structure. The first is the eukaryotic group and contains PPiases from Saccharomyces cerevisiae (Y-PPiase) [20], Kluyveromyces lactis [21], Arabidopsis thaliana [22], and the bovine retina [23]. The other is the prokaryotic group and includes PPiases from E. coli [24], Thermus thermophilus (Tth PPiase) [25], Bacillus stearothermophilus (Bst PPiase) [26], Rhodospirillum rubrum [27], thermophilic bacterium PS-3 [20,28], archaeon Methanobacterium thermoautotrophicum (strainDH) [29], Sulfolobus acidocaldarius strain 7 [30], and Sulfolobus sp. strain 7 [31]. Eubacterial and archaeal enzymes are homotetramers or hexamers comprising 19- to 23-kDa subunits [1,2,10], and eukaryotic enzymes are homodimers comprising 32- to 35-kDa subunits [20,32,33]. Thus, Pho PPiase resembles the bacterial and archaeal type [34], based on the molecular mass of a single subunit. All known PPiases, both cytoplasmic and membrane-bound, require a divalent metal ion, with Mg2+ conferring the highest activity. Previous kinetic studies of PPi hydrolysis and Pi/PPi exchange have suggested that Mg2+ has a dual role: free magnesium ion activates the enzyme, whereas magnesium pyrophosphate is the substrate for soluble PPiase [17]. The active site structure of soluble PPiases is remarkably similar to that of membrane-bound PPiases, supporting the notion that they share a common catalytic mechanism [27]. Despite similar requirements for all types of PPiases, the differences in their kinetic properties could be important in their regulation [35]. Pho PPiase was dependent on the presence of divalent cations for catalytic activity, with the highest activity in the presence of Mg2+. In particular, a drastic increase in enzyme activity was observed in the presence of Mg2+ (0–2 mM). The enzyme also retained high levels of activity in the presence of up to 8 mM Mg2+. Ca2+ and K+ had little impact on enzyme activity for catalysis. Other cations (Mn2+) might be able to replace Mg2+, but their impact needs to be studied further. PPiases reported to date are relatively thermostable, especially in the presence of divalent metal cations, as reported for the other PPiases [26]. However, most become inactive at high temperatures (80–100 °C). Pho PPiase was particularly thermostable and did not become inactive at 100 °C even in the absence of Mg2+. In addition, Pho PPiase was stable against various denaturants, retaining some activity in 8 M urea. Urea was found to inhibit the enzyme at very high concentrations. The inhibitory effect of urea on enzyme activity is probably due to some subtle conformational change or direct interaction of urea with the active site of the enzyme. Further studies are needed to determine why there is residual enzyme activity after a 5 h incubation in 8 M urea. Pho PPiase showed optimal activity at alkaline pH and at a higher optimal temperature than PPiases from other archaea such as M. thermoautotrophicum (7.7 and 70 °C, respectively) [29], S. acidocaldarius (6.0 and 75 °C, respectively) [36], and T. acidophilum (6.7 and 85 °C, respectively). Therefore, it is likely to have widespread applications. PPiases are becoming an important model for enzymatic hydrolysis and synthesis of polyphosphates [37]. The present method of
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genetic engineering provides an effective means of producing diverse folding patterns and enzymatic properties. Therefore, desired enzymes have become more accessible. Attention has recently been directed to the mechanism and regulation of active transport systems in PPiases, including proton transport systems in the membrane. One example is the study of the tomoplast PPiase from the mung bean [38]. PPiases may also have an important role in evolution by affecting the accuracy with which DNA molecules are copied during chromosome duplication [39]. Further study of the structure and function of PPiases is therefore extremely important. In conclusion, P. horikoshii is a hyperthermophilic archaea, and Pho PPiase is thus an excellent model for studies of thermostability and folding dynamics. Large-scale production of highly thermostable enzymes for industrial use may become a practical application. Acknowledgments This work was supported by Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University. The authors would like to thank Enago (www.enago.cn) for the English language review. References [1] D.A. Ware, J.R. Postgate, Physiological and chemical properties of a reductantactivated inorganic pyrophosphatase from Desulfovibrio desulfuricans, J. Gen. Microbiol. 67 (1971) 145–160. [2] N. Tomonaga, T. Mori, Purification and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans, J. Biochem. 81 (1977) 477–483. [3] R. Lahti, T. Niemi, Purification and some properties of inorganic pyrophosphatase from Streptococcus faecalis, J. Biochem. 90 (1981) 79–85. [4] A. Hachimori, A. Takeda, M. Kaibuchi, N. Ohkaeara, T. Samejima, Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus, J. Biochem. 77 (1975) 1177–1183. [5] J. Josse, Constitutive inorganic pyrophosphatase of Escherichia coli I. Purification and catalytic properties, J. Biol. Chem. 241 (1966) 1938–1947. [6] M.Y. Liu, J. Le Gall, Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibrio vulgaris: rubrerythrin and a new, highly active, enzyme, Biochem. Biophys. Res. Commun. 171 (1990) 313–318. [7] J.A. Verhoeven, K.M. Schenck, R.R. Meyer, J.M. Trela, Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus, J. Bacteriol. 168 (1986) 318–321. [8] M.S.M. Jetten, T.J. Fluit, A.J.M. Stams, A.J.B. Zehnder, A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii, Arch. Microbiol. 157 (1992) 284–289. [9] O.M.H. Richter, G. Schäfer, Purification and enzymatic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum, Eur. J. Biochem. 209 (1992) 343–349. [10] G.J.W.M. van Alebeek, J.T. Keltjens, C. van der Drift, Purification and characterization of inorganic pyrophosphatase from Methanobacterium thermoautotrophicum (strain delta H), Biochim. Biophys. Acta 1206 (1994) 231–239. [11] M. Maeshima, Vacuolar H+-pyrophosphatase, Biochim. Biophys. Acta 1465 (2000) 37–51. [12] S.S. Jiang, L.L. Fan, S.J. Yang, S.Y. Kuo, R.L. Pan, Purification and characterization of thylakoid membrane-bound inorganic pyrophosphatase from Spinacia oleracia, L, Arch. Biochem. Biophys. 346 (1997) 105–112. [13] M. Zancani, F. Macri, A.D.B. Peruffo, A. Vianello, Isolation of the catalytic subunit of a membrane-bound H+-pyrophosphatase from pea stem mitochondria, Eur. J. Biochem. 228 (1995) 138–143. [14] M. Lundin, H. Baltscheffsky, H. Ronne, Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function, J. Biol. Chem. 266 (1991) 12168–12172. [15] T. Sivula, A. Salminen, A.N. Parfenyev, P. Pohjanjoki, A. Goldman, B.S. Cooperman, A.A. Baykov, R. Lahti, Evolutionary aspects of inorganic pyrophosphatase, FEBS Lett. 454 (1999) 75–80.
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