Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT 3

pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability of PhPPase was enhanced in the presence of Mg. A divalent cation was absolutely required for enzyme activity, Mg being most effective; Zn, Co and Mn efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Km for pyrophosphate and Mg 2+ were 113 and 303 μM, respectively; and maximum velocity, Vmax, was estimated at 930 U mg.


Introduction
Inorganic pyrophosphatases (PPases, EC 3.6.1.1)are widely distributed in all living cells and catalyze the hydrolysis of pyrophosphate (PP i ) into two orthophosphates (P i ) (Chen et al. 1990, Lundin et al. 1991).In many organisms, the bulk of PPase activity is soluble, localized in the cytosol and serves mainly to remove the PP i formed in biosynthetic reactions driven by nucleotide triphosphates (Kornberg 1962).Pyrophosphatases from the Bacteria, Archaea and Eukarya have been characterized.Bacterial and archaeal PPases exist as either homotetramers or homohexamers, with a subunit molecular mass of about 20 kDa.In contrast, eukaryotic PPases form mostly homodimers, with a subunit molecular mass of about 28-35 kDa.To date, the soluble PPases have been classified into Family I and Family II PPases (Shintani et al. 1998, Young et al. 1998).Family I PPases have a highly conserved active site structure formed by 14-16 amino acid residues and three to four Mg 2+ ions and have similar catalytic properties (Harutyunyan et al. 1996, Heikinheimo et al. 1996, Baykov et al. 1999).Family II PPases are activated by Mn 2+ and display 10-20 times greater activity than Family I PPases (Shintani et al. 1998, Parfenyev et al. 2001).
Enzymes derived from microorganisms growing at extreme temperatures are of biotechnological use as highly thermostable biocatalysts.Thermostable PPases are commonly used to suppress pyrophosphorolysis in DNA sequencing reactions with DNA polymerases (Tabor andRichardson 1990, Vander et al. 1997).Some extremely thermostable PPases have been isolated and characterized from archaea (Sulfolobus (Wakagi et al. 1992), Methanobacterium (van Alebeek et al. 1994), Methanococcus (Kuhn et al. 2000), Thermus (Satoh et al. 1998) and Thermoplasma (Richter and Schafer 1992)).However, no information is available on the biochemical properties of thermostable PPase from hyperthermophilic archaeon Pyrococcus spp.
In the genome database of the hyperthermophilic archaeon Pyrococcus horikoshii OT3, we found the homologous PPase gene.The putative PhPPase gene has been classified as a member of the Family I PPases (Young et al. 1998, Shintani et al. 1998), and the structure of P. horikoshii PPase has already been reported (Liu et al. 2004).In this study, we report on the cloning of P. horikoshii PPase and compare the properties of the recombinant PhPPase with those of other microbial PPases.

Enzyme construction and expression
Chromosomal DNA of P. horikoshii OT3 was prepared as reported previously (Gonzalez et al. 1998).The gene (PH1907) was amplified by PCR using the chromosomal DNA as the template and two primers, PP1 (forward): 5′-AGGGGGTA CATATGAACCCGTTCCACGACCT-3′; and PP2 (reverse): 5′-AATAAAGTCGACTCACTCCTTCTTGCCGA-3′, which was designed based on an open reading frame (ORF) sequence for a protein of 178 amino acids.Amplification by PCR was carried out with KOD DNA polymerase at 94 °C for 30 s, 55 °C for 2 s, 74 °C for 30 s for 30 cycles.An amplified 0.53-kbp DNA fragment was digested with restriction enzymes NdeI and SalI (the NdeI and SalI sites are underlined in primers PP1 and PP2, respectively) and inserted into the pET-21a vector.The nucleotide sequences of the inserted genes were confirmed with an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Wellesley, MA).Escherichia coli BL21(DE3) cells containing the expression plasmid were grown in 2YT medium (1% yeast extract, 1.6% tryptone, 0.5% NaCl) containing ampicillin (100 µg ml -1 ) to an OD 600 of approximately 0.6.Gene expression was then induced with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 4 h at 37 °C.

Purification of the recombinant proteins
Escherichia coli cells containing the expressed recombinant enzyme were centrifuged and frozen at −70 °C.The thawed cells were then disrupted by sonication in buffer A (50 mM Tris-HCl, pH 8.0, 15 mM MgCl 2 , 0.1 mM EDTA).The suspension of disrupted cells was centrifuged at 27,000 g for 30 min and the supernatant fraction was heat-treated at 85 °C for 30 min, followed by recentrifugation.The supernatant was loaded on a HiTrap Q column (Amersham Biosciences, Piscataway, NJ) equilibrated in buffer A, and the bound protein was eluted with a linear gradient of NaCl (0 to 1.0 M in the same buffer).The protein solution was concentrated using a centricon 10 filter from Amicon (Millipore, Bedford, MA) and dialyzed against buffer B (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 15 mM MgCl 2 ).The dialyzed solution was loaded on a HiPrep Sephacryl S-200 HR 26/60 column (Amersham Biosciences) and eluted with buffer B. The purity of the recombinant protein was assessed by SDS-PAGE.Protein concentration was determined using a Bio-Rad protein assay system (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.

Enzyme assay
In the standard assay, PPase activity was determined by the formation of inorganic phosphate at 60 °C in the reaction mix-ture containing 50 mM Tris-HCl (pH 7.5 at 60 °C), 5 mM MgCl 2 and 1 mM sodium phosphate.The reaction was started by the addition of enzyme, and 100-µl samples were taken at 30-s time intervals and put on ice.The phosphate present was determined by adding 1 ml of phosphate reagent (1% ammonium heptamolybdate (w/v), 0.83 M sulfuric acid, 8% iron (II) sulfate (w/v)) and the absorbance of the phosphomolybdate complex was measured at 660 nm after 30 min (van Alebeek et al. 1994).Activities were developed with 0.1 µmol of standard sodium phosphate.The activity towards other substrates such as sodium tripolyphosphate (PPP i ), phosphoenolpyruvate (PEP), p-nitrophenyl phosphate (pNP), ATP, ADP and AMP was determined by the same method as above except that PP i was replaced by each substrate.One unit of activity is defined as the amount of enzyme required to hydrolyze 1 µmol PP i per min at 60 °C.
The ORF (PH1907) was amplified by PCR from a P. horikoshii genomic DNA, cloned and sequenced to confirm the sequences in the database.The gene was expressed in E. coli cells, and the recombinant protein was purified as described in the Materials and methods.Expression and subsequent purification yielded 3.2 mg of PhPPase from a 1-l culture.

Molecular properties
The molecular mass of the protein from PH1907 was estimated to be about 24.5 kDa by SDS-PAGE (Figure 1).This value is inconsistent with the size (20,833 Da) calculated from the amino acid sequence.This discrepance may be explained by the many positively charged amino acid residues of PhPPase.Recently, a three-dimensional crystal structure of PhPPase showed that the enzyme is an oligomeric hexamer (Liu et al. 2004).

Catalytic properties
We measured the activity of the recombinant PhPPase in the presence of 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 and 1 mM sodium pyrophosphate.The recombinant PhPPase showed maximal activity in the temperature range from 60 to 75 °C, with a temperature optimum of 70 °C (Figure 2a).The optimal pH for the PhPPase was 7.5 at 60 °C.Heat inactivation of the enzyme was estimated by measuring the residual PPase activity after heat treatment at two different temperatures in the presence or absence of 5 mM Mg 2+ (Figure 2b).The recombinant PhPPase showed high thermostability, and the half-life of heat inactivation was about 50 min at 105 °C.The heat stabil-ity of PhPPase was much higher than that reported previously for other thermostable PPases from Aquifex aeolicus (90 min at 95 °C) (Hoe et al. 2001) and Sulfolobus acidocaldarius (150 min at 95 °C) (Hansen et al. 1999).Furthermore, the heat stability of PhPPase was enhanced by the addition of Mg 2+ to the incubation mixture, as reported for the other PPases (Hachimori et al. 1979, Ichiba et al. 1988).The difference between the optimum temperature of PhPPase and the optimum growth temperature of P. horikoshii suggests that PhPPase requires co-factors for its thermophilicity in cells.

Divalent cation requirement
Family I PPase has shown strong metal cation-dependency, with Mg 2+ conferring the highest efficiency (Cooperman 1982) and sensitivity to inhibition by Ca 2+ (Yang and Wensel 1992).In contrast, Family II PPase prefers Mn 2+ over Mg 2+ (Parfenyev et al. 2001).Pyrocococcus horikoshii pyrophosphatase is dependent on the presence of divalent cations for catalytic activity, with the highest activity in the presence of Mg 2+ .Other cations (Zn 2+ Co 2+ Mn 2+ ) could efficiently replace Mg 2+ , but the effectiveness of the latter cations is limited to a narrow range of concentrations (0.05-0.5 mM) (Figure 3).Neither Ni 2+ nor Ca 2+ could activate the enzyme for catalysis.These results support the conclusion that PhPPase belongs to the Family I PPases.The enzyme responded sensitively to changes in the cation concentration of the reaction mixture.In particular, a drastic decrease in enzyme activity was observed at Zn 2+ , Co 2+ and Mn 2+ concentrations above 0.5 mM (Figure 3).The inhibition at high cation concentrations is probably due to the presence of free cations in the assay (Celis and Romero 1987).

Substrate specificity
The substrate specificity of PhPPase was investigated (Table 1) and found to be high with respect to PP i .The release activities of inorganic phosphate (P i ) from PPP i , ATP and ADP were determined to be 2.7, 5.9 and 2.9% of that from PP i , respectively.Hydrolysis of AMP, PEP and pNP by PhPPase was not observed.
The reaction rate of PhPPase at different PP i and Mg 2+ concentrations did not follow normal Michaelis-Menten kinetics.Therefore, kinetic data were analyzed according to the procedure of Hill by the EZ-Fit program (Perrella 1988).At pH 7.5 and 60 °C, the values of K m and maximum velocity (V max ) for PP i were determined to be 113 ± 12 µM and 930 ± 40 U mg -1 , respectively, which corresponds to a k cat of 744 s -1 .An apparent K m for Mg 2+ was determined to be 303 ± 9 µM.
In conclusion, we have identified a gene that codes for PPase protein from the P. horikoshii genome and also compared the properties of recombinant PhPPase with those of other PPases.The PhPPase is more similar to bacterial and archaeal enzymes than to eukaryotic enzymes, based on the molecular mass of a single subunit.Cytoplasmic PPase has no function in energy conservation.Its function seems to keep the cellular PP i concentration low, through the hydrolysis of PP i .Whether PhPPase is located in the cytoplasm has not been reported.This work increased our understanding of the metabolism of a hyperthermophilic archaeon.The hyperthermostability of PhPPase suggests that it can be used to suppress pyrophosphorolysis in sequencing reactions using thermostable DNA polymerases (Tabor andRichardson 1990, Vander Horn et al. 1997).
Table 1.Substrate specificity of the Pyroccocus horikoshii pyrophosphatase (PhPPase).Activities were determined as described in Materials and methods.Each substrate was tested at a concentration of 1 mM in the presence of 5 mM Mg 2+ .

Figure 2 .
Figure 2. Thermophilicity and thermostability of Pyrococcus horikoshii pyrophosphatase (PhPPase).(a) The PPase activity was determined at the indicated temperatures, as described in the Materials and methods.Negative control reactions in the absence of the enzyme were performed in parallel.(b) Enzyme (1 µM PhPPase in 50mM Tris-HCl, pH 7.5) was incubated at 100 °C in the presence (᭺) or absence (᭹) of Mg 2+ and at 105 °C in the presence (ᮀ) or absence () of Mg 2+ , and the residual PPase activity of samples was measured at 60 °C.

Figure 4 .
Figure 4. Inhibition of PhPPase activity by NaF.The specific activity was determined at the indicated NaF concentrations, as described in Materials and methods.