• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Nov 1999; 181(22): 7107–7114.
PMCID: PMC94187

Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from Prevotella intermedia

Abstract

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an Mr of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.

Prevotella intermedia, an anaerobic, gram-negative, rod-shaped bacterium, has been reported to be associated with advanced adult periodontal disease (4, 39), acute necrotizing ulcerative gingivitis (21), and pregnancy gingivitis (16, 32). Although the exact role of oral microbes in the etiology of periodontal disease remains unknown, they have been shown to produce a variety of potential virulence factors, including high phosphatase activity (38). To date, alkaline phosphatases (ALPases) of both P. intermedia and Porphyromonas gingivalis have been characterized (3, 48). A neutral phosphatase gene was also isolated from the oral spirochete Treponema denticola, which is associated with chronic periodontal disease (14). In addition, acid phosphatases (ACPases) are widely found in oral gram-negative bacteria (22), although only limited information regarding their role in periodontal disease or in the bacterial life cycle is currently available. For this reason, we have initiated a study of ACPase activities of the oral pathogens. P. intermedia was selected as the bacterium of choice, since ACPase activities of P. intermedia strains isolated from active sites in patients were significantly higher than those from healthy subjects (23), suggesting a possible clinical correlation. While studying the properties of the ACPase from P. intermedia, we found that purified enzyme exhibited substantial activities against O-phospho-l-tyrosine as well as phosphotyrosine-containing peptides, compounds that act as substrates for phosphotyrosyl phosphatases (PTPases).

In general, protein tyrosine phosphorylation is associated with alterations in receptor activity, cellular proliferation, and modulation of the cell cycle (18). Eukaryotic PTPases have been shown to constitute a family of enzymes that contain a number of conserved motifs, such as HCXAGXXR. However, prokaryotic PTPases appear to constitute novel families that are devoid of these motifs but that contain unique signature motifs of their own. For instance, small, acidic PTPases contain N-terminally located, highly conserved active sites (FVCXGNICRSPXAEAXF) (20). Moreover, PiALP, an ALPase from P. intermedia, appeared to represent a new family of alkaline PTPases that showed significant homology with the predicted primary structures of PhoD from Zymomonas mobilis and PhoV from Synechococcus spp., although PTPase activities of PhoD and PhoV were not investigated (3).

In the present study, we have purified an ACPase containing a PTPase activity, designated PiACP, from P. intermedia ATCC 25611 and isolated and sequenced its corresponding gene. The gene encoded a protein exhibiting significant homology to the class A bacterial ACPases. This is the first report that one of the class A bacterial ACPases possesses PTPase activity. Furthermore, preliminary mutational analysis of the enzyme was carried out to ascertain the essential role of conserved amino acid residues in enzymatic activity.

MATERIALS AND METHODS

Bacterial strain and growth conditions.

P. intermedia ATCC 25611 was obtained from the American Type Culture Collection and grown anaerobically (5% [vol/vol] CO2, 10% [vol/vol] H2, 85% [vol/vol] N2) at 37°C in brain heart infusion broth (Difco) supplemented with yeast extract (0.5%), hemin (5 μg/ml), and menadione (0.5 μg/ml). Escherichia coli JM109 and E. coli BL21(DE3) and BL21(DE3)/pLysS were used in subcloning and expression experiments. All E. coli strains were grown on Luria-Bertani (LB) agar plates or in LB broth in the presence of appropriate antibiotics (ampicillin, 50 μg/ml; chloramphenicol, 34 μg/ml).

Enzyme assays.

Standard phosphatase reaction mixtures contained 50 mM sodium acetate (pH 4.9), 50 mM NaCl, 5% glycerol, 1.0 to 10 ng of enzyme (depending on activity), and p-nitrophenylphosphate (pNPP) (10 mM) as a substrate. The pNPP reactions were carried out in 0.5 ml, terminated by the addition of 0.5 ml of 1 N NaOH, and quantified by measuring absorbance at 420 nm.

The PTPase activity of PiACP on the synthetic phosphopeptide A-E-N-A-E-Y(P)-L-R-V (corresponding to human epidermal growth factor [EGF] receptor [EGFR]) was determined with 0.2 mM phosphopeptide in 50 mM sodium acetate (pH 4.9) buffer. The phosphopeptides were purchased from Sawady (Tokyo, Japan) as previously described (40). The reactions (25-μl reaction mixtures) were started by adding the purified PiACP (0.2 to 5 ng); the reaction mixtures were incubated at 37°C for 15 min, and the reactions were terminated by the addition of 100 μl of malachite green solution (Upstate Biotechnology Inc., Lake Placid, N.Y.), followed by incubation at room temperature for an additional 15 min. The free inorganic phosphate released from the peptide(s) was determined by measuring the A650 according to the manufacturer’s instructions.

Routine measurements of phosphatase activity were carried out by the pNPPase assay, in which one unit of activity was defined as that hydrolyzing 1 μmol of substrate per min at 37°C. All values for enzyme activity represent means of three replicate determinations. The effects of various divalent metal ions on enzyme activity were examined by using 100 mM sodium acetate buffer (pH 4.9) containing 10 mM pNPP and 2 or 5 mM concentrations of one of the following: ZnCl2, MgCl2, MnCl2, BaCl2, CaCl2, or CuSO4. The optimal pH was determined with 100 mM concentrations of the following buffers at the appropriate pH range: sodium acetate buffer (pH 5.0 to 6.5), Tris-HCl buffer (pH 6.5 to 8.5), and glycine-NaOH buffer (pH 8.5 to 10.0). The kinetics of the enzyme with pNPP and EGFR peptides as the substrate were determined at 37°C and at optimum pH, i.e., pH 4.9. For the determination of Km and maximum velocity (Vmax), pNPP and EGFR were used at concentrations in the ranges 0.25 to 10 and 0.04 to 0.4 mM, respectively.

When the phosphatase activities of the enzyme were examined by using the esters pNPP, 3′-AMP, 5′-AMP, β-glycerophosphate, glucose-6-phosphate, ATP, α-naphthyl acid phosphate, O-phosphotyrosine, O-phosphoserine, and O-phosphothreonine, the amount of inorganic phosphate released was estimated by the method of Fiske and Subbarow (9).

Purification of PiACP.

Unless otherwise mentioned, all the procedures were performed at 0 to 4°C. P. intermedia ATCC 25611 cells (20 g) were harvested from 8 liters of culture by centrifugation at 10,000 × g for 20 min. The cells were washed twice with 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl, suspended in 50 mM Tris-HCl (pH 7.5) containing 5% glycerol (buffer A) and 50 mM NaCl, and then broken by sonic disruption with 1-min pulses of a sonic disrupter. To the resulting lysate, Triton X-114 was added to a final concentration of 1%, and the mixture was stirred for 90 min at 4°C and then centrifuged at 100,000 × g for 60 min. The supernatant fraction was collected as the crude enzyme extract and applied to a DEAE Bio-Gel (Bio-Rad Laboratories, Hercules, Calif.) column (6 by 20 cm) equilibrated with buffer A containing 50 mM NaCl. The column was washed with the equilibration buffer until no protein was detected in the effluent by measurement of A280. The flowthrough (unbound) fraction was concentrated by filtration through a Centricon-10 ultrafiltration device (Millipore, Bedford, Mass.) and applied to a carboxymethyl Bio-Gel (Bio-Rad) column (2 by 40 cm) equilibrated with buffer A containing 50 mM NaCl. The flowthrough fraction was concentrated as described above and applied to a phosphocellulose (Sigma, St. Louis, Mo.) column (2 by 20 cm) equilibrated with buffer A containing 50 mM NaCl. The flowthrough fraction was dialyzed against buffer A containing 0.5 M NaCl and loaded onto a phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden) column (1 by 7 cm) equilibrated with 0.5 M NaCl in buffer A. The enzyme was eluted with 50 mM Tris-HCl (pH 7.5) containing 50% glycerol. The active fractions were pooled and concentrated as described above. The purified enzyme was frozen and stored in small portions at −80°C.

To determine the molecular weight of PiACP, the purified enzyme was subjected to high-performance liquid chromatography with TSK gel G3000SWxl (TOSOH, Tokyo, Japan) preequilibrated with 200 mM Tris-HCl (pH 7.5) and calibrated with molecular weight standards (Oriental Yeast).

Dephosphorylation of phosphotyrosine proteins in A431 lysate.

A 2-μl aliquot of purified PiACP was incubated with 10 μl of lysate of the human epidermoid carcinoma cell line A431 that contained phosphotyrosine EGFR (Upstate Biotechnology) in the presence of 50 mM sodium acetate (pH 4.9)–50 mM NaCl–5% glycerol at 37°C for 1, 3, or 15 h. Proteins were resolved in 10% polyacrylamide gels containing sodium dodecyl sulfate (SDS) (17), followed by transfer to Immobilon-P membranes (Millipore) for immunoblot (Western) analysis by using RC20H antibodies (1:1,000) and the ECL method (Amersham). In multiple trials, 1 to 5 min of exposure to X-ray films was sufficient to visualize bands.

Determination of partial amino acid sequences.

The purified samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred electrophoretically to Immobilon-P membranes as described above, and then stained with Coomassie blue R-250 (Sigma). The stained bands were excised, and the absorbed proteins were sequenced with automatic gas phase sequencer HP G1005A (Hewlett-Packard, Palo Alto, Calif.) by Takara Shuzo (Tokyo, Japan). Another sample was digested with Staphylococcus aureus V8 protease (Sigma). The peptides generated were resolved by SDS-PAGE (18% polyacrylamide), electrophoretically transferred to Immobilon-P membranes, and stained with Coomassie blue R-250. Portions of the membranes containing the two most visually prominent bands were excised and subjected to automatic gas phase sequencing.

Oligonucleotides and DNA amplification by PCR.

For amplification of DNA fragments encoding the partial amino acid sequences from the purified PiACP, two synthetic oligonucleotide primers were designed on the basis of the amino acid sequences of two internal regions of the purified enzyme. The primers TA42 (5′-TNYTNCCNACNCCNCCNCAR-3′) and TA43 (5′-GTRTGNCCNSWNGGRTANSWNCC-3′) (N denotes complete degeneracy) were synthesized by Takara Shuzo. Amplification of the DNA fragments was carried out with these primers in standard PCR buffer that included 2.5 mM MgCl2, 200 μM (each) dATP, dTTP, dCTP, and dGTP, and 2 U of Taq polymerase (Promega, Madison, Wis.). The thermal cycle parameters were 94°C for 1 min (denaturation), 34°C for 2 min (annealing) and 72°C for 1 min 10 s (extension), for 35 cycles. In addition, time delays of 2 min at 94 and 72°C were incorporated at the beginning and end, respectively. The PCR product was purified by use of a gel extraction kit (Qiagen) and cloned into the pGEM-T vector (Promega). Sequencing the resulting clones led to the identification of one encoding the anticipated amino acid sequence of PiACP. The DNA insert of this clone was then used as a probe for further screening to obtain the full-length PiACP clone.

Construction of a genomic library and screening.

Genomic DNA from P. intermedia ATCC 25611 was isolated as previously described (1). Standard procedures for recombinant DNA manipulations were carried out as described by Sambrook et al. (34). For construction of the genomic library, P. intermedia chromosomal DNA was digested with HincII and HindIII and was ligated to pBluescript SK(+) and KS(+), respectively, that had been cut with the corresponding restriction enzymes and treated with ALPase. The oligonucleotide probe (~400 bp of PCR product) was labeled with digoxigenin by using a labeling kit (Boehringer Mannheim, Indianapolis, Ind.) according to the manufacturer’s instructions. Colony hybridization was performed as previously described (1). Positive clones were detected with the reagents and procedures of the same kit.

DNA sequencing.

Plasmid DNA of the pBluescript clones obtained above was prepared with the Wizard Miniprep system (Promega) and sequenced by the dideoxy method of Sanger et al. (35) with a dye terminator sequencing kit (Applied Biosystems) together with a synthetic oligonucleotide primer. The sequence was determined with an Applied Biosystems model 373S automated DNA sequencer. The nucleotide sequences were analyzed with the computer software package DNA Strider, version 1.2 (24). Amino acid homology searches and comparisons were done with GENETYX-Mac software (Software Development Co., Ltd., Tokyo, Japan) and BLAST network services of DDBJ. Sequence alignments were optimized with the CLUSTAL W program (45).

Expression of PiACP gene in E. coli.

The putative PiACP gene open reading frame (ORF) (792 bp) was subcloned in the T7-based bacterial expression plasmid pET3a as follows. The gene was amplified by the following primers (corresponding to the 5′ and 3′ ends of the gene, respectively): 5′-GGAGTTGCATATGACAAAAAAGACTTTACTTGTCGG-3′ and 5′-GGAGTGGATCCTTAGTTTGCTGCCTTGAAAGTG-3′ (the NdeI and BamHI sites, respectively, are underlined). The PCR product was restricted with NdeI and BamHI and cloned into the same sites of pET3a as described previously (25). The resulting clone, pET3a-PiACP, was confirmed by DNA sequencing and introduced into E. coli BL21(DE3)/pLysS. Growth of the transformant, induction with isopropyl-1-thio-β-d-galactopyranoside (IPTG), and lysis with lysozyme were carried out essentially as described previously (2). SDS-PAGE analysis of proteins was performed as described by Laemmli (17) with an 18% acrylamide (acrylamide/bisacrylamide ratio, 30:0.4) gel. Site-directed mutagenesis and deletion of the cloned PiACP gene were performed by the PCR-based “megaprimer” method as described previously (5).

Purification of recombinant PiACP.

Identical procedures were used for the purification of wild-type and three mutant recombinant PiACPs. In brief, the total extract of 1 g of induced E. coli BL21(DE3)/pLysS cells containing the pET3a-PiACP plasmid was prepared as described above. After growth for 24 h at 37°C with vigorous shaking, the cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 2 mg of lysozyme per ml), and lysed by sonication in ice. The lysate was centrifuged at 100,000 × g for 20 min. The pellet, which contained nearly all of the expressed PiACP protein, was dissolved in 6 ml of buffer A containing 6 M guanidine hydrochloride with the aid of a homogenizer under cold conditions and incubated in ice for another 1 h. The solution was centrifuged at 100,000 × g for 20 min. The supernatant was dialyzed against 2 liters of buffer A containing 50 mM NaCl with two changes. The dialyzed material was loaded onto a phosphocellulose column (1 by 6 cm) equilibrated with buffer A containing 50 mM NaCl. An NaCl gradient in buffer A was used to subsequently elute the enzyme at an NaCl concentration of about 200 mM. The phosphocellulose fraction, already highly pure, was concentrated by Centricon-10 ultrafiltration and further purified by gel filtration chromatography through TSK gel G3000SWxl, from which it was eluted as an apparent monomer (data not shown).

Nucleotide sequence accession number.

The GenBank accession number for the nucleotide sequence of the PiACP gene reported in this paper is AB017537.

RESULTS

Purification of PiACP.

The purification of PiACP from Triton X-114 extract is summarized in Table Table1.1. The enzyme was purified 194-fold with a final specific activity of 132.1 U/mg, and the overall yield of the activity was 23.5%. The molecular mass of native enzyme from P. intermedia was estimated by gel filtration to be about 28 kDa. On an SDS-PAGE gel, ~4 μg of purified PiACP was analyzed; as shown (Fig. (Fig.1,1, lane B), the preparation contains a single major band with a molecular mass of ~30 kDa, with no single contaminant being more than 5% of the total protein. These results suggested that the enzyme was a monomer of an ~30-kDa polypeptide.

TABLE 1
Summary of purification of PiACP from P. intermedia
FIG. 1
SDS-PAGE (16% polyacrylamide) analysis of the purified PiACP. Lane A, molecular mass markers; lane B, purified PiACP (4 μg). The positions and molecular masses of standard proteins are indicated at the left.

Enzymatic activity of purified PiACP.

The optimal temperature and pH for PiACP were determined in vitro by using pNPP as a substrate; essentially similar results were obtained by using a phosphorylated peptide (EGFR) (data not shown). The purified enzyme had the highest activity at around 46°C and was inactive at 60°C. The optimum pH value for the activity was approximately 4.9. To characterize PiACP further, the effect of inhibitors on the activity of the enzyme was investigated. As shown in Table Table2,2, sodium molybdate and sodium orthovanadate, which are known inhibitors of PTPases (18), and sodium fluoride were found to inhibit the activity of PiACP. On the other hand, the activity was not inhibited by okadaic acid and microcystin-LR, which are known to be specific inhibitors of serine/threonine protein phosphatases of the PP1 and PP2A families (37). Sulfhydryl-modifying reagents, such as N-ethylmaleimide, iodoacetic acid, and phenylarsine oxide (PAO), which are known to inhibit PTPases of eukaryotic origin, had no significant effect. None of the divalent metal ions stimulated the activity, while only Cu2+ and Zn2+ inhibited the activity. Like PhoN of Salmonella typhimurium, PiACP was also resistant to the ACPase inhibitors EDTA and tartrate (15). The purified PiACP dephosphorylated p-Tyr strongly and p-Ser and p-Thr weakly. In addition, it dephosphorylated the EGFR phosphopeptide containing p-Tyr (40). A commercial phosphopeptide, H2N-T-S-T-E-P-Q-Y(P)-Q-P-G-E-N-L-COOH, containing the C-terminal phosphorylation site of c-src, was also efficiently dephosphorylated by PiACP (data not shown). As shown in Fig. Fig.2,2, the Km for purified PiACP with EGFR as a substrate was determined to be 0.83 mM (pH 4.9; 37°C) and the Vmax was 8.44 μmol/min · mg of enzyme. The Km and Vmax for purified PiACP with pNPP as the substrate were determined to be 0.24 mM (pH 4.9; 37°C) and 32.46 μmol/min · mg, respectively. These values were similar to those for the rat liver low-molecular-weight PTPases with the same substrate (40). Furthermore, the enzyme strongly dephosphorylated a 170-kDa protein (molecular size corresponds to that of EGFR) in A431 cell lysate which was phosphorylated on tyrosine (Fig. (Fig.3).3).

TABLE 2
Effect of potential substrates and inhibitors on PiACP activity
FIG. 2
Substrate dependence curves for PiACP. Purified PiACP was assayed at 37°C in 50 mM sodium acetate (pH 4.9) buffer with a synthetic peptide corresponding to human EGFR, either A-E-N-A-E-Y(P)-L-R-V [0.04 to 0.4 mM; Y(P), phosphotyrosine] ...
FIG. 3
Dephosphorylation of tyrosine-phosphorylated proteins in A431 lysate by PiACP. A 2-μl aliquot of purified PiACP was incubated with 10 μl of lysate of the human epidermoid carcinoma cell line A431 for 1 h (lane b), 3 h (lane c), or 15 h ...

Partial amino acid sequence of PiACP.

The N-terminal amino acid sequence of the purified PiACP polypeptide, determined by Edman degradation, was Lys-Lys-Ile-Lys-Asp-Ala-Arg-Thr-Asn-Pro-Asp-Leu-Tyr-Tyr-Leu-Gln-Asp-Gly. Two internal sequences obtained from N-terminal sequencing of V8 protease fragments were Leu-Leu-Pro-Thr-Pro-Pro-Gln-Pro-Gly-Ser-Ile-Gln-Phe-Leu-Tyr-Asp-Glu-Ala-Gln-Tyr and Leu-Ser-Thr-Asn-Gly-Ser-Tyr-Pro-Ser-Gly-His-Thr-Ala-Ile-Gly-Trp-Ala-Thr-Ala-Leu. Interestingly, these two internal amino acid sequences were found to contain the conservative motifs in class A bacterial ACPases, thus providing preliminary evidence that the primary structure of PiACP may be similar to those of other ACPases, a conclusion further supported by sequence analysis of the complete gene as described below.

Cloning of PiACP.

On the basis of the internal amino acid sequences of PiACP, oligonucleotide primers were synthesized and PCR was performed to obtain a partial clone of the enzyme gene as described in Materials and Methods, which was then used to screen the P. intermedia genomic DNA library to identify the full-length clone. Southern blot analysis indicated that P. intermedia genomic DNA may not produce convenient restriction fragments that will contain a complete structural gene for PiACP. Since the partial region for the PiACP gene contained HincII and HindIII sites, the upstream region of the PiACP gene was screened from a HincII library and the downstream region was screened from a HindIII library of P. intermedia genomic DNA. Two positive clones from the HincII library and one from the HindIII library were independently obtained from a total of 2,000 transformants by colony hybridization. Southern blot analysis indicated that the two HincII clones contained identical inserts. The results of Southern blotting as well as restriction mapping also indicated that the HincII and HindIII clones contained overlapping DNA fragments as expected (data not shown). The clones from HincII and HindIII libraries were designated pAC1 and pAC2, respectively.

Sequence analysis of the phosphatase gene.

In order to identify and characterize the gene for PiACP, both HindIII and HincII clones were sequenced and the gene sequences were assembled and analyzed. Since the coding region was sequenced and confirmed for both strands by using overlapping fragments, sequencing errors were ruled out. This total sequence contained one complete ORF and two incomplete ORFs. Furthermore, the N-terminal and two internal sequences described earlier could be identified in a long ORF (Fig. (Fig.4).4). The PiACP gene contained 792 bp coding for a putative polypeptide of 264 amino acids with a calculated molecular mass of 29,164 Da and an estimated pI of 8.39. A potential ribosome-binding site was not identified since little information is known about P. intermedia gene control at present. A sequence with hyphenated dyad symmetry with the potential to form a stem-loop structure in the RNA was located downstream from the translational termination codon TAA, suggesting that this sequence may act as a transcriptional terminator. The overall G+C content of the gene (46.9%) agrees with that estimated for the chromosomal DNA from P. intermedia strains (41 to 44%) (36). The first 20 amino acid residues appeared to have a hydrophobic region characteristic of a signal sequence including a potential cleavage site (between Ala-20 and Gln-21), as defined by the criteria of von Heijne (47). This indicates that PiACP may be secreted across the cytoplasmic membrane by using the signal peptide.

FIG. 4
Nucleotide and predicted amino acid sequences of PiACP. The underlined areas indicate areas of the protein that were previously analyzed by amino acid sequencing. The predicted site of proteolytic cleavage of the putative signal sequence is indicated ...

The deduced sequence of the PiACP protein was compared to those of all proteins in the SwissProt database with the GENETYX-Mac program (Software Development). A significant degree of sequence homology between this enzyme and the class A bacterial ACPases, such as PhoC (the principal ACPase of Morganella morganii) (43), Apy ATP-diphosphohydrolase (6) PhoN (a nonspecific ACPase [46] of Shigella flexneri), the PhoN ACPase of Providencia stuartii (unpublished results; EMBL accession no. X64820), the nonspecific PhoN ACPase of S. typhimurium (15), and the PhoC ACPase of Z. mobilis (30), was found. The result of multiple-sequence alignment analysis within this family of enzymes showed the existence of conserved motif KX6RP-(X12–54)-PSGH-(X31–54)-SRX5HX3D, as summarized by Stukey and Carman (42), which is also shared among lipid phosphatases and mammalian glucose-6-phosphatases (G6Pases) (Fig. (Fig.5).5). The overall amino acid identities were found to be 63.6, 60.8, 38.4, and 25.3% when the PiACP protein was compared with the M. morganii, P. stuartii, S. typhimurium, and Z. mobilis enzymes, respectively. Amino acid identities with Apy and PhoN of S. flexneri sequences were 41.2 and 59.0%, respectively, confirming the class A identity of PiACP.

FIG. 5
Comparison of the deduced amino acid sequence of PiACP with those of the class A bacterial ACPases. ACP-Sfl, ACPase from S. flexneri; ACP-Pst, ACPase from P. stuartii; ACP-Mm, ACPase from M. morganii; Apy-Sfl, apyrase from S. flexneri; ACP-Sty, ACPase ...

Expression of recombinant PiACP in E. coli.

To ascertain that the PiACP gene indeed codes for a phosphatase, full-length PiACP was overexpressed in E. coli. Upon induction of E. coli BL21(DE3)/pLysS containing pET3a-PiACP with IPTG, a polypeptide with an Mr of ~30,000 was produced (Fig. (Fig.6),6), in good agreement with the calculated molecular mass of the predicted amino acid sequence (29,164 Da). Recombinant PiACP was purified as described in Materials and Methods. During purification, the ACPase activity, with either pNPP or EGFR as the substrate, always cochromatographed with the 30-kDa protein. The final preparation (Fig. (Fig.6,6, lane 2) was judged at least 90% pure. Bacterial extract obtained from uninduced cells contained very little ACPase activity, while the activity of an extract of E. coli BL21(DE3)/pLysS containing the pET3a vector was undetectable (data not shown).

FIG. 6
Overexpression of recombinant PiACP in E. coli. The ORFs for PiACP and its mutants were cloned and expressed in pET3a, as described in Materials and Methods. BL21(DE3)/pLysS cells containing the following recombinant PiACP clones were induced with (lane ...

To gain a preliminary insight into the nature of the amino acids important for ACPase activity, we treated purified recombinant PiACP with reagents that covalently modify specific acid chains (49). As shown in Fig. Fig.7,7, PiACP was strongly inactivated by diethyl pyrocarbonate (DEPC), a histidine modifier, and phenylglyoxal, an arginine modifier, at pH 4.9. However, PAO, a cysteine modifier, caused a slow and gradual loss of activity. Thus, His and Arg residues appeared to be important determinants of enzyme activity.

FIG. 7
Chemical modification of recombinant PiACP in E. coli. The residual activity was measured at 37°C (pH 4.9) with pNPP as the substrate after the following treatments: PAO (10 mM [□] and 20 mM [■]), ...

Mutagenesis of recombinant PiACP.

The invariant His and Arg residues are shared among class A bacterial ACPases, a neutral phosphatase from T. denticola, and lipid phosphatases and G6Pases (42). In G6Pases, the mutation of two conserved His residues resulted in the loss of activity (19, 29). In this study, to further confirm the role of His residues, we mutated specific His residues in recombinant PiACP and investigated the effect of the mutations on enzyme activity. A comparison between PiACP and class A bacterial ACPases revealed two conserved regions containing His residues, one of which is the peptide stretch GSYPSGHT (residues 164 to 171) of PiACP. A mutant clone in which the nucleotides corresponding to this stretch were deleted by in vitro mutagenesis was constructed. In addition, two mutants were constructed by site-specific mutagenesis of pET3a-PiACP DNA by PCR, viz., H170Q and H209T mutants. Figure Figure66 shows that the GSYPSGHT deletion mutant (lane 3) expressed a protein that migrated faster than the wild type (lane 2), indicating a molecular mass difference of ~0.8 kDa, whereas proteins expressed by H170Q (lane 4) and H209T (lane 5) mutant clones were ~2 kDa bigger than the wild type. The three mutant proteins were purified to near homogeneity, and their phosphatase activities were tested by using pNPP as the substrate. All three mutants were completely devoid of activity (data not shown). These results not only confirm the 30-kDa protein as the phosphatase but in addition suggest an essential role for these specific amino acid residues in enzyme activity.

DISCUSSION

In the present study, we isolated, cloned and analyzed a novel ACPase, PiACP, in molecular detail and discovered that it contained PTPase activity. Phosphatase activities and gene sequences in several periodontopathogenic bacteria, including P. intermedia, have been reported (3, 14, 48). However, limited information about these potentially important enzymes is available. In this study, comparison of the sequence of PiACP with the sequences of other proteins clearly indicated that PiACP was closely related to the class A bacterial ACPase family (6, 15, 30, 43, 46). Class A bacterial ACPases are further classified into two major subgroups: classes A1 and A2 (44). Class A1 enzymes are resistant to fluoride and contain a slightly smaller polypeptide component (~25 kDa, such as PhoC of M. morganii), while class A2 enzymes are inhibited by fluoride and contain a slightly larger polypeptide component (~27 kDa, such as PhoN of S. typhimurium). Very recently, a new subclass, viz., class A3, has been recognized (33); this class consists of monomeric enzymes that are inhibited by fluoride, O-vanadate, and various divalent cations, including Cu2+ and Zn2+. An example of a class A3 phosphatase is the apyrase of S. flexneri, whose activity is strongly inhibited by 1 mM O-vanadate, 5 mM sodium fluoride, 5 mM Zn2+, and 5 mM Cu2+ to less than 5% of its initial value, although it was resistant to a high concentration of EDTA (20 mM) (6, 7). Based on these diagnostic criteria, we propose that PiACP may be classified as a class A3 enzyme rather than a class A2 enzyme.

Stukey and Carman (42) have recently recognized a conserved sequence motif, KX6RP-(X12–54)-PSGH-(X31–54)-SRX5HX3D, that is shared among class A bacterial ACPases, a neutral phosphatase from T. denticola, and mammalian phosphatases (such as G6Pases and lipid phosphatases), although their overall amino acid identities were low. Of these phosphatases, G6Pases have been well investigated by structure-function analysis, which has shown that two His residues participate in the catalytic mechanism and that His-176 could act as the phosphoryl acceptor in catalysis (19, 29). Curiously, two His residues were also shown to be essential for activity in our mutational studies of PiACP (i.e., H170 and H209), although the exact role of each His residue was not determined. On the other hand, sequence alignment analysis also revealed the presence of two Arg residues conserved between PiACP and G6Pase: Arg-142 and Arg-203 in PiACP and Arg-83 and Arg-170 in G6Pase, respectively. Structure-function studies suggest that Arg-83 in G6Pase is involved in stabilizing the phosphoryl enzyme intermediate formed during catalysis (19). A detailed mutational analysis of specific PiACP residues, including Arg residues, will be needed to define their roles in the PiACP catalytic mechanism. This is in progress.

A phylogenetic tree depicting the evolutionary relationships among the primary structures of the above-described enzymes is shown in Fig. Fig.8.8. In particular, PiACP was found to be closely related to the enzyme of the enterobacteria, such as M. morganii and S. flexneri. Interestingly, it has been suggested that the phoN gene of S. typhimurium, a well-studied enterobacterium, was acquired by lateral transmission from another species, since the overall base composition of phoN was different from that of the Salmonella chromosome (11). However, this does not appear to be the case with the PiACP gene, because the G+C content of the PiACP gene (46.9%) is almost consistent with the overall G+C content (41 to 44%) of P. intermedia (36). These observations raise interesting questions about the evolution and origin of these phosphatases, since an ancestral relatedness between P. intermedia and several enterobacteria, such as Morganella species, seems to exist. In this group of phosphatase genes, only the product of the PiACP gene has thus far been shown to contain in vitro PTPase activity (this paper). It will be interesting to see if other ACPases of this group also exhibit PTPase activity.

FIG. 8
Phylogenetic tree of the class A bacterial ACPase family and other phosphatases. The unrooted tree was constructed with NjPlot of the CLUSTAL W software package. All of these phosphatases contain the motif KXXXXXXRP-(X12–54)-PSGH-(X31–54 ...

To date, several prokaryotic PTPases have been genetically and biochemically identified. For instance, IphP from the cyanobacterium Nostoc commune UTEX 584 has been cloned and characterized (31). The enzyme displayed both protein phosphoserine and PTPase activities, thus showing significant similarity with VH1 (13). Another bacterial PTPase previously described, YopH, was found to be an important virulence determinant in Yersinia spp. (12). These PTPases contain the highly conserved motif HCXAGXXR (both Cys and Arg are essential for activity) in the active domains of the enzymes (41). On the other hand, a PTPase without this motif, viz., small, acidic PTPase, has recently been identified (20) and has been found to contain another motif, FVCXGNICRSPXAEAXF, near the N terminus. Upon examination of the complete sequences of class A bacterial ACPases including PiACP, however, we were not able to find either the HCXAGXXR or the FVCXGNICRSPXAEAXF motifs. Class A bacterial ACPases also shared no overall sequence with the prokaryotic PTPases described above. Nevertheless, PiACP was inhibited by sodium orthovanadate and sodium molybdate, known inhibitors of PTPases. Furthermore, it did exhibit substantial activity against protein tyrosine phosphates, such as those present in growth factor receptors containing EGFR. These results suggest that one can consider the class A bacterial ACPase group including PiACP as constituting a novel family of acidic PTPases significantly different from other known PTPases. It is also tempting to speculate that the class A bacterial ACPase family belongs to a novel widespread PTPase family containing several mammalian phosphatases, such as G6Pases.

In our study, the effects by Cys modifier PAO on PiACP activity were weaker than those of the His modifier DEPC and the Arg modifier phenylglyoxal (Fig. (Fig.7).7). Considering that catalysis by all PTPases reported to date has been shown to proceed through the formation of a covalent phosphorus- and sulfur-containing intermediate involving Cys, the mechanism of PiACP activity, as well as that of class A bacterial ACPases, must be different from that of the known PTPases.

It is currently not known why the H170Q and H209T mutant proteins migrated more slowly than the wild type in the SDS-PAGE gel and exhibited an apparent molecular mass of 32 kDa, which is 2 kDa higher than that of the wild-type enzyme. However, because this size difference corresponds approximately to the molecular weight of the PiACP signal peptide, it seemed possible that the replacement of His-170 or His-209 had an effect on the processing of the PiACP precursor and that this somehow resulted in the secretion of unprocessed enzyme, as demonstrated for the ACPase enzyme of E. coli (27).

Finally, the biological function of PiACP is currently unknown. In view of the pathogenesis of periodontal disease, the ability of PiACP to dephosphorylate the phosphopeptide corresponding to EGFR may play a significant role. A clinical isolate of P. intermedia was recently found to invade a human oral epithelial cell line (8). Polypeptide growth factors, such as EGF, are biological mediators of cellular functions, such as differentiation, motility, and matrix synthesis (10), and have been shown to play important roles in the regenerative response (28). Considering that EGFRs were expressed at high levels on the cell surfaces of basal cell layers of the gingival epithelium (26), it can be speculated that PiACP released from this bacterium inhibits EGF promotion of cell proliferation. Additional studies are needed to ascertain the exact nature and physiological function of PiACP.

ACKNOWLEDGMENTS

We thank H. Shima, Hokkaido University, Sapporo, Japan, for critical comments.

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

REFERENCES

1. Ansai T, Yamashita Y, Awano S, Shibata Y, Wachi M, Nagai K, Takehara T. A murC gene in Porphyromonas gingivalis 381. Microbiology. 1995;141:2047–2052. [PubMed]
2. Ansai T, Dupuy L C, Barik S. Interactions between a minimal protein serine/threonine phosphatase and its phosphopeptide substrate sequence. J Biol Chem. 1996;271:24401–24407. [PubMed]
3. Ansai T, Awano S, Chen X C, Fuchi T, Arimoto T, Akifusa S, Takehara T. Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia. FEBS Lett. 1998;428:157–160. [PubMed]
4. Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol. 1996;11:266–273. [PubMed]
5. Barik S. Site-directed mutagenesis by PCR; substitution, insertion, deletion, and gene fusion. Methods Neurosci. 1995;26:309–323.
6. Berlutti F, Casalino M, Zagaglia C, Fradiani P A, Visca P, Nicoletti M. Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and the VirF and VirB regulatory cascade. Infect Immun. 1998;66:4957–4964. [PMC free article] [PubMed]
7. Bhargava T, Datta S, Ramachandran V, Ramakrishnan R, Roy R K, Sankaran K, Subrahmanyam Y V B K. Virulent Shigella codes for a soluble apyrase: identification, characterization and cloning of the gene. Curr Sci. 1995;68:293–300.
8. Dorn B R, Leung K-P, Progulske-Fox A. Invasion of human oral epithelial cells by Prevotella intermedia. Infect Immun. 1998;66:6054–6057. [PMC free article] [PubMed]
9. Fiske C H, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem. 1925;66:375–400.
10. Graves D T, Cochran D L. Mesenchymal cell growth factors. Crit Rev Oral Biol Med. 1990;1:17–36. [PubMed]
11. Groisman E A, Saier M H, Jr, Ochman H. Horizontal transfer of a phosphatase gene as evidence for mosaic structure of the Salmonella genome. EMBO J. 1992;11:1309–1316. [PMC free article] [PubMed]
12. Guan K-L, Dixon J E. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science. 1990;249:553–556. [PubMed]
13. Guan K-L, Broyles S S, Dixon J E. A tyr/ser protein phosphatase encoded by vaccinia virus. Nature (London) 1991;350:359–362. [PubMed]
14. Ishihara K, Kuramitsu H K. Cloning and expression of a neutral phosphatase gene from Treponema denticola. Infect Immun. 1995;63:1147–1152. [PMC free article] [PubMed]
15. Kasahara M, Nakata A, Shinagawa H. Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase. J Bacteriol. 1991;173:6760–6765. [PMC free article] [PubMed]
16. Kornman K S, Loesche W J. The subgingival microbial flora during pregnancy. J Periodontal Res. 1980;15:111–122. [PubMed]
17. Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–685. [PubMed]
18. Lau K-H W, Farley J R, Baylink D J. Phosphotyrosyl protein phosphatases. Biochem J. 1989;257:23–36. [PMC free article] [PubMed]
19. Lei K-J, Pan C-J, Liu J-L, Shelly L L, Chou J Y. Structure-function analysis of human glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a. J Biol Chem. 1995;270:11882–11886. [PubMed]
20. Li Y, Strohl W R. Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2) J Bacteriol. 1996;178:136–142. [PMC free article] [PubMed]
21. Loesche W J, Syed S A, Laughon B E, Stoll J. The bacteriology of acute necrotizing ulcerative gingivitis. J Periodontol. 1981;53:223–230. [PubMed]
22. Lo Storto S, Silvestrini G, Bonucci E. Ultrastructural localization of alkaline and acid phosphatase activities in dental plaque. J Periodontal Res. 1992;27:161–166. [PubMed]
23. Maeda N, Okamoto M, Kondo K, Ishikawa H, Osada R, Tsurumoto A, Fujita H. Incidence of Prevotella intermedia and Prevotella nigrescens in periodontal health and disease. Microbiol Immunol. 1998;42:583–589. [PubMed]
24. Marck C. ‘DNA strider’: a ‘C’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988;16:1829–1836. [PMC free article] [PubMed]
25. Mazumder B, Adhikary G, Barik S. Bacterial expression of human respiratory syncytial viral phosphoprotein P and identification of Ser237 as the site of phosphorylation by cellular casein kinase II. Virology. 1994;205:93–103. [PubMed]
26. Nordlund L, Hormia M, Saxén L, Thesleff I. Immunohistochemical localization of epidermal growth factor receptors in human gingival epithelia. J Periodontal Res. 1991;26:333–338. [PubMed]
27. Ostanin K, Harms E H, Stevis P E, Kuciel R, Zhou M-M, Van Etten R L. Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. J Biol Chem. 1992;267:22830–22836. [PubMed]
28. Oxford G E, Nguyen K H T, Alford C E, Tanaka Y, Humphreys-Beher M G. Elevated salivary EGF levels stimulated by periodontal surgery. J Periodontol. 1998;69:479–484. [PubMed]
29. Pan C-J, Lei K-J, Annabi B, Hemrika W, Chou J Y. Transmembrane topology of glucose-6-phosphatase. J Biol Chem. 1998;273:6144–6148. [PubMed]
30. Pond J L, Eddy C K, Mackenzie K F, Conway T, Borecky D J, Ingram L O. Cloning, sequencing, and characterization of the principal acid phosphatase, the phoC+ product, from Zymomonas mobilis. J Bacteriol. 1989;171:767–774. [PMC free article] [PubMed]
31. Potts M, Sun H, Mockaitis K, Kennelly P J, Reed D, Tonks N K. A protein tyrosine/serine phosphatase encoded by the genome of the cyanobacterium Nostoc commune UTEX 584. J Biol Chem. 1993;268:7632–7635. [PubMed]
32. Raber-Durlacher J E, van Steenbergen T J M, van der Velden U, de Graaff J, Abraham-Inpijn L. Experimental gingivitis during pregnancy and post-partum: clinical, endocrinological, and microbiological aspects. J Clin Periodontol. 1994;21:549–558. [PubMed]
33. Rossolini G M, Schippa S, Riccio M L, Berlutti F, Macaskie L E, Thaller M C. Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology. Cell Mol Life Sci. 1998;54:833–850. [PubMed]
34. Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory; 1989.
35. Sanger F, Nicklen S, Coulson A R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463–5467. [PMC free article] [PubMed]
36. Shah H N, Gharbia S E. Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov. Int J Syst Bacteriol. 1992;42:542–546. [PubMed]
37. Shenolikar S. Protein serine/threonine phosphatases—new avenues for cell regulation. Annu Rev Cell Biol. 1994;10:55–86. [PubMed]
38. Slots J. Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system. J Clin Microbiol. 1981;14:288–294. [PMC free article] [PubMed]
39. Slots J, Listgarten M A. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. J Clin Periodontol. 1988;15:85–93. [PubMed]
40. Stefani M, Caselli A, Bucciantini M, Pazzagli L, Dolfi F, Camici G, Manao G, Ramponi G. Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes. FEBS Lett. 1993;326:131–134. [PubMed]
41. Streuli M, Krueger N X, Tsai A Y M, Saito H. A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proc Natl Acad Sci USA. 1989;86:8698–8702. [PMC free article] [PubMed]
42. Stukey J, Carman G M. Identification of a novel phosphatase sequence motif. Protein Sci. 1997;6:469–472. [PMC free article] [PubMed]
43. Thaller M C, Berlutti F, Schippa S, Lombardi G, Rossolini G M. Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii. Microbiology. 1994;140:1341–1350. [PubMed]
44. Thaller M C, Berlutti F, Schippa S, Iori P, Passariello C, Rossolini G M. Heterogeneous patterns of acid phosphatases containing low-molecular-mass polypeptides in members of the family Enterobacteriaceae. Int J Syst Bacteriol. 1995;45:255–261.
45. Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. [PMC free article] [PubMed]
46. Uchiya K I, Tohsuji M, Nikai T, Sugihara H, Sasakawa C. Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase. J Bacteriol. 1996;178:4548–4554. [PMC free article] [PubMed]
47. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983;133:17–21. [PubMed]
48. Yamashita Y, Toyoshima K, Yamazaki M, Hanada N, Takehara T. Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381. Infect Immun. 1990;58:2882–2887. [PMC free article] [PubMed]
49. Zhang Z-Y, Davis J P, Van Etten R L. Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase. Biochemistry. 1992;31:1701–1711. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...