Characterization of a Novel Acid Phosphatase from Embryonic Axes of Kidney Bean Exhibiting Vanadate-Dependent Chloroperoxidase Activity

(cid:0) (cid:0) (cid:0) A novel colorless acid phosphatase (KeACP), which was distinct from the kidney bean purple acid phosphatase, was purified to apparent homogeneity and cloned from embryonic axes of kidney bean ( Phaseolus vulgaris L. Ohfuku) during germination. When ortho-vanadate (VO 43- ) is added to the apo-form of the enzyme, KeACP uniquely exhibits the chloroperoxidase activity with loss of phosphatase activity. This is the first demonstration that KeACP is vanadate-dependent chloroperoxidase in plants to be characterized and suggests that KeACP may play a role in modifying a wide variety of chlorinated compounds that are present in higher plants. The enzyme is a dimer that presents three forms made up of combination of the dominant 56-kDa and the minor 45-kDa subunits, and both subunits contain carbohydrate. The full-length cDNA of the KeACP gene is 1,641 nucleotides, and this sequence is predicted to encode a protein having 457 amino acid residues (52,865 Da), including a signal peptide. The complete nucleotide sequence of the genomic DNA (3,228 bp) of KeACP consists of seven exons and six introns. Northern blot analysis demonstrated that the KeACP gene was expressed specifically in embryonic axes of the kidney bean, and its expression coincided with elongation of the embryonic axis during germination.


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The enzyme solution was applied to a DEAE Toyopearl 650S column (2.6 × 40 cm) (Tohso, Tokyo, Japan) equilibrated with 20 mM Tris-HCl (pH 7.5). The adsorbed materials were eluted with a linear gradient of NaCl concentration from 0 to 0. 5  PAGE and protein sequencing -Non-denaturating (native) PAGE was carried out at 4˚C using a 5% polyacrylamide gel (pH 8.8). ACP activity staining was carried out by incubating the gels with acetate buffer (pH 5.0) containing 0.1 mg/ml Fast Black K salt (Sigma, St. Louis, MO) and 1 mg/ml α-naphthylphosphate in the dark at room temperature until colored bands appeared. SDS-PAGE was performed using a 10% polyacrylamide gel in the presence and absence of 2-ME. The separated protein bands were stained with Coomassie Brilliant Blue R-250 (CBB). The size markers were phosphorylase b (97 kDa), BSA (66 kDa), egg ovalbumin (45 kDa), carbonic anhydrase (30 kDa) and soybean trypsin inhibitor (20.1 kDa). Glycoprotein staining of the gel was performed according to a periodic acid-Schiff (PAS) staining protocol.
For the N-terminal amino acid sequence, the proteins separated by SDS-PAGE were electroblotted onto a PVDF membrane using a semi-dry apparatus according to the method described previously (17). The peptide fragments were generated by limited proteolysis with Staphylococcal V8 protease and Achromobacter lyticuis lysyl endopeptidase during electrophoresis. After the PVDF membranes were briefly stained with CBB, the stained bands were excised. The N-terminal amino acid sequences were determined using an automated protein sequence analyzer (Model 492HT, Applied Biosystems, Foster City, CA). Deglycosylation of KeACP -Deglycosylation of the purified KeACP was performed using N-glycosidase F Deglycosylation Kit (Roche, Penzberg, Germany) according to the manufacturer's instruction. The purified enzyme (50 µg) was 9 pH of the enzyme was around pH 6.0, and it showed activity in the range of pH 5 to 8 (data not shown). The routine assay was performed at 30˚C by adding 50 µl of enzyme solution to 0.5 ml of reaction mixture consisting of 50 mM p-NPP and 200 mM Tris-maleate (pH 6.0). One enzyme unit was defined as 1 µmole of p-NPP hydrolyzed per 1 min.
Substrate specificity of the KeACP was estimated by incubating the purified enzyme with various substrates (17 kinds). The assay was carried out at 30˚C by adding 50 µl of the enzyme solution to a reaction mixture containing 10 mM target substrate and 50 mM Tris-maleate (pH 6.0), and phosphorus released during the 2 min incubation was measured according to a method described previously (18). In all assays one unit of enzyme activity was defined as 1 µmole of Pi released per 1 min.
Assays to examine the effects of metal ions and chemical agents (9 kinds) on ACP activity were carried out as above, by incubating p-NPP at 30˚C for 30 min with native enzyme or enzyme that was dialyzed against buffer containing EDTA. All measurements were repeated five times and the results represent the arithmetic means.
Assay for chloroperoxidase (CPO) activity -Vanadate-substituted enzyme (V-CPO) was prepared by incubating the enzyme with various concentration of o-vanadate in 100 mM Tris-HCl (pH 7.5) at 30˚C for 30 min. The purified enzyme was then dialyzed against 100 mM Tris-HCl (pH 7.5) containing 20 mM EDTA. The CPO activity was measured by following the increase in the absorbance at 290 nm due to the chlorination of monochlorodimedon (MCD) in the presence of hydrogen peroxide (11).
The standard assay was carried out at 30˚C in a reaction mixture containing 2 mM H 2 O 2 , Cloning and nucleotide sequencing of the KeACP gene -RNA was isolated from kidney bean embryonic axes elongated through 48 hrs after germination. Embryonic axes (0.1 g) were ground in liquid nitrogen. Total RNA was extracted with RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) according to "clean-up" recommendations of the manufacturer. The cDNA library was constructed using a SMART™ RACE cDNA amplification Kit (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's instruction. The primers used to amplify sequences were designed based on the partial nucleotide sequence of the KeACP gene and the A. thaliana putative ACP mRNA (GenBank Accession Number AY091415). The primers used were SAK01 for the 5'-RACE (5'-tgcacatcacgcaaggtcac-3') and HAK01 for the 3'-RACE Total genomic DNA was extracted from fine meal of the kidney bean seed according to the plant DNA extraction method, as described previously (19). To isolate the KeACP gene from the total genomic DNA by PCR, primers were designed and synthesized based on the cDNA sequence of KeACP that was determined in this study, as listed in Table I. The PCR products were sequenced on both strands using an ABI BigDye-Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The dye-labeled extension products were analyzed with an automated DNA sequencer (Model 377, Applied Biosystems). Nucleotide sequences were analyzed using MacVector sequence analysis software (Oxford Molecular, Ltd). The sequence data have been submitted to the GenBank database under accession numbers AB116719 for full-length cDNA and AB116720 for genomic DNA of KeACP.
Northern blot analysis -Total RNAs were extracted from 0.1 g of embryonic axes of the kidney bean at different periods of growth on the agar plate at 25˚C in darkness until 72 hrs after the beginning of imbibition. RNAs were also extracted from 0.1 g of roots, leaf and stem after two weeks of growth. The total RNAs extracted (30 µg) using the RNeasy Plant Mini kit were separated by 1% formaldehyde agarose gel electrophoresis and transferred to a nylon membrane (Hybond N+, Pharmacia Biotech).
For dot blot analysis, 60 µg of total RNAs from each tissue were transferred to a nylon membrane as well. A DNA probe (245 bp) encoding a partial region of KeACP cDNA was labeled using a PCR-based DIG-Labeling Mix (Roche) with primer pair SAK01 and HAK01 (Table I). Northern hybridization was performed by high stringency hybridization at 60˚C overnight.

RESULTS
Purification and molecular characteristics of KeACP from embryonic axes of kidney bean - Table II presents a summary of the purification procedure parameters.
The enzyme was isolated from embryonic axes that elongated during germination of the kidney bean seed. KeACP was purified 852-fold to near homogeneity by anion-exchange DEAE column chromatography followed by Con A affinity and hydrophobic column chromatography. The Con A purification step effectively served to remove a significant amount of contaminating proteins, indicating that KeACP may be a glycoprotein. Through all column chromatography purification steps, a single chromatographic peak of KeACP activity was observed, and the final preparation was colorless. This suggests that neither purple color ACP, which has usually been found in dry seeds (20), nor multiple ACP forms are present in embryonic axes of kidney beans.
The pH dependence of phosphatase activity was also investigated for KeACP, yielding a curve with a pH optimum at pH 6.0 (data not shown) that was almost identical to that of other ACPs.
The apparent molecular mass of the purified enzyme was estimated to be around 96 kDa by analytical gel filtration on a Superdex 200 HR 10/30 column (Fig. 1).
SDS-PAGE of the purified preparation both in the presence and in the absence of 2-ME revealed the presence of 56-and 45-kDa polypeptides with different staining intensities by CBB and PAS in a 3:1 ratio, respectively ( Fig. 2A and B). This suggests that the native enzyme has a dimeric structure with no intersubunit disulfide linkages. indicating that native KeACP presents three forms of subunit structure. Both bands on the gel were stained with a PAS reagent, and the enzyme retained on Con A column was eluted with D-(+)-mannose, strongly suggesting that they are glycosylated.
Deglycosylation of the KeACP with N-glycosidase F under the denauration condition was partially succesful. After 12 hrs incubation, decreases in molecular mass of 2 and 1.4 kDa for dominant and minor subunits, respectively, on SDS-PAGE were observed ( Fig. 2C). However, both bands were stained by PAS, indicating that oligosaccaride chain had still not been removed. The N-terminal amino acid sequences of both bands were determined to be NH 2 -SEWPAVDIPLDHEAFAVPKG (Fig. 2B), implying that two subunits have identical N-terminal sequences, but one probably has an excision of more than 100 amino acids in its C-terminal region, or/and has a different glycosylation pattern.
Substrate specificity -Because the physiological relevant substrate(s) for KeACP is still unknown, the purified enzyme was tested for its activity against a variety of phosphorylated substrates (Table III). The activity against p-NPP was taken to be 100%.
Relatively high activity was observed with phosphoenolpyruvate and this was comparable to pyrophosphate, phosphorylated-Tyr, β-naphtylphosphate, glucose-1-phosphate and ADP, with more than 50% activity. Several other compounds were also dephosphorylated by KeACP at a much lower rate compared with p-NPP.
Bis-p-NPP, phytate and 5'-AMP were apparently poor substrates. As a result, KeACP shows a wide variety of substrate specificity, but not a preference for other substrates over p-NPP. However, it is noteworthy that KeACP catalyzed dephosphorylation of phosphorylated-Ser, -Thr and -Tyr under the conditions employed. Table IV KeACP uniquely catalyzes the chloroperoxidation reaction. Figure 3A shows that more than 100 µM vanadate was required to obtain full chloroperoxidase activity, and that ACP activity disappeared at rates depending upon the concentration of vanadate added.

Effects of various chemicals on KeACP activity -As shown in
A modified Hill plot (21)  protease and lysyl endopeptidase were analyzed for their N-terminal amino acid sequences. The sequences determined derived from two major fragments, from Asp-237 to Phe-248, and from Arg-304 to Met-317. These were in complete agreement with those deduced from the nucleotide sequence of the KeACP cDNA (data not shown).
The deduced amino acid sequence of the enzyme was analyzed using the SignalP (Ver. 2) program (22), along with the iPSORT and TargetP algorithms. The results predict that the N-terminal region of the precursor protein has characteristics of a signal peptide, with a probable cleavage site between Ala-28 and Gly-29, as shown in Fig. 4.
However, the N-terminal amino acid sequences of both subunits begin at Ser-37 but not Gly-29 (Fig. 4) showed that all intron sequences were removed from the fully processed cDNA.
Northern blot analysis -The accumulation level of the KeACP mRNA (1.6 kb) was analyzed by northern blots using total RNA extracted from elongated embryonic axes of germinated kidney bean at selected time intervals after the beginning of imbibition. Northern blots indicated that KeACP gene expression appeared soon after the imbibition and increased until 18 hrs imbibition, at which time the radicle germinates from the seed coats. Subsequently, the level of expression was constant (Fig.   5A). Dot blot analysis indicated the same tendency. On the other hand, for other tissues, such as roots, leaf and stem, the levels of mRNAs after two weeks of cultivation were very low compared with that of embryonic axes (72 hrs after imbibition), as shown in Fig. 5B. Accordingly, this suggests that the KeACP protein is specifically localized in embryonic axes tissue during elongation that occurs after the beginning of germination.

DISCUSSION
KeACP was purified to near homogeneity as a single activity peak from kidney bean embryonic axes by using ion-exchange, Con A-Sepharose and hydrophobic column chromatography. As shown in Table II However, both subunits were still stained by PAS reagent, indicating that the partial fucosylation of some oligosaccharide core may make inacceessible to endoglycosidase employed. Using gel filtration chromatography, the molecular mass of purified KeACP was determined apparently to be around 96 kDa, demonstrating the dimeric structure of the enzyme. However, the molecular mass of the enzyme by gel filtration was somewhat lower than the expected 100-110 kDa by sum of combination of subunit masses, probably due to specific interactions between glycosyl residues and gel filtration resin.
The N-terminal sequences (20 residues) of both polypeptides were identical, suggesting that the 45-kDa polypeptide is derived from deletion of about 100 amino acids from C-terminal of the 56-kDa polypeptide. Both CBB and enzyme activity staining on native-PAGE showed three bands corresponding to each form. Accordingly, the dimer of KeACP is composed of two non-covalently bound subunits, and seems to exist as a mixture of three dimeric forms, a homodimer of the 56-kDa subunit, or the 45-kDa subunit and a heterodimer of each subunit. Red kidney bean ACP (7)  The precursor protein has characteristics of a signal peptide at its N-terminal region, with a probable cleavage site between Ala-28 and Gly-29, as shown in Fig. 4. Comparative analysis of the structures of purple ACPs from higher plants has allowed the identification of conserved sequence and structural motifs in this type of enzyme from many eukaryotic species (2,9). Generally, plant purple ACPs belong to the metallo-phosphoesterase family of proteins, indicating that the composition of dinuclear center is Fe 3+ -Zn 2+ in red kidney bean (7) and soybean (6) haloperoxidases. These non-heme vanadate-containing peroxidases were first isolated from seaweed brown macro-algae (30), and have also been found in fungi (31)  mM for Cl -, depending upon pH, reported for a representative CPO from the fungus Curvularia inaequalis (34). It is well known that soils contain approximately 2 mM vanadate (31), which is likely enough to obtain a fully activated CPO in the plant in vivo.
However, the fact that CPO activity has not been detected earlier in kidney bean homogenates may be related to the fact that addition of vanadate to the apo-form of the enzyme is necessary for CPO activity. Indeed, a general property of V-bromoperoxidase is that vanadate can be removed by dialysis against phosphate-containing buffers, and the CPO activity may be restored only by addition of vanadate (35).
The CPOs are defined by their ability to oxidize electrophilic halide species, Cl -, Brand I -, to the corresponding hypohalous species in the presence of H 2 O 2 . However, V-CPO derived from KeACP specifically oxidized Clto hypochlorite but was inactive against other halides. This property of vanadate-KeACP is distinct from other V-CPOs from various organisms. Assuming that V-CPO carries out its function in vivo, the physiological role of this enzyme has been unclear, as it produces a strong oxidizing agent, hypochlorite, at an early stage of elongation of the embryonic axis during germination. There is increasing evidence suggesting that the H 2 O 2 producing-reaction locates in photosynthetic or respiratory electron transport systems in plants, and a few reports using soybeans suggested that seed germination is accompanied by a generation of reactive oxygen species containing H 2 O 2 in the embryonic axis (36,37). Since seed germination represents the developmental period that is most sensitive for pathogen infection (38), it is more likely that hypochlorite produced in this stage plays an important role in protecting the embryo against attack by pathogens or parasitic organisms. Another possible explanation is that hypochlorite produced by V-CPO may promote seed germination by decomposing antioxidants, which are derivatives of well-known germination inhibitors present in plant seeds (39,40).
The full-length KeACP gene was most closely related to the soybean purple (6), KeACP develops no purple color or CPO activity after the addition of vanadate.
Furthermore, northern blot data presented here demonstrated that the KeACP gene transcript specifically accumulated during rapid elongation of embryonic axes of the kidney bean, but not other tissues (Fig. 5). However, it is interesting that the soybean purple ACP-like GmPAP3 gene, which has a sequence similarity to KeACP, was induced by salt stress through its involvement in reactive oxygen species (41). Further studies are needed to demonstrate the subcellular localization of KeACP in embryonic axes, and how induction of the KeACP gene expression responds to various stresses, including oxidative stresses and pathogens (43), at the germination stage.
The data presented here demonstrate that KeACP is a novel ACP that is clearly distinct from previously reported ACPs in plants. However, it might be premature to speculate on possible functions of KeACP. Further characterization of its precise structure and physiological substrates will be required to clarify its possible roles in kidney bean embryonic axes cells.