Characteristics and sequence of phosphoglycolate phosphatase from a eukaryotic green alga Chlamydomonas reinhardtii.

Phosphoglycolate phosphatase (PGPase), a key enzyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The enzyme was an approximately 65-kDa homodimer with a pI value of 5.1 composed of approximately 32-kDa subunits not connected by any S-S bridges. It was also highly specific for phosphoglycolate with a K(m) value of 140 microm and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl(2), and it recovered competitively following the addition of MgCl(2) or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl(2), indicating that the enzyme binds to Ca(2+). The N-terminal region of amino acid sequence deduced from cDNA sequence that was not contained in the purified PGPase had similar characteristics to those of typical stroma-targeting transit peptides in C. reinhardtii. The following region of the deduced sequence containing 302 amino acid residues was similar to p-nitrophenylphosphatase-like proteins, although the purified PGPase did not hydrolyze p-nitrophenylphosphate. Genomic DNA fragments from wild type containing the sequence homologous to the cDNA for PGPase complemented the PGPase-deficient mutant pgp1. Possible regulatory mechanisms during adaptation to limiting CO(2) were discussed based on the characteristics of the purified PGPase and the deduced amino acid sequence.

PGPase has been partially purified from several species of plants and algae including tobacco (8,9), spinach (8,10), maize (11), pea (12), Halimeda cylindracea (13), C. reinhardtii (14), and Coccochloris peniocystis (7). PGPase has also been partially purified from components of animal tissues such as human red blood cells (15). The remarkable similarity in the kinetic characteristics of these animal PGPases to those of plant PGPases has been described previously (16). No information, however, is available on the molecular structure (e.g. amino acid sequences) in eukaryotes.
The regulatory mechanism of PGPase activity is still not clear in any organisms, although some observations suggest environmental regulation by such factors as CO2 concentration (3,17,18) and light (19). PGPase also seems to have an important role in animals by affecting the phosphoglycolate level. In human red blood cells, for example, phosphoglycolate is an effective activator of the bisphosphoglycerate shunt, which is a major modifier of the oxygen affinity of hemoglobin (16). However, the regulatory mechanism of PGPase activity in animals is not clear.
In this report, we purified PGPase from C. reinhardtii. This is the first homogeneous PGPase purified from a eukaryotic organism. We determined the complete nucleotide sequence of the PGPase cDNA and found characteristics expected to be involved in its regulatory mechanism to help elucidate the physiological importance of PGPase in plants.

EXPERIMENTAL PROCEDURES
Enzyme Purification-All purification steps were carried out at 4 °C unless otherwise specified. C. reinhardtii 2137 mt+ cells were grown photoautotrophically under ambient air (aerated with air) at 25 °C as described previously (18) and collected by centrifugation. The cells were disrupted by sonication in 20 mM MES-KOH buffer, pH 6.3, containing 5mM MgCl2 and1mM phenylmethanesulfonyl fluoride and then centrifuged at 150,000 * g for 30 min. The supernatant was incubated at 50 °C for 5 min, and the precipitate was removed by centrifugation at 45,000 g for 20 min. The proteins precipitated between 50 -60%saturated (NH4)2SO4 were collected and dissolved in 2.5 ml of 20 mM MOPS-KOH buffer, pH 6.3, containing 5 mM MgCl2. After being passed through a Sephadex G-25 column (PD-10, Amersham Pharmacia, Uppsala, Sweden) equilibrated with the same buffer, the fraction was applied to a DEAE-Sephacel column (1 * 7 cm) (Amersham Pharmacia Biotech) equilibrated with the same buffer. Proteins were eluted by changing the buffer to 20 mM Tris-maleate buffer, pH 6.3, containing 5 mM MgCl2. The fractions with PGPase activity were collected and concentrated to approximately 1 ml using a centrifugal concentrator (Ultrafree-15, Millipore, Bedford, MA) and dialyzed overnight against 1 liter of 20 mM MOPS-KOH buffer, pH 7.1, containing 1 mM MgCl2. Solid potassium chloride and EGTA were then added slowly to attain final concentrations of 1.0 M and 1mM, respectively. After centrifugation (10,000 * g, 10 min), the supernatant was applied to a phenyl-Sepharose column (1 7 cm) (6FF high sub, Amersham Pharmacia Biotech) equilibrated with 20 mM MOPS-KOH buffer, pH 7.1, containing 1 M potassium chloride, 1 mM MgCl2,and1mM EGTA at 25 °C. After the column was washed with 25 ml of the same buffer, proteins were eluted with 20 mM MOPS-KOH buffer, pH 7.1, containing 0.8 M KCl, 1 mM MgCl2,and 1mM EGTA, and the fractions with PGPase activity were collected. After the addition of Tween 20 (final concentration, 0.001%, v/v), the preparation was concentrated to approximately 0.5 ml using Ultrafree-15 and stored at -20 °C until use. The eluate was loaded onto a Q-Sepharose column (1 * 7 cm) (Amersham Pharmacia Biotech) equilibrated with 20 mM bistrispropane-HCl buffer, pH 7.5, containing 1mM MgCl2 and eluted with a 75-ml linear gradient of KCl (0 -50 mM). Alternatively, the eluate from phenyl-Sepharose was applied to a 5-20% gradient-native PAGE (20). PGPase was detected using activity staining (9) as a white band after the incubation of the gels for 15 min at 25 °Cin20mM MES-bistrispropane buffer, pH 8.3, containing 5 mM MgCl2,5mM CaCl2,and4mM phosphoglycolate. The unstained band corresponding to the white band was cut out and homogenized either in 20 mM MOPS-KOH buffer, pH 7.1, containing 5 mM MgCl2 to collect the purified PGPase or in Tris-HCl, pH 6.8, containing 1% SDS, 2% mercaptoethanol, and 0.001% bromphenol blue to apply SDS-PAGE using a 12.5% polyacrylamide slab gel (20).
Determination of the N-terminal Sequence-The separated proteins on SDS-PAGE gel were transferred onto a polyvinylidene difluoride membrane using a semidry transfer apparatus (TransBlot SD, Bio-Rad) after washing the gel with water for 5 min. After staining the membrane with 0.025% Coomassie Brilliant Blue R-250 in 40% methanol, the peptide band was excised and subjected to determination of N-terminal amino acid sequence by Edman degradation using an Applied Biosystems Model 477A sequencing system (Applied Biosystems, Foster City, CA).
DNA Sequence-DNA were sequenced by using the BigDye Terminator DNA Sequencing Kit and an ABI PRISM 377 DNA sequencer (Applied Biosystems).
Transformation of Chlamydomonas Cells-C. reindardtii was transformed with plasmid DNA by electroporation (21) with the slight modification of HS medium being replaced with min-70 medium (4).
Immunoblotting-Proteins in the crude extract were separated on SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane as described above. The blots were blocked overnight in a Trisbuffered saline buffer containing 5.5% (w/v) nonfat skim milk, 2% bovine serum albumin, 0.1% (v/v) Tween 20, and 0.02% (w/v) sodium azide at 4 °C, washed with Tris-buffered saline containing 0.1% Tween 20 and 5.5% nonfat skim milk (BT), and incubated with the primary antibody for 180 min. After being washed four times with BT, the membrane was incubated for 60 min with anti-rabbit IgG biotin conjugate (1:1000 dilutions in BT, Sigma). After being washed three times with BT and 1 time with Trisbuffered saline containing 0.1% Tween 20, the membrane was incubated with streptavidin-horseradish peroxidase conjugate (1:1000 dilution in Tris-buffered saline containing 0.1% Tween 20, Life Technologies, Inc.). After being washed five times with Tris-buffered saline containing 0.1% Tween 20 and two times with 20 mM Tris-HCl, pH 7.5, the peptides that reacted with the primary antibody were visualized in 20 mM Tris-HCl, pH 7.5, containing 0.025% 3.3 -diaminobenzidine tetrahydrochloride, 0.03% hydrogen peroxide, and 0.3% CoCl2.
Preparation of the Primary Antibody-A synthetic peptide AS1787, corresponding to the N-terminal 17amino acid sequence of purified PGPase subunit with cysteine substituted for the N-terminal Ser, was generated by Takara Shuzo Co., Ltd. (Kusatsu, Japan). The antibody against AS1787 was generated and purified by using rProtein A-Sepharose FF (Amersham Pharmacia Biotech) and CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) coupled with AS1787 by the same company.
Estimation of Molecular Mass-The PGPase preparation after DEAE-Sephacel was applied to the HiLoad 16/60 Superdex 200-pg column (Amersham Pharmacia Biotech) equilibrated with 20 mM MOPS-KOH, pH 8.0, 5 mM MgCl2, 100 mM KCl, and 1 mM EDTA at the rate of 1 ml min -1 for the molecular mass determination.
Enzyme Assay and Protein Determination-PGPase activity was determined by measuring the phosphoglycolate-dependent release of inorganic phosphate (23) as described previously using the assay mixture containing 20 mM MES-bistrispropane, pH 8.0, 5 mM MgCl2,and 4mM phosphoglycolate (18). Protein was quantified by the method of Bradford (24) using bovine serum albumin as a standard.
Chlorophyll Content Determination-Chlorophyll content was determined after extraction with 96% (v/v) ethanol (25). Table I summarizes the purification steps for PGPase from C. reinhardtii. PGPase eluted from the DEAE-Sephacel column by changing 20 mM MOPS-KOH in the buffer to 20 mM Tris-maleate had a higher specific activity than that of the final preparation reported by Husic and Tolbert (14). The next step involving phenyl-Sepharose column chromatography was most effective in attaining a specific activity comparable to that from higher plants (Table I), although PGPase was still not the major protein after this step (Fig. 1, lane 1). PGPase activity was stable after the phenyl-Sepharose step for at least 6 months at -20 °C. The single peptide band with a molecular mass of 32 kDa was obtained by SDS-PAGE after the next purification step, either Q-Sepharose column chromatography or native PAGE (Fig. 1). The purified PGPase after Q-Sepharose chromatography had a specific activity of 86.5 μmol Pi (mg protein)

Purification of PGPase-
, a 1300-fold increase from that of the sonicated cell suspension (Table  I).
Molecular Mass, pI Value, Km Value, and Substrate Specificity-The molecular mass was estimated to be ~65 kDa for the native PGPase by Superdex 200 (Fig. 2) and ~32 kDa for the subunit by SDS-PAGE (Fig. 1), suggesting PGPase to be a homodimer. The isoelectric point was estimated to be 5.1 (data not shown). The Km value for phosphoglycolate was ~140 μM (see Fig. 9). Consistent with the partially purified PGPases from higher plants (9,11), the purified PGPase from C. reinhardtii was also highly specific for phosphoglycolate. After the Q-Sepharose step, the enzyme did not at all hydrolyze 3-phosphoglycerate, D-ribose-5-phosphate, phosphoenolpyruvate, O-phospho-l-serine, or p-nitrophenylphosphate, although it hydrolyzed ribulose-5-phosphate and fructose-1, 6-bisphosphate  at a rate of 1-4% of that of phosphoglycolate hydrolysis (data not shown).
Amino Acid Sequence-The N-terminal amino acid sequence of the purified PGPase was determined up to 21 residues, SARPIATNEQKLELLKKVESF. By searching the expressed sequence tag (EST) data base for C. reinhardtii strain C9 (26), we found an EST encoding the 21-residues amino acid sequence of PGPase with the except that the 20th residue was a cysteine, which was hardly distinguishable from serine by the amino acid sequencer. The complete nucleotide sequence (GenBank TM accession number AB052169) of the EST clone contained an open reading frame encoding a polypeptide consisting of 330 amino acids. The deduced amino acid sequence of the EST clone was similar to that of p-nitrophenylphosphatase (NPPase) in yeast and the relatives in various other organisms (Fig. 3). A close similarity was observed especially to an NPPase-like protein in A. thaliana with a 52% identity for the entire sequence (Fig. 3) (Fig. 3), were well conserved among PGPases in prokaryotes at the similar positions (data not shown).
The first 28 N-terminal amino acid residues, which were not contained in the purified PGPase protein, have the characteristics of a transit peptide of C. reinhardtii. The amino acid sequence was rich in Arg and Ala and contained no acidic residue. Four Arg residues spaced by two or three neutral amino acids in the middle region were just within the random coil region between two amphiphilic helical motifs consisting of five or six residues (Fig. 4). The amino acid sequence just upstream of the N-terminal of the purified enzyme, VAAQA, was similar to the motif VXA (27). These characteristics are consistent with the typical features of a stroma transit peptide (27,28), indicating that PGPase in C. reinhardtii is a stroma protein (14) as reported in higher plants (11). The predicted molecular mass and isoelectric point of the mature subunit of 302 amino acids were approximately 33kDa and 5.6, respectively, consistent with those determined for the purified PG-Pase as described above.
Although 425 proteins containing the motif FDXDG were found in the Swiss-Prot data base at the ExPASy site (www. expasy.org), they were mainly Ca Immunodetection of PGPase Subunit-The affinity-purified antibody raised against a synthetic peptide AS1787 (see under "Experimental Procedures") reacted strongly with the subunit of purified PGPase in the immunoblot analysis (Fig. 5, lane 6). Several other bands were also detected by the antibody in crude extract of wild-type 2137 (lanes 8-10), but they were also detected by the preimmune serum (lanes 3-5). The 32-kDa peptide corresponding to the PGPase subunit seems to be the only band detected specifically by the antibody and was not detected in a PGPase-deficient mutant pgp1-18-7F, in which the activity to hydrolyze p-glycolate was marginal (3,17,18) and was not caused by the PGPase with the optimal pH of approximately 8 (18). These findings suggest that the PGPase we purified is the PGPase essential for growth under ambient air from which activity is missing in the mutant. It was also reported that PGPase activity in 2137 increased by 60%5h after transfer 5% CO2-grown cells to air (17). However, not so much difference was observed between the immunoblot profiles of 5% CO2-grown and 5-h air-adapting cells of 2137, although PGPase activity increased by 50% (Fig. 5).
Complementation of pgp1 Mutation-Four clones in a genomic DNA library of C. reinhardtii C9 (21) showed strong signals in two separate Southern blotting experiments probed with independent digoxigeninlabeled polymerase chain reac  (39) at the DDBJ site (www.ddbj.nig.ac.jp). Arg residues matching well with the characteristics of stroma transit peptides are in boldface. A motif similar to VXA that is often found just before the processing site of stroma-targeting proteins is underlined. Closed triangle, the processing site suggested by N-terminal sequence of purified PGPase. The residues matching between PGP1 and C. reinhardtii CaM are double underlined. Boxed residues match with the consensus motif in EF-hand region of Ca 2 -binding proteins; H, residues predicted to form a -helix; E, residues predicted to form β-strand; C, residues predicted to form random-coil structure. tion products of the genomic DNA from a wild-type 2137 by using primers designed based on the cDNA sequence of PGP1 (data not shown). The DNA fragments from these clones were used for the transformation (21) of N142, a wall-less progeny of the pgp1 mutant after crossing 18-7F with cw15 strains. The genomic clones 57B9 and 60C10 successfully restored the growth of the pgp1 mutant under air. In a transformant with 57B9, the growth rate under air was approximately 40% of that of wild type, and PGPase activity was approximately 25% of that of wild type. These results support that the high CO2requiring phenotype of pgp1 is caused by the deficiency in PGPase activity, and that the sequenced cDNA represents a physiologically functional PGPase.
mRNA Levels During the Adaptation to Air-We observed an increase in mRNA level of PGPase 5 h after transferring the wild-type cells from 5% CO2 to air (Fig. 6). Although there was not so much increase in the peptide level of PGPase (Fig. 5), the increase in the activity of PGPase 5 h after transfer to air is probably expected to be a result of a posttranscriptional regulation.
Optimal pH-Husic and Tolbert (14) reported a broad pH optimum with the maximal activity approximately pH 6.3 for partially purified PGPase from C. reinhardtii. However, this profile was uncertain, because they used a very complicated buffer system using 20 mM acetate, ~pH 6.3. We observed a 50% inhibition of PGPase activity by 20 mM acetate (data not shown). We determined the pH profile using 20 mM 3,3-dimethylglutarate-KOH as the buffer for acidic pH and 20 mM MESbistrispropane for basic pH (Fig. 7). The optimal pH was between 8 and 9, and the activity at pH 6.3 was less than half that at pH 8 in the enzyme purified with DEAE-Sephacel, which was quite similar to that reported in H. cylindracea (13), although the activity was relatively higher between pH 5 and 7 in the crude extract (Fig. 7).
Effect of Calcium-It was reported that PGPase from maize was inhibited very strongly by Ca 2 + , and this was reversed by Mg 2 + (11). We also observed the reversible inhibition in the purified PGPase from C.
reinhardtii (data not shown). The inhibition increased with decreasing concentration of Mg 2 + in the reaction mixture (Fig. 8), and the IC50 value for CaCl2 was ~50 FIG.5. Immunoblotting analysis of PGPase before and after 5-h adaptation of 5% CO 2 -grown cells to air. C. reinhardtii wild-type 2137 cells grown under 5% CO2 in six flasks containing 250 ml of min-74 liquid medium were combined, mixed, and distributed into six flasks again. Three were used for the RNA analysis (see Fig. 6), and the other three were exposed to three different conditions,0 h in air (350 ppm of CO2),5hin air, and5hin5%CO2. and 175 μM with 0.5 and 1 mM MgCl2, respectively (data not shown). The addition of 5 mM EGTA also resulted in 100% recovery after inhibition by CaCl2, whereas EGTA did not affect the activity of PGPase in the absence of CaCl2 (Table II) even after preincubation with 5 mM EGTA (data not shown). Fig. 9 compares the double reciprocal plots of PGPase activity at different concentrations of CaCl2. The affinity for phosphoglycolate was not affected by Ca 2 + , indicating it to be a non-competitive inhibition with respect to its substrate. The subunit of PGPase pretreated with Ca 2 + migrated faster in SDS-PAGE than PGPase pretreated only with EGTA (Fig. 10), which is characteristic of Ca 2 + -binding proteins (29).
Effect of Other Compounds- Table II shows the effect of some other compounds on the activity of purified PGPase from C. reinhardtii. The activity was strongly inhibited by PMB with an IC50 of 0.45 M, although only a slow inhibition by Nethylmaleimide was reported in tobacco PGPase (30). The activity recovered ~90% following the addition of 10 mM DTT, whereas 10 mM DTT did not affect the activity without PMB treatment even after 2-h treatment at 30 °C. These results suggest that at least one of the SH groups of four Cys residues (Fig. 3) has a potential to modulate the activity. Treatment with 10 mM DTT at 30 °C for 2 h did not change the mobility of PGPase in the native PAGE (data not shown). Mobility was not changed in SDS-PAGE either with or without 2-mercaptoethanol (data not shown). These results indicate that there is no S-S bond between the subunits. Activity was inhibited by Ophenanthroline, a metal complex reagent, but the IC50 value of 1mM was higher than in ordinary metaloenzymes. PGPase activity was not sensitive to sodium azide, suggesting that the enzyme does not contain heme.

DISCUSSION
PGPase is essential for all autotrophic organisms and is also important for the function of human red blood cells (16). However, there is little information about its regulation and no information about the molecular structure of eukaryotic PG-Pases. Here we present the first report on the purification of a eukaryotic PGPase with high enough purity to enable the determination of its N-terminal amino acid sequence of up to 21 residues, which allowed us to determine the complete nucleotide sequence of cDNA. We concluded that PGPase from C. reinhardtii is a homodimer with a molecular mass of ~65 kDa consisting of two identical 32-kDa subunits whose molecular masses were predicted to be 33 kDa from the cDNA sequence. Eukaryotic PG-Pases were reported to be homodimers with almost the same native molecular mass among a wide variety of organisms, such as maize (11), tobacco (9), spinach (31), and human (15). Earlier reports proposed a much higher molecular mass of PGPases from plants and algae, 92 kDa as the native molecular mass for the partially purified enzyme from C. reinhardtii (14), 93 kDa for spinach, and 81-86 kDa (tetramer) for tobacco (30). However, such values were shown to be quite unlikely, at least in spinach and tobacco (9), and the inconsistency may be partly a consequence of the insufficient purity in those PGPase preparations. The pI value of 5.1 for the purified PGPase from C. reinhardtii was consistent with those reported among a wide variety of organisms, 4.9 for PGPase from maize leaves (11), 4.4 for PGPase from pea (12), 4.2 and 5.5 for two isoforms from french bean (32), and 5.0 for PGPase from human red blood cells (15). On the other hand, the reported kinetic characteristics of PGPase vary widely among the different species. The Km value for phosphoglycolate was reported to be 26 μM in spinach (10) and tobacco (30), 222 μM in the cyanobacteria C. peniocystis (7), 570 μM in maize (11), and 800 μM in the brown algae H. cylindracea (13). Much larger values of 2 mM have been reported in pea (12) and bean (32). The Km value of 140 μM determined for C. reinhardtii in our study was similar to that of C. peniocystis, intermediate to that of spinach and maize, although the value of 23 μM reported earlier for C. reinhardtii (14) was as small as that in spinach and tobacco. Such diversity may be a result of some differences in experimental conditions, because the values can be affected by the concentrations of inorganic phosphate, ribose-5-phosphate, and MgCl 2 (8,16,33).

Ca
2 + may have an important role in the regulation of PG-Pase activity in vivo. Our results indicated that C. reinhardtii PGPase was a Ca 2 + -binding protein and was strongly but reversibly inhibited by Ca 2 + , although the effective concentration appeared higher than that usually observed in cells. It is very probable that at least the first EF-hand-like region in C. reinhardtii PGPase (Fig. 4) is responsible for Ca 2 + binding.
The region was more similar than the second one to the EF-hand motif and was observed at almost the same position from the FIG.7. Effect of pH on the activity of PGPase from C. reinhardtii strain 2137. The pH profiles of the enzyme preparation after DEAE-Sephacel and the crude enzyme preparation were compared. The activity in the crude preparation represents the sum of activities in both supernatant and precipitate after sonicated cells were centrifuged at 150,000 * g (18).