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Characterization of phytochelatin synthase-like protein encoded by alr0975 from a prokaryote, Nostoc sp. PCC 7120
BBRC Biochemical and Biophysical Research Communications 315 (2004) 751–755 www.elsevier.com/locate/ybbrc
Characterization of phytochelatin synthase-like protein encoded by alr0975 from a prokaryote, Nostoc sp. PCC 7120 Naoki Tsuji,a Shingo Nishikori,b Osamu Iwabe,a Kentaro Shiraki,b Hitoshi Miyasaka,c Masahiro Takagi,b Kazumasa Hirata,a,* and Kazuhisa Miyamotoa a
b
Environmental Biotechnology Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 1-6 Yamadaoka, Osaka 565-0871, Japan School of Materials Science, Japan Advanced Instituted of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan c The Kansai Electric Power Co., Inc., Technical Research Center, 11-20 Nakouji, 3 Amagasaki, Hyogo 671-0974, Japan Received 27 January 2004
Abstract Phytochelatins (PCs) are well known as the heavy metal-detoxifying peptides in higher plants, eukaryotic algae, fungi, and nematode. In contrast, neither PCs nor PC synthase genes have ever been identified in any prokaryotes. The genome sequences for the cyanobacterium Nostoc sp. PCC 7120 were recently completed and allowed us to identify a gene encoding a PC synthase-like protein, termed alr0975. The predicted product of alr0975 contains the conserved N-terminal domain but not the variable C-terminal domain found in eukaryotic PC synthases. The recombinant alr0975 protein strongly catalyzed the first step of PC synthesis, in which glutathione (GSH) is converted to c-glutamylcysteine (c-EC), although the protein only weakly catalyzed the second step of PC synthesis, namely the transfer of c-EC moiety to an acceptor GSH molecule to form PC2 . These results suggest alr0975 protein may be a more primitive form of the PC synthases found in eukaryotes. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Phytochelatins; Phytochelatin synthase; Nostoc sp. PCC 7120; Cyanobacterium; Heavy metal; c-Glutamylcysteine
Phytochelatins (PCs) are well known as the principal heavy metal-detoxifying peptides in the plant kingdom [1–3]. These peptides, of the general structure [(c-GluCys)n -Gly], have also been found in some eukaryotic microalgae, fungi, and surprisingly, the worm Caenorhabditis elegans. PCs are enzymatically synthesized from the substrate glutathione (GSH) in response to heavy metal exposure [4]. The enzyme catalyzing the biosynthesis of PCs is phytochelatin synthase (PC synthase, EC2.3.2.15), and the gene for which has been identified already in several eukaryotes including some higher plants, fission yeast, and C. elegans [5–8]. It is known that the expression of this gene generally occurs constitutively and is not induced in the presence of heavy metals in higher plants [6]. PC synthesis is proposed to have two distinct steps: (Step 1) formation of c-EC * Corresponding author. Fax: +81-06-6879-8239. E-mail address: [email protected] (K. Hirata).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.122
concomitant with the cleavage of Gly from GSH; and (Step 2) transfer of the c-EC unit to an acceptor GSH molecule or an oligomeric PC peptide (PCn ) [4]. In higher plants, the synthesis of PCs occurs in vivo and in vitro only in the presence of heavy metals. Neither PCs nor the PC synthase gene has ever been identified in any prokaryotes; this includes the cyanobacteria, even though they, like higher plants and eukaryotic microalgae, are photoautotrophic. Recently, the entire genome sequences for several cyanobacteria have been completed [9–11]. Using Cyanobase (http://www.kazusa.or.jp/cyano; established by Kazusa DNA Research Institute), we found among these sequences a gene encoding a PC synthase-like protein, termed alr0975, in the genome of one cyanobacterium, Nostoc sp. PCC 7120. In this report, we purified recombinant protein from alr0975-transformed Escherichia coli and characterized it as a PC synthase.
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Materials and methods Bacterial strain. Escherichia coli BL21(DE3) (Novagen, Madison, WI, USA) was used for recombinant protein expression. E. coli cells containing expression were grown in Luria broth (LB) medium containing 50 lg/ml carbenicillin. Plasmid construction. The coding region of the PC synthase-like gene alr0975 was amplified from genomic DNA extracted from Nostoc sp. PCC 7120 obtained from the Pasteur Culture Collection (PCC) using the primers NsF1 (50 -CTTCATATGATAGTTATGAAACTC TTTATC-30 ) and NsR1 (50 -ATCGGATCCTAATCTTGTGTTTTA CTTACG-30 ) carrying NdeI and BamHI sites, respectively. The fragments were digested with NdeI and BamHI, and ligated into the pET25b(+) vector (Novagen, Madison, WI, USA). The resultant plasmid, pET25b-alr0975, was transformed into E. coli BL21 (DE3) cells. Purification of the recombinant alr0975 protein. Recombinant alr0975 protein was expressed in E. coli containing pET25b-alr0975 at 25 °C. At mid-log phase, protein expression was induced by the addition of isopropyl-b-D -thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and the cells were cultured for a further 6 h. The
cells were harvested by centrifugation (1500g for 10 min at 4 °C) and disrupted by sonication in 100 mM Tris–HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM b-mercaptoethanol. The supernatant obtained after centrifugation (15,000g for 30 min at 4 °C) was dialyzed overnight against 20 mM Tris–HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM b-mercaptoethanol. The dialyzed sample was applied to a DEAE-Toyopearl column (5 cm 15 cm; Tosho, Tokyo, Japan) equilibrated with 20 mM Tris–HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM b-mercaptoethanol. The flow-through fraction was dialyzed overnight against 20 mM phosphate buffer (pH 6.0) containing 1 mM EDTA. The sample was then applied to a HiTrap SP € column (Pharmacia Biotech, Uppsala, Sweden) attached to an AKTA fast protein liquid chromatography system equilibrated with 20 mM phosphate buffer (pH 6.0) containing 1 mM EDTA at 4 °C. The sample was eluted with a linear gradient of 0–10 mM NaCl in 20 mM phosphate buffer (pH 6.0) and 1 mM EDTA, at a flow rate of 1.0 ml/min. Homogeneity of the purified protein was confirmed by SDS–PAGE. Protein concentration was measured according to the method of Bradford [12]. Enzyme assay. PC synthase activity was measured according to the method of Oven et al. [13]. The reaction mixture (125 ll) contained
A
B
Fig. 1. (A) Alignment of the deduced amino acid sequences of alr0975 (Accession No. AP003584) and N-terminal domains of PC synthases: AtPCS1 (A. thaliana, AF135155), GmPCS1 (Glycine max, AF411075), CePCS (C. elegans, AF299333), and SpPCS (Schizosaccharomyces pombe, Z68144). The alignment was prepared using the GENETYX-MAC Multi-sequences Program Ver. 10.1.1 (Software Development, Tokyo). The conserved five Cys residues are indicated as asterisk. (B) Phylogenetic analysis of PC synthases was performed with the Neighbor-Joining method using the GENETYX-MAC Multi-sequences Program Ver. 10.1.1 (Software Development, Tokyo).
N. Tsuji et al. / Biochemical and Biophysical Research Communications 315 (2004) 751–755 200 mM Tris–HCl buffer (pH 8.0), 10 mM b-mercaptoethanol, 10 mM glutathione, 0.5 mM CdCl2 , and 2 lg recombinant alr0975 protein. The reaction mixture was incubated at 35 °C for 60 min and terminated by addition of 125 ll of 3.6 N HCl. The supernatant obtained after centrifugation (10,000g for 10 min at room temperature) was analyzed by HPLC as described previously [14]. Mass spectrometry. MALDI–TOF mass spectra of negatively charged ions were acquired on a Voyager-DE STR (Applied Biosystems, Framingham, MA, USA) operated in the linear mode. Ten milligrams per milliliter of a-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA/MeCN + H2 O (1:1) solution was used as a matrix solution. One microliter of sample solution and 10 ll of matrix solution were mixed and 0.5 ll of the mixture obtained was applied.
Results Sequence characteristics of alr0975 protein Recently, the complete genomic sequence of Nostoc sp. PCC 7120 has been determined [10]. Cyanobase sequence data for Nostoc sp. PCC 7120 indicate the
Fig. 2. (A) SDS–PAGE of purified recombinant alr0975 protein. Lane 1, molecular weight standard; lane 2, 1.2 lg purified recombinant alr0975 protein. (B) PC synthase assay using recombinant alr0975 protein. The reaction mixture was incubated for 0 or 60 min as described in “Materials and methods,” and the resultant thiol compounds were determined by HPLC. This figure shows the HPLC profile of the reaction mixture incubated for 0 min (a) and 60 min (b).
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presence of a predicted gene, alr0975 (DDBJ/EMBL/ GenBank Accession No. AP003584), which is similar to PC synthase genes found in eukaryotes. This gene encodes a putative protein having 36% identity at the amino acid level to AtPCS1 (Accession No. AF085230) derived from Arabidopsis thaliana. The alr0975 protein consists of 243 amino acid residues. The sequence is homologous to the N-terminal conserved domain of the PC synthase family, but is missing the C-terminal variable domain (Fig. 1). This unexpected finding indicates the possible occurrence of a PC synthase-like protein in a cyanobacterium, Nostoc sp., even though PCs had never previously been identified in any prokaryotes. Function of the recombinant alr0975 protein To investigate the ability of the alr0975 protein to function as a PC synthase, alr0975 was cloned into the T7 promoter-based expression vector pET25b(+). The pET25b-alr0975 construct was transformed into E. coli BL21 (DE3). The soluble protein fraction was extracted from the transformed cells and recombinant protein was purified by ion-exchange chromatography. The recombinant protein obtained was analyzed by SDS– PAGE to confirm its purity and molecular weight (Fig. 2A). A single clear band was obtained with a molecular weight of about 26 kDa, corresponding to the predicted size of the amino acid sequence encoded by alr0975. PC synthase activity of the recombinant protein was assayed using GSH as a substrate in the presence of 0.5 mM CdCl2 and 10 mM b-mercaptoethanol. In the presence of 2 lg purified protein, 1.25 lmol GSH was almost completely converted to c-EC within 1 h, and the production of PC2 and c-EC2 was also detected (Fig. 2B). However, the concentration of PC2 was very small and equivalent to 4.9% of the c-EC synthesized. These three reaction products were identified by MALDI–TOF-MS in comparison with the results from their standards (Fig. 3). These results suggest that the protein encoded by alr0975 acts as PC synthase in vitro, but the ability to catalyze Step 2 in PC synthesis is much
Fig. 3. Mass spectra of alr0975 protein reaction products. MALDI–TOF mass spectra of positively charged ions (A, GSH and c-EC mixture) and negatively charged ions (B, PC2 and c-EC2 mixture) isolated from PC synthase assay using alr0975 protein were acquired on a Voyager-DE STR (Applied Biosystems, Framingham, MA, USA).
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less than that for Step 1. Surprisingly, even in the absence of Cd2þ , the recombinant protein showed almost the same c-EC and PC2 -forming activity as it did in the presence of Cd2þ . Furthermore, the protein synthesized 74% as much c-EC in the absence both of Cd2þ and b-mercaptoethanol as it did in the presence of these reagents.
Discussion Among photoautotrophic organisms, PCs have been found throughout the plant kingdom from higher plants to eukaryotic microalgae [1–3], but never in any cyanobacteria. Recently the genes encoding PC synthase have been identified [5–8], and AtPCS1, from A. thaliana, has been particularly well studied [13,15]. PC synthase typically has a conserved N-terminal domain and a variable C-terminal domain, both of which are cysteine-rich [16]. PC synthesis is proposed to have two distinct steps: (Step 1) formation of c-EC from GSH; and (Step 2) transfer of the c-EC unit to GSH or PCn to form PC2 or PCnþ1 [4]. In higher plants, heavy metal ions are necessary for PC synthesis in vivo; in particular, Cd2þ strongly activates PC synthesis [17]. The nucleotide sequences of the entire genome of several cyanobacteria have been determined [9–11]. Among them, we found a gene, alr0975, which encodes a PC synthase-like protein in Nostoc sp. PCC 7120. As shown in Fig. 2B, purified recombinant protein from alr0975-transformed E. coli could convert 76% of the GSH added as a substrate to c-EC in 1 h under the conditions used in this study. Furthermore, the synthesis
of PC2 was detected, although its yield from GSH, 3.7%, was much less than that of c-EC. These results strongly suggest that the alr0975 protein functions as PC synthase, but the catalytic activity for (Step 2) is much less than that for (Step 1), resulting in very little synthesis of PCs. Surprisingly, unlike AtPCS1, alr0975 does not require heavy metals for (Step 1), indicating that the alr0975 protein can catalyze at least (Step 1) as well as AtPCS1 even in the absence of heavy metals. In the reaction of the alr0975 protein, we detected a byproduct and identified it as c-EC2 (3.4% yield from GSH) (Fig. 3). In the case of AtPCS1, c-EC2 was not detected as a reaction product [15]. Two possible mechanisms can be proposed for the synthesis of c-EC2 : (i) Gly was cleaved from PC2 synthesized in the reaction; or (ii) a binding reaction of c-EC moieties synthesized in (Step1) occurred. Kinetic analysis of the PC synthesis assay for the alr0975 protein showed that c-EC2 appeared with a noticeable lag behind PC2 . In addition, synthesis of c-EC2 was observed when PC2 was used as a substrate instead of GSH (data not shown). Therefore, the alr0975 protein may cleave Gly not only from GSH but also from PC2 . The cad1-5 mutant of A. thaliana, which lacks the C-terminal domain of AtPCS1, accumulates 33% of the wild-type levels of PCs in vivo [6]. Based on this information, Cobbett hypothesized that the N-terminal domain of PC synthase is the catalytic domain, whereas the C-terminal domain is a regulatory site and not absolutely required for catalysis [16]. Maier et al. [18] reported recently that five Cys residues in the N-terminal domain were essential for heavy metal binding and activation of PC synthase. The fact that the alr0975 pro-
Fig. 4. Alignment of the deduced amino acid sequences of alr0975 (Nostoc sp. PCC 7120, Accession No. AP003584), Npun1799 (N. punctiforme, AF135155), PMT1013 (P. marinus MIT9313, BX572098), and Tery3140 (T. erythraeum IMS101, AF299333). The alignment was prepared using the GENETYX-MAC Multi-sequences Program Ver. 10.1.1 (Software Development, Tokyo).
N. Tsuji et al. / Biochemical and Biophysical Research Communications 315 (2004) 751–755
tein has only one of the 5 Cys residues conserved among the PC synthases of higher plants may explain why the cyanobacterial protein showed very weak PC synthase activity, even though its sequence is of sufficient length to catalyze Steps 1 and 2. In any prokaryotes, unlike higher plants, neither PCs nor PC synthase genes have ever been identified. In also Nostoc sp. PCC7120, no accumulation of PCs occurred in the cells treated with heavy metals. Furthermore, detectable expression of alr0975 was not observed by Northern analysis in either untreated cells or those treated with heavy metals (data not shown). On the other hand, in the alr0975-transformed E. coli cells, low level of PC2 synthesis occurred dependent on cadmium (data not shown). Using NCBI-BLAST (http://www.ncbi.nlm.nih.gov/ blast/) to search recently completed prokaryotic genome sequences, three additional genes encoding PC synthaselike proteins have been also found in the cyanobacteria Nostoc punctiforme, Trichodesmium erythraeum IMS101, and Prochlorococcus marinus MIT. The alr0975 protein and the deduced proteins encoded by these genes have common characteristics: high homology to the N-terminal domain of PC synthase found in eukaryotes and absence of four out of the five conserved Cys residues in the sequences (Fig. 4). In this study, it was found that the alr0975 protein functions as a PC synthase (Fig. 2B), but does not perform this role in Nostoc cells challenged with heavy metals. These results suggest that the proteins encoded by the cyanobacterial genes are progenitor or primitive forms of PC synthase and represent an early stage in the evolution of the enzyme in photoautotrophic organisms, predating the acquisition of PC-based heavy metal resistance mechanisms. Moreover, the C-terminal domain of PC synthase varies much more widely among plant species than the N-terminal domain. Therefore, it is expected that the full-length PC synthase may have evolved from the cyanobacterial protein by acquiring more Cys residues and by fusion with the C-terminal domain from their own genomes or that of other species. Acknowledgments We are grateful to Dr. S. Kajiyama (Graduate School of Engineering, Osaka University) for identification of thiol compounds by MALDI–TOF MS. This work was supported by the Sasakawa Scientific Research Grant from The Japan Science Society and Grants-inAid for Scientific Research (No. 11132240 and No. 12019241) from the Ministry of Education, Culture, Sports, Science and Technology.
References [1] W. Gekeler, E. Grill, E.-L. Winnacker, M.H. Zenk, Algae sequester heavy metals via synthesis of phytochelatin complexes, Arch. Microbiol. 150 (1988) 197–202.
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[2] W. Gekeler, E. Grill, E.-L. Winnacker, M.H. Zenk, Survey of the plant kingdom for the ability to bind heavy metals through phytochelatins, Z. Naturforsch 44c (1989) 361–369. [3] E. Grill, E.-L. Winnacker, M.H. Zenk, Phytochelatins: the principal heavy-metal complexing peptides of plants, Science 230 (1985) 674–676. [4] E. Grill, S. L€ offler, E.-L. Winnacker, M.H. Zenk, Phytochelatins, the heavy-metal binding peptides of plants, are synthesized from glutathione by specific c-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase), Proc. Natl. Acad. Sci. USA 86 (1989) 6838–6842. [5] S. Clemens, E.J. Kim, D. Neumann, J.I. Chroeder, Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast, EMBO J. 18 (1999) 3325–3333. [6] S.-B. Ha, A.P. Smith, R. Howden, W.M. Dietrich, S. Bugg, M.J. O’Connell, P.B. Goldsbrough, C.S. Cobbett, Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe, Plant Cell 11 (1999) 1153–1163. [7] O.K. Vatamaniuk, S. Mari, Y.-P. Lu, P.A. Rea, AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution, Proc. Natl. Acad. Sci. USA 96 (1999) 7110– 7115. [8] O.K. Vatamaniuk, E.A. Bucher, J.T. Ward, P.A. Rea, A new pathway for heavy metal detoxification in animals, J. Biol. Chem. 276 (2001) 20817–20820. [9] T. Kaneko, S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N. Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, et al., Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions, DNA Res. 3 (1996) 109–136. [10] T. Kaneko, Y. Nakamura, C.P. Wolk, T. Kuritz, S. Sasamoto, A. Watanabe, M. Iriguchi, A. Ishikawa, K. Kawashima, T. Kimura, et al., Complete genomic sequence of the filamentous nitrogenfixing cyanobacterium Anabaena sp. strain PCC 7120, DNA Res. 8 (2001) 205–213. [11] Y. Nakamura, T. Kaneko, S. Sato, M. Ikeuchi, H. Katoh, S. Sasamoto, A. Watanabe, M. Iriguchi, K. Kawashima, T. Kimura, et al., Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, DNA Res. 9 (2002) 123–130. [12] M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Anal. Biochem. 72 (1976) 248–254. [13] M. Oven, J.E. Page, M.H. Zenk, T.M. Kutchan, Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase, J. Biol. Chem. 277 (2002) 4747–4754. [14] K. Hirata, Y. Tsujimoto, T. Namba, T. Ohta, N. Hirayanagi, H. Miyasaka, M.H. Zenk, K. Miyamoto, Strong induction of phytochelatin synthesis by Zn in marine green alga, Dunaliella tertiolacta, J. Biosci. Bioeng. 92 (2001) 24–29. [15] O.K. Vatamaniuk, S. Mari, Y.-P. Lu, P.A. Rea, Mechanism of heavy metal ion activation of phytochelatin (PC) synthase, J. Biol. Chem. 275 (2000) 31451–31459. [16] C. Cobbett, P. Goldsbrough, Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis, Annu. Rev. Plant Biol. 53 (2002) 159–182. [17] M.H. Zenk, Heavy metal detoxification in higher plants—a review, Gene 179 (1996) 21–30. [18] T. Maier, C. Yu, G. K€ ullertz, S. Clemens, Localization and functional characterization of metal-binding sites in phytochelatin synthases, Planta 218 (2003) 300–308.
Characterization of phytochelatin synthase-like protein encoded by alr0975 from a prokaryote, Nostoc sp. PCC 7120 Naoki Tsuji,a Shingo Nishikori,b Osamu Iwabe,a Kentaro Shiraki,b Hitoshi Miyasaka,c Masahiro Takagi,b Kazumasa Hirata,a,* and Kazuhisa Miyamotoa a
b
Environmental Biotechnology Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 1-6 Yamadaoka, Osaka 565-0871, Japan School of Materials Science, Japan Advanced Instituted of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan c The Kansai Electric Power Co., Inc., Technical Research Center, 11-20 Nakouji, 3 Amagasaki, Hyogo 671-0974, Japan Received 27 January 2004
Abstract Phytochelatins (PCs) are well known as the heavy metal-detoxifying peptides in higher plants, eukaryotic algae, fungi, and nematode. In contrast, neither PCs nor PC synthase genes have ever been identified in any prokaryotes. The genome sequences for the cyanobacterium Nostoc sp. PCC 7120 were recently completed and allowed us to identify a gene encoding a PC synthase-like protein, termed alr0975. The predicted product of alr0975 contains the conserved N-terminal domain but not the variable C-terminal domain found in eukaryotic PC synthases. The recombinant alr0975 protein strongly catalyzed the first step of PC synthesis, in which glutathione (GSH) is converted to c-glutamylcysteine (c-EC), although the protein only weakly catalyzed the second step of PC synthesis, namely the transfer of c-EC moiety to an acceptor GSH molecule to form PC2 . These results suggest alr0975 protein may be a more primitive form of the PC synthases found in eukaryotes. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Phytochelatins; Phytochelatin synthase; Nostoc sp. PCC 7120; Cyanobacterium; Heavy metal; c-Glutamylcysteine
Phytochelatins (PCs) are well known as the principal heavy metal-detoxifying peptides in the plant kingdom [1–3]. These peptides, of the general structure [(c-GluCys)n -Gly], have also been found in some eukaryotic microalgae, fungi, and surprisingly, the worm Caenorhabditis elegans. PCs are enzymatically synthesized from the substrate glutathione (GSH) in response to heavy metal exposure [4]. The enzyme catalyzing the biosynthesis of PCs is phytochelatin synthase (PC synthase, EC2.3.2.15), and the gene for which has been identified already in several eukaryotes including some higher plants, fission yeast, and C. elegans [5–8]. It is known that the expression of this gene generally occurs constitutively and is not induced in the presence of heavy metals in higher plants [6]. PC synthesis is proposed to have two distinct steps: (Step 1) formation of c-EC * Corresponding author. Fax: +81-06-6879-8239. E-mail address: [email protected] (K. Hirata).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.122
concomitant with the cleavage of Gly from GSH; and (Step 2) transfer of the c-EC unit to an acceptor GSH molecule or an oligomeric PC peptide (PCn ) [4]. In higher plants, the synthesis of PCs occurs in vivo and in vitro only in the presence of heavy metals. Neither PCs nor the PC synthase gene has ever been identified in any prokaryotes; this includes the cyanobacteria, even though they, like higher plants and eukaryotic microalgae, are photoautotrophic. Recently, the entire genome sequences for several cyanobacteria have been completed [9–11]. Using Cyanobase (http://www.kazusa.or.jp/cyano; established by Kazusa DNA Research Institute), we found among these sequences a gene encoding a PC synthase-like protein, termed alr0975, in the genome of one cyanobacterium, Nostoc sp. PCC 7120. In this report, we purified recombinant protein from alr0975-transformed Escherichia coli and characterized it as a PC synthase.
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N. Tsuji et al. / Biochemical and Biophysical Research Communications 315 (2004) 751–755
Materials and methods Bacterial strain. Escherichia coli BL21(DE3) (Novagen, Madison, WI, USA) was used for recombinant protein expression. E. coli cells containing expression were grown in Luria broth (LB) medium containing 50 lg/ml carbenicillin. Plasmid construction. The coding region of the PC synthase-like gene alr0975 was amplified from genomic DNA extracted from Nostoc sp. PCC 7120 obtained from the Pasteur Culture Collection (PCC) using the primers NsF1 (50 -CTTCATATGATAGTTATGAAACTC TTTATC-30 ) and NsR1 (50 -ATCGGATCCTAATCTTGTGTTTTA CTTACG-30 ) carrying NdeI and BamHI sites, respectively. The fragments were digested with NdeI and BamHI, and ligated into the pET25b(+) vector (Novagen, Madison, WI, USA). The resultant plasmid, pET25b-alr0975, was transformed into E. coli BL21 (DE3) cells. Purification of the recombinant alr0975 protein. Recombinant alr0975 protein was expressed in E. coli containing pET25b-alr0975 at 25 °C. At mid-log phase, protein expression was induced by the addition of isopropyl-b-D -thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and the cells were cultured for a further 6 h. The
cells were harvested by centrifugation (1500g for 10 min at 4 °C) and disrupted by sonication in 100 mM Tris–HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM b-mercaptoethanol. The supernatant obtained after centrifugation (15,000g for 30 min at 4 °C) was dialyzed overnight against 20 mM Tris–HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM b-mercaptoethanol. The dialyzed sample was applied to a DEAE-Toyopearl column (5 cm 15 cm; Tosho, Tokyo, Japan) equilibrated with 20 mM Tris–HCl buffer (pH 8.0) containing 1 mM EDTA and 1 mM b-mercaptoethanol. The flow-through fraction was dialyzed overnight against 20 mM phosphate buffer (pH 6.0) containing 1 mM EDTA. The sample was then applied to a HiTrap SP € column (Pharmacia Biotech, Uppsala, Sweden) attached to an AKTA fast protein liquid chromatography system equilibrated with 20 mM phosphate buffer (pH 6.0) containing 1 mM EDTA at 4 °C. The sample was eluted with a linear gradient of 0–10 mM NaCl in 20 mM phosphate buffer (pH 6.0) and 1 mM EDTA, at a flow rate of 1.0 ml/min. Homogeneity of the purified protein was confirmed by SDS–PAGE. Protein concentration was measured according to the method of Bradford [12]. Enzyme assay. PC synthase activity was measured according to the method of Oven et al. [13]. The reaction mixture (125 ll) contained
A
B
Fig. 1. (A) Alignment of the deduced amino acid sequences of alr0975 (Accession No. AP003584) and N-terminal domains of PC synthases: AtPCS1 (A. thaliana, AF135155), GmPCS1 (Glycine max, AF411075), CePCS (C. elegans, AF299333), and SpPCS (Schizosaccharomyces pombe, Z68144). The alignment was prepared using the GENETYX-MAC Multi-sequences Program Ver. 10.1.1 (Software Development, Tokyo). The conserved five Cys residues are indicated as asterisk. (B) Phylogenetic analysis of PC synthases was performed with the Neighbor-Joining method using the GENETYX-MAC Multi-sequences Program Ver. 10.1.1 (Software Development, Tokyo).
N. Tsuji et al. / Biochemical and Biophysical Research Communications 315 (2004) 751–755 200 mM Tris–HCl buffer (pH 8.0), 10 mM b-mercaptoethanol, 10 mM glutathione, 0.5 mM CdCl2 , and 2 lg recombinant alr0975 protein. The reaction mixture was incubated at 35 °C for 60 min and terminated by addition of 125 ll of 3.6 N HCl. The supernatant obtained after centrifugation (10,000g for 10 min at room temperature) was analyzed by HPLC as described previously [14]. Mass spectrometry. MALDI–TOF mass spectra of negatively charged ions were acquired on a Voyager-DE STR (Applied Biosystems, Framingham, MA, USA) operated in the linear mode. Ten milligrams per milliliter of a-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA/MeCN + H2 O (1:1) solution was used as a matrix solution. One microliter of sample solution and 10 ll of matrix solution were mixed and 0.5 ll of the mixture obtained was applied.
Results Sequence characteristics of alr0975 protein Recently, the complete genomic sequence of Nostoc sp. PCC 7120 has been determined [10]. Cyanobase sequence data for Nostoc sp. PCC 7120 indicate the
Fig. 2. (A) SDS–PAGE of purified recombinant alr0975 protein. Lane 1, molecular weight standard; lane 2, 1.2 lg purified recombinant alr0975 protein. (B) PC synthase assay using recombinant alr0975 protein. The reaction mixture was incubated for 0 or 60 min as described in “Materials and methods,” and the resultant thiol compounds were determined by HPLC. This figure shows the HPLC profile of the reaction mixture incubated for 0 min (a) and 60 min (b).
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presence of a predicted gene, alr0975 (DDBJ/EMBL/ GenBank Accession No. AP003584), which is similar to PC synthase genes found in eukaryotes. This gene encodes a putative protein having 36% identity at the amino acid level to AtPCS1 (Accession No. AF085230) derived from Arabidopsis thaliana. The alr0975 protein consists of 243 amino acid residues. The sequence is homologous to the N-terminal conserved domain of the PC synthase family, but is missing the C-terminal variable domain (Fig. 1). This unexpected finding indicates the possible occurrence of a PC synthase-like protein in a cyanobacterium, Nostoc sp., even though PCs had never previously been identified in any prokaryotes. Function of the recombinant alr0975 protein To investigate the ability of the alr0975 protein to function as a PC synthase, alr0975 was cloned into the T7 promoter-based expression vector pET25b(+). The pET25b-alr0975 construct was transformed into E. coli BL21 (DE3). The soluble protein fraction was extracted from the transformed cells and recombinant protein was purified by ion-exchange chromatography. The recombinant protein obtained was analyzed by SDS– PAGE to confirm its purity and molecular weight (Fig. 2A). A single clear band was obtained with a molecular weight of about 26 kDa, corresponding to the predicted size of the amino acid sequence encoded by alr0975. PC synthase activity of the recombinant protein was assayed using GSH as a substrate in the presence of 0.5 mM CdCl2 and 10 mM b-mercaptoethanol. In the presence of 2 lg purified protein, 1.25 lmol GSH was almost completely converted to c-EC within 1 h, and the production of PC2 and c-EC2 was also detected (Fig. 2B). However, the concentration of PC2 was very small and equivalent to 4.9% of the c-EC synthesized. These three reaction products were identified by MALDI–TOF-MS in comparison with the results from their standards (Fig. 3). These results suggest that the protein encoded by alr0975 acts as PC synthase in vitro, but the ability to catalyze Step 2 in PC synthesis is much
Fig. 3. Mass spectra of alr0975 protein reaction products. MALDI–TOF mass spectra of positively charged ions (A, GSH and c-EC mixture) and negatively charged ions (B, PC2 and c-EC2 mixture) isolated from PC synthase assay using alr0975 protein were acquired on a Voyager-DE STR (Applied Biosystems, Framingham, MA, USA).
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less than that for Step 1. Surprisingly, even in the absence of Cd2þ , the recombinant protein showed almost the same c-EC and PC2 -forming activity as it did in the presence of Cd2þ . Furthermore, the protein synthesized 74% as much c-EC in the absence both of Cd2þ and b-mercaptoethanol as it did in the presence of these reagents.
Discussion Among photoautotrophic organisms, PCs have been found throughout the plant kingdom from higher plants to eukaryotic microalgae [1–3], but never in any cyanobacteria. Recently the genes encoding PC synthase have been identified [5–8], and AtPCS1, from A. thaliana, has been particularly well studied [13,15]. PC synthase typically has a conserved N-terminal domain and a variable C-terminal domain, both of which are cysteine-rich [16]. PC synthesis is proposed to have two distinct steps: (Step 1) formation of c-EC from GSH; and (Step 2) transfer of the c-EC unit to GSH or PCn to form PC2 or PCnþ1 [4]. In higher plants, heavy metal ions are necessary for PC synthesis in vivo; in particular, Cd2þ strongly activates PC synthesis [17]. The nucleotide sequences of the entire genome of several cyanobacteria have been determined [9–11]. Among them, we found a gene, alr0975, which encodes a PC synthase-like protein in Nostoc sp. PCC 7120. As shown in Fig. 2B, purified recombinant protein from alr0975-transformed E. coli could convert 76% of the GSH added as a substrate to c-EC in 1 h under the conditions used in this study. Furthermore, the synthesis
of PC2 was detected, although its yield from GSH, 3.7%, was much less than that of c-EC. These results strongly suggest that the alr0975 protein functions as PC synthase, but the catalytic activity for (Step 2) is much less than that for (Step 1), resulting in very little synthesis of PCs. Surprisingly, unlike AtPCS1, alr0975 does not require heavy metals for (Step 1), indicating that the alr0975 protein can catalyze at least (Step 1) as well as AtPCS1 even in the absence of heavy metals. In the reaction of the alr0975 protein, we detected a byproduct and identified it as c-EC2 (3.4% yield from GSH) (Fig. 3). In the case of AtPCS1, c-EC2 was not detected as a reaction product [15]. Two possible mechanisms can be proposed for the synthesis of c-EC2 : (i) Gly was cleaved from PC2 synthesized in the reaction; or (ii) a binding reaction of c-EC moieties synthesized in (Step1) occurred. Kinetic analysis of the PC synthesis assay for the alr0975 protein showed that c-EC2 appeared with a noticeable lag behind PC2 . In addition, synthesis of c-EC2 was observed when PC2 was used as a substrate instead of GSH (data not shown). Therefore, the alr0975 protein may cleave Gly not only from GSH but also from PC2 . The cad1-5 mutant of A. thaliana, which lacks the C-terminal domain of AtPCS1, accumulates 33% of the wild-type levels of PCs in vivo [6]. Based on this information, Cobbett hypothesized that the N-terminal domain of PC synthase is the catalytic domain, whereas the C-terminal domain is a regulatory site and not absolutely required for catalysis [16]. Maier et al. [18] reported recently that five Cys residues in the N-terminal domain were essential for heavy metal binding and activation of PC synthase. The fact that the alr0975 pro-
Fig. 4. Alignment of the deduced amino acid sequences of alr0975 (Nostoc sp. PCC 7120, Accession No. AP003584), Npun1799 (N. punctiforme, AF135155), PMT1013 (P. marinus MIT9313, BX572098), and Tery3140 (T. erythraeum IMS101, AF299333). The alignment was prepared using the GENETYX-MAC Multi-sequences Program Ver. 10.1.1 (Software Development, Tokyo).
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tein has only one of the 5 Cys residues conserved among the PC synthases of higher plants may explain why the cyanobacterial protein showed very weak PC synthase activity, even though its sequence is of sufficient length to catalyze Steps 1 and 2. In any prokaryotes, unlike higher plants, neither PCs nor PC synthase genes have ever been identified. In also Nostoc sp. PCC7120, no accumulation of PCs occurred in the cells treated with heavy metals. Furthermore, detectable expression of alr0975 was not observed by Northern analysis in either untreated cells or those treated with heavy metals (data not shown). On the other hand, in the alr0975-transformed E. coli cells, low level of PC2 synthesis occurred dependent on cadmium (data not shown). Using NCBI-BLAST (http://www.ncbi.nlm.nih.gov/ blast/) to search recently completed prokaryotic genome sequences, three additional genes encoding PC synthaselike proteins have been also found in the cyanobacteria Nostoc punctiforme, Trichodesmium erythraeum IMS101, and Prochlorococcus marinus MIT. The alr0975 protein and the deduced proteins encoded by these genes have common characteristics: high homology to the N-terminal domain of PC synthase found in eukaryotes and absence of four out of the five conserved Cys residues in the sequences (Fig. 4). In this study, it was found that the alr0975 protein functions as a PC synthase (Fig. 2B), but does not perform this role in Nostoc cells challenged with heavy metals. These results suggest that the proteins encoded by the cyanobacterial genes are progenitor or primitive forms of PC synthase and represent an early stage in the evolution of the enzyme in photoautotrophic organisms, predating the acquisition of PC-based heavy metal resistance mechanisms. Moreover, the C-terminal domain of PC synthase varies much more widely among plant species than the N-terminal domain. Therefore, it is expected that the full-length PC synthase may have evolved from the cyanobacterial protein by acquiring more Cys residues and by fusion with the C-terminal domain from their own genomes or that of other species. Acknowledgments We are grateful to Dr. S. Kajiyama (Graduate School of Engineering, Osaka University) for identification of thiol compounds by MALDI–TOF MS. This work was supported by the Sasakawa Scientific Research Grant from The Japan Science Society and Grants-inAid for Scientific Research (No. 11132240 and No. 12019241) from the Ministry of Education, Culture, Sports, Science and Technology.
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