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J. Biol. Chem., Vol. 277, Issue 7, 4747-4754, February 15, 2002

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Molecular Characterization of the Homo-phytochelatin Synthase of Soybean Glycine max

RELATION TO PHYTOCHELATIN SYNTHASE^*

Matjaz Oven, Jonathan E. Page, Meinhart H. Zenk^§, and Toni M. Kutchan^¶

From the  Leibniz-Institut für Pflanzenbiochemie, Weinberg 3 and the ^§ Biozentrum der Universität Halle, Weinbergweg 22, Halle/Saale 06120, Germany

Received for publication, August 27, 2001, and in revised form, October 30, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)·metal complexes, various other metal·thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451-31459).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phytochelatins (PCs)¹ of the general formula (Glu-Cys)_n-Gly (n = 2-11) are the principal heavy metal-detoxifying compounds in the plant kingdom (1-4). These peptides are linear polymers of the -glutamyl-cysteinyl (-Glu-Cys) portion of glutathione (GSH). Iso-phytochelatins are isoforms of phytochelatins (2) in which the terminal amino acid consists of serine (5), glutamic acid (6), glutamine (7), or in the case of the homo-phytochelatins, -alanine (8, 9). Although the enzymology of phytochelatin formation by a constitutive phytochelatin synthase was clarified early on (10), the gene encoding this enzyme was discovered only recently (11-13). Remarkably, phytochelatin synthase activity has been even found to occur in a nematode Caenorhabditis elegans (14).

Although the biosynthesis of phytochelatins seems to be clear on the enzymological and molecular level, there is little information available on the formation of iso-phytochelatins (2). Because iso-phytochelatins occur in only those plants that contain glutathione isomers, e.g. homoglutathione (hGSH) (8), hydroxymethyl-glutathione (5), or -glutamyl-cysteinyl-glutamic acid (6), it could be supposed that these substances are involved in iso-phytochelatin biosynthesis. Furthermore, it was shown that several leguminaceous plants contain hGSH but not GSH (8, 15-17), and it could be, therefore, assumed that hGSH is the substrate for homo-phytochelatin biosynthesis. However, it has been reported that a crude enzyme preparation from Pisum catalyzes the formation of homo-phytochelatin from homoglutathione in the presence of GSH (9). In addition, using the same enzyme preparation from Pisum, and in the presence of GSH, the synthesis of (-Glu-Cys)_n-Ser from the corresponding glutathione isomer containing serine could be observed. This indicated for the first time that Pisum may either have a phytochelatin synthase with broad substrate specificity or that several enzymes with differing substrate specificity are present in the crude plant extracts (9). The explanation put forward by these authors was that phytochelatin synthase has a -Glu-Cys donor binding site that is specific for glutathione and a -Glu-Cys acceptor binding site that is less specific and accepts various tripeptides such as glutathione, homoglutathione, and hydroxymethyl-glutathione. However, conclusive evidence for the incorporation of GSH in iso-phytochelatins was not demonstrated.

We chose Glycine max (L.) Merr. (soybean) as experimental material because this plant has been shown to contain homoglutathione (16) and produces high amounts of homo-phytochelatins, but not phytochelatins, when exposed to non-toxic doses of Cd²⁺ ions (8). Using the nucleotide sequence of the cDNA encoding phytochelatin synthase (11-13), we isolated a cDNA encoding homo-phytochelatin synthase by homology-based cloning. Our goal was to express and biochemically characterize the G. max homo-phytochelatin synthase enzyme and compare its properties to the Arabidopsis thaliana phytochelatin synthase using GSH as substrate. Our hypothesis, which was put forward early on (8), was that, although phytochelatin synthase uses glutathione as a substrate for the -glutamylcysteine dipeptidyl transpeptidase (EC 2.3.2.15), homo-phytochelatin synthase from the order Fabales would use homoglutathione as substrate.

We suggested previously that phytochelatin synthase is a metal-activated enzyme that uses GSH or PCs for its substrate (10). We showed that the addition of EDTA or apoPCs into the reaction mixture containing phytochelatin synthase, cadmium ions and GSH resulted in the immediate inactivation of the enzyme (18). We suggested that the phytochelatin synthase-catalyzed reaction terminated immediately after the metal ions were chelated by EDTA or apoPCs and concluded that the activity of phytochelatin synthase is regulated by the reaction product, i.e. PCs (18). However, a significantly different model for phytochelatin biosynthesis was presented recently (19). The authors of this model suggested that metal·GS₂, rather than free metal ions, activate phytochelatin synthase. Furthermore, these authors suggested that metal·GS₂ as well as metal·PC complexes are active substrates for phytochelatin synthase (19). Some of our previously published results, most notably the results of our studies on the termination of phytochelatin synthase catalyzed reaction (18), cannot be explained by this new model. If this model would be correct, then the addition of apoPCs into the reaction mixture containing phytochelatin synthase, metal ions, and GSH should not result in the immediate reaction termination, because metal·PC complexes should have been used as substrates for phytochelatin synthase during phytochelatin chain elongation. Hence, such a reaction should proceed ad infinitum. The authors of the newest model suggested that PC biosynthesis terminates when GSH or apoPCs compete with thiolates for the high affinity site of the synthase or when higher order substrate-inactive metal·PC complexes are formed, or when metal·PC complexes are removed from the cytosolic pool into the vacuole (19). We addressed the question of phytochelatin synthase activation and the enzyme-catalyzed reaction in the second part of the presented study.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant Material-- Cultured suspension cells of G. max (soybean) were provided by the cell culture laboratory of the Leibniz-Institut für Pflanzenbiochemie in Halle. Cultures were grown in 1-liter Erlenmeyer flasks containing 400 ml of Linsmaier-Skoog medium (20) over 7 days at 23 °C on a gyratory shaker (100 rpm) in diffuse light (750 lux). Differentiated soybean plants were grown under greenhouse conditions at 24 °C.

Extraction of Plant Suspension Cells-- Suspension cells were vacuum-filtered, and 400 mg of filtered cells was extracted in 600 µl of 1 N NaOH that contained NaBH₄ (1 mg ml¹). Following sonication (3 × 10 s) and centrifugation (5 min, 10,000 × g, room temperature), 500 µl of supernatant was transferred into a new tube and acidified by the addition of 100 µl of 3.6 N HCl. After 5-min incubation on ice (protein precipitation), samples were once more centrifuged (5 min, 10,000 × g, room temperature) and analyzed by HPLC.

Amplification of a Phytochelatin Synthase Partial Clone from G. max-- Total RNA was isolated from 4-day-old suspension cultured cells of G. max (21) using TRIzol reagent (Invitrogen). mRNA was purified from total RNA using an Oligotex kit (Qiagen), and first-strand cDNA was synthesized using a cDNA synthesis system (Invitrogen). Partial cDNA fragments encoding phytochelatin synthases were amplified using Taq polymerase and degenerate primers corresponding to conserved regions in known phytochelatin synthases. The primer sequences were 5'-TGG AA(A/G) GG(A/C/G/T) (C/T)C(A/C/G/T) TGG (A/C)G(A/C/G/T) TGG-3', corresponding to the WKGPWRW motif, and 5'-GG(A/G) TA(C/T) TT(A/G) AA(A/C/G/T) C(T/G)(A/C/G/T) GC(A/C/G/T) AC(A/G) TC-3', corresponding to the DVARFKY motif. cDNA fragments were amplified by PCR in a GeneAmp PCR 9700 thermal cycler (PE Applied Biosystems) using the following program: 3 min at 94 °C, 30 cycles of 94 °C, 30 s; 42 °C, 1 min; 72 °C, 30 s; followed by an extension step at 72 °C for 10 min. The amplified DNA was resolved by agarose gel electrophoresis (21). DNA was isolated from a band of the expected length (ca. 330 bp) and cloned into pGEM-T Easy (Promega) prior to nucleotide sequence determination. DNA sequencing of partial and full-length clones on both strands was performed at GATC (Konstanz, Germany) and MWG (Ebersberg, Germany).

Full-length Cloning of Homo-phytochelatin Synthase from G. max-- The full-length homo-phytochelatin synthase cDNA GmhPCS1 was obtained by screening a G. max  ZAP cDNA library (Stratagene) using the G. max phytochelatin synthase-like PCR product as a probe. The probe was -³²P-labeled using the RadPrime DNA Labeling System (Invitrogen), and phage plaques hybridizing with the labeled probe were identified using autoradiography. The identified cDNA was in vivo excised from the phage in pBluescript phagemid and sequenced.

To express the cDNA in Escherichia coli cells, the open reading frame was cloned into the pET-14b vector (Novagen), which contains a hexahistidine N-terminal fusion tag. PCR primers were designed to introduce a NotI restriction enzyme site at the start codon (5'-TAT CCA TAT GGC GAC GGC GGG G-3') and an XhoI restriction enzyme site downstream of the stop codon (5'-TAT CCT CGA GTC AAG AGA GAG GAG C-3'). PCR amplification was performed in 50 µl using 2.5 units of Pfu polymerase with the following conditions: 3 min at 94 °C, then 25 cycles of 94 °C for 30 s, 55 °C for 1 min 30 s, and 72 °C for 30 s; followed by an extension step at 72 °C for 10 min. After restriction enzyme digestion of the PCR product and vector, the GmhPCS1 open reading frame was cloned into pET-14b to give construct pET:GmhPCS1. Cloning was confirmed by sequencing with the T7 primer. Similarly, the Arabidopsis phytochelatin synthase cDNA, AtPCS1, was amplified by PCR using primers containing recognition sites for the restriction endonucleases EcoRI (5'-TAT CGA ATT CAT GGC TAT GGC GAG TTT A-3') and XhoI (5'-TAT CCT CGA GCT AAT AGG CAG GAG CAG C-3'), appropriate for subcloning into pET-28a (Novagen).

Heterologous Expression and Enzyme Purification-- The G. max and Arabidopsis phytochelatin synthase clones were expressed in E. coli BL21(DE3) (Novagen). The expression of recombinant proteins was performed according to the manufacturer's instructions. Briefly, bacteria were grown at 37 °C to A₆₀₀ 0.6, and the expression of recombinant proteins was induced by the addition of IPTG (final IPTG concentrations: 0.4 mM for bacteria transformed with pET:GmhPCS1 and 1 mM for bacteria transformed with pET:AtPCS1). The expression of recombinant proteins was allowed to proceed overnight at 18 °C. Bacteria were lysed in buffer containing 50 mM Tris-HCl, pH 7.0, 500 mM NaCl, 2.5 mM imidazole, 10% glycerol, 1% Tween 20, and 750 µg/ml lysozyme, and the recombinant proteins were purified from the bacterial extracts using a cobalt metal affinity resin (Talon, CLONTECH) as described by the manufacturer. To remove the hexahistidine tag, protein eluting from the Talon column was loaded onto a PD10 column (Amersham Biosciences, Inc.) equilibrated with buffer containing 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 2.5 mM imidazole, 10% glycerol, 10 mM -mercaptoethanol, 3 mM CaCl₂, and 5 mM MgCl₂. The protein was incubated with 20 units of thrombin protease (Amersham Biosciences, Inc.) overnight at 4 °C. After digestion, the protein solution was again passed over a Talon column, and the flow-through, containing phytochelatin synthase lacking the hexahistidine tag, was collected. The homogeneity of the purified enzymes was confirmed by SDS-PAGE analysis.

Enzyme Assay-- The phytochelatin synthase activity was measured as described previously (10). The standard 125-µl reaction mixture contained 200 mM Tris-HCl, pH 8.0, 10 mM -mercaptoethanol, 0.5 mM CdCl₂, 10 mM GSH, and 0.1 µg of recombinant enzyme. Homo-phytochelatin synthase activity assays were performed under the same experimental conditions except that 10 mM GSH was replaced by 10 mM hGSH, or by 10 mM hGSH and 0.5 mM GSH in the assay. Optionally, in the experiments where the role of thiols in the phytochelatin synthase assay was studied, -mercaptoethanol was omitted or replaced by other thiols, and 10 mM GSH was replaced by 10 mM S-methyl-GSH. The influence of pH on enzyme activity was monitored in sodium citrate (pH 4-6), sodium phosphate (pH 6-7), Tris-HCl (pH 7-9), and glycine/NaOH (pH 9-10.5). The assay mixtures were incubated at 35 °C for 5 min. Prior to HPLC analysis, samples were acidified by the addition of 125 µl of 3.6 N HCl and centrifuged (5 min, 10,000 × g, room temperature).

Identification of Thiols and S-Methyl-thiolate Compounds-- The determination of thiols occurring in plant cell suspension cultures, as well as thiols and S-methyl-thiolate compounds that resulted from a PC synthase assay, was performed by HPLC analysis. In all cases, 100 µl of acidified sample was injected onto a reversed-phase HPLC system (Knauer HPLC; solvent A: 99.9% water, 0.1% trifluoroacetic acid; solvent B: 79.9% water, 20% acetonitrile, 0.1% trifluoroacetic acid; flow rate: 2 ml min¹; column Knauer B6 Y535 Eurospher-100 C18 with precolumn), optionally with DTNB post-column derivatization as previously described (10). The HPLC was calibrated using GSH in various concentrations (0.1-100 nmol injected). Samples used for mass spectrometric analysis were isolated using HPLC with the DTNB derivatization omitted. The isolated compounds were submitted to mass spectrometric analysis as described previously (15).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Occurrence of Homoglutathione and Glutathione and the Respective Phytochelatins in G. max-- Control cell cultures of G. max were analyzed by HPLC for the occurrence of GSH and hGSH. These control cells contained 650 nmol·g (dry weight) dwt¹ hGSH, whereas the concentration of GSH was in the order of 50 nmol·g dwt¹. Traces of hPC₂ (20 nmol·g dwt¹), resulting from the presence of Zn²⁺ and Cu²⁺ ions in the growth medium, were detected (22) (Fig. 1A). The exposure of the cultivated G. max cells to 50 µM CdCl₂ (Fig. 1B) resulted in the formation of hPC₂ and hPC₃ (1290 and 630 nmol·g dwt¹, respectively). PC₂ and PC₃ were not observed under these conditions. G. max was, therefore, a suitable plant material for the isolation of a putative homo-phytochelatin synthase-encoding cDNA.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   HPLC analysis of thiol-containing compounds in G. max suspension cells. Cells were grown (A) in LS medium or (B) in LS medium supplemented with 50 µM CdCl2 for 4 days. The identification of homo-phytochelatins resulted from the comparison with previously isolated hPC2 and hPC3 from V. angularis (15). PCs could not be detected in G. max suspension cells.

Amplification of Phytochelatin Synthase cDNA Homologs by PCR-- A comparison of the amino acid sequences of known PC synthases (A. thaliana, Triticum aestivum, Schizosaccharomyces pombe, and C. elegans) revealed that all four proteins have a conserved N-terminal end, whereas the C-terminal end of these proteins is more variable. The design of degenerate PCR primers was based on the conserved regions at the protein N-terminal end. The PC synthase-like cDNA fragments were PCR-amplified, cloned, and sequenced. Sequence analysis showed that only one PC synthase homolog was amplified. This 334-bp cDNA fragment had high identity to known plant PC synthases (87 and 84% identity at the amino acid level to Arabidopsis and T. aestivum PC synthases, respectively). The isolated fragment was -³²P-labeled and used as a probe in screening of a G. max cDNA library. The screening resulted in the isolation of a full-length cDNA clone, GmhPCS1, that showed high identity to Arabidopsis and T. aestivum PC synthases (74 and 68% at the amino acid level) (Fig. 2).

View larger version (90K): [in this window] [in a new window]   Fig. 2.   Amino acid sequence alignment of GmhPCS1 and PC synthases from A. thaliana (AtPCS1) and T. aestivum (TaPCS1). Identical amino acids are boxed in black. The alignment was generated using the ClustalX program (31).

Purification of Recombinant Homo-phytochelatin Synthases-- GmhPCS1 and AtPCS1 were cloned in T7 promoter-based expression vectors and expressed in E. coli BL21(DE3). His-tagged proteins were purified from the crude bacterial extracts using a cobalt-containing resin. PC synthase activity of the individual purification steps was determined by the standard PC synthase assay using GSH as a substrate (Table I). The protein content during the purification was monitored by SDS-PAGE. Thrombin protease removal of the hexahistidine tag was a yield-limiting step; however, it was included in the protein purification to prevent a possible interaction of histidine with heavy metals in the PC synthase assays.

View this table: [in this window] [in a new window]   Table I Purification of recombinant GmhPCS1 from E. coli Activity was determined using 10 mM GSH, 0.5 mM CdCl2, and 10 mM -mercaptoethanol in 200 mM Tris-HCl, pH 8.0.

The expression of GmhPCS1 and AtPCS1 differed substantially in E. coli. Using GSH as a substrate and crude soluble protein from the expression cultures, GmhPCS1 showed a specific activity of 22 pmol substrate converted/s (pkat)·µg protein¹, whereas that of AtPCS1 was 230 pkat·µg protein¹. It is suspected that different codon usage in each gene might effect expression level. By comparing the codon usage of GmhPCS1 and AtPCS1 it was observed that AGG, one of the E. coli low frequency arginine codons (1.4 codons used per 1000 codons), was very abundant in GmhPCS1 cDNA (18 codons used per 1000 codons) and significantly less abundant in AtPCS1 cDNA (6.2 codons used per 1000 codons). To verify whether the codon usage was the limiting factor in GmhPCS1 expression, each GmhPCS1 and AtPCS1 was expressed in the E. coli BL21-Codon Plus (DE2)-RIL strain (Stratagene), which contains additional AGG tRNAs. However, the heterologous expression of the two cDNAs in this bacterial strain did not increase the yield of GmhPCS1, but rather decreased the yield of AtPCS1. The reason for the different expression levels of GmhPCS1 and AtPCS1 remains therefore unclear.

Characterization of Recombinant Homo-phytochelatin Synthases-- Homogenous homo-phytochelatin synthase from G. max was tested in the presence of 10 mM hGSH and found to produce hPC at a low rate (hPC synthase activity 2.2 pkat·µg¹ enzyme). Increasing the protein concentration showed a linear relationship in hPCs synthesized. When tested with GSH, however, the soybean enzyme showed high PC-synthesizing activity (1210 pkat·µg¹ enzyme). In contrast, the recombinant enzyme from Arabidopsis was completely inactive with hGSH as substrate (hPC-synthesizing activity within the range of experimental error), but showed normal PC-synthesizing activity with GSH in the range of 1510 pkat·µg¹ enzyme. Surprisingly, however, when 0.5 mM GSH was added to the GmhPCS1 assay mixture containing 10 mM hGSH, where only low hPC synthase activity was observed as given above, the hPC synthase activity increased almost 100-fold (210 pkat·µg¹ enzyme; Fig. 3). Although the predominant reaction product of this experiment was hPC₂ (230 nmol·µg¹ enzyme after 25 min), a small amount of PC₂ was simultaneously synthesized under these conditions as well (21 nmol·µg¹ enzyme after 25 min). A similar increase in hPC-synthesizing activity could also be observed with AtPCS1 when 0.5 mM GSH was applied together with 10 mM hGSH in the reaction mixture. The hPC-synthesizing activity of AtPCS1 was determined to be 190 pkat·µg¹ enzyme.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Time course of enzymatic hPC2 formation in the presence and absence of GSH. A 125-µl assay containing 0.1 µg of purified GmhPCS1, 200 mM Tris-HCl, pH 8.0, 0.5 mM CdCl2, 10 mM -mercaptoethanol, and 10 mM hGSH resulted in low hPC2 synthesis (2.5 nmol · µg1 enzyme at 25 min, ). The addition of 0.5 mM GSH after 7.5 min resulted in a rapid hPC2 synthesis (230 nmol · µg1 enzyme at 25 min, ).

The identity of the reaction product was verified as hPC₂ using MS and MS/MS (Fig. 4A). The observed result suggested that hGSH acts only as an acceptor molecule for -Glu-Cys units being generated from GSH by the homo-phytochelatin synthase. To test this hypothesis, hGSH was incubated in a GmhPCS1 assay, and S-methyl-GSH was added instead of GSH. S-Methyl-GSH has been previously reported to be a substrate for PC synthase (19). Under these conditions, in addition to the expected S-methyl-PC₂ and S-methyl-PC₃, another new metabolite was synthesized, which by mass spectral analysis was identified as S-methyl-homo-PC₂ (Fig. 4B). The incorporation of --Glu-Cys(SCH₃)- into the newly formed pentapeptide proved that GSH is a co-substrate in the synthesis of homo-phytochelatins. Furthermore, the position of the methyl group on the first cysteine residue demonstrated that the pentapeptide was synthesized by a condensation of a -Glu-Cys(SCH₃)- unit, originating from S-methyl-GSH, and -Glu-Cys--Ala (i.e. hGSH). The absence of a peptide with the structure -Glu-Cys--Glu-Cys(SCH₃)--Ala containing a Z₂ fragment with a mass of 319 Da or a possible structure of a mass of 233 Da should be noted. The peptide with a possible structure -Glu-Cys--Glu-Cys(SCH₃)--Ala was, therefore, not formed.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Mass spectra of GmhPCS1 reaction products. MS/MS spectrum of the reaction product isolated from an hPC synthase assay with 0.1 µg of purified GmhPCS1, 200 mM Tris-HCl, pH 8.0, 0.5 mM CdCl2, 10 mM -mercaptoethanol, and 10 mM hGSH and (A) 0.5 mM GSH (product: hPC2) or (B) 0.5 mM S-methyl-GSH (product: pentapeptide -Glu-Cys(SCH3)--Glu-Cys--Ala).

A comparison of AtPCS1 and GmhPCS1 with regard to their pH optima showed within experimental error that there is no difference between the two enzymes (pH 8.2 ± 0.2). The temperature optimum is in both cases 35 °C. These values correspond to the previously determined parameters for the partially purified native PC synthase from Silene cucubalus (10) (pH optimum 7.9 and temperature optimum 35 °C). The K_m value for GSH was determined for GmhPCS1 as 15 mM and for AtPCS1 as 11 mM.

Specificity of Metal Activation of Homo-phytochelatin Synthase from G. max and Phytochelatin Synthase from A. thaliana-- An absolute heavy metal ion requirement for phytochelatin synthesizing activity has frequently been demonstrated (9, 12, 23). In vitro chelation of heavy metals, such as Cd²⁺, by metal-free phytochelatin or by EDTA instantaneously terminates phytochelatin formation (18). These results support the hypothesis that phytochelatin synthesis proceeds until the metal ions are complexed and, thereby, no longer accessible to the enzyme. The activation of constitutive phytochelatin synthase by different heavy metal ions has been reported both for formation of phytochelatins (23) as well as for homo-phytochelatins (8) both in vivo and in vitro (10).

Surprisingly, the repetition of the heavy metal activation experiments with recombinant Arabidopsis phytochelatin synthase (19) led to considerable differences as compared with our previous results (10, 18). It was reported that S-alkyl-phytochelatins can be synthesized from the GSH homolog S-alkyl-glutathione even in the absence of free heavy metal ions (19). These authors proposed that a blocked thiol group of glutathione is sufficient for enzyme activation. According to their model of the mechanism of phytochelatin synthase activity, the activating metal·glutathionato complex or alkylated glutathione serve as a substrate in phytochelatin synthesis (19). Furthermore, these authors reported that phytochelatin synthase is activated by Mg²⁺, Ni²⁺, and Co²⁺, i.e. by metal ions that were previously reported to fail to activate PC synthase (2). A comparison of the metal activation of both Arabidopsis and G. max recombinant synthases yielded the results shown in Table II. The concentrations of metal ions used were previously optimized for each individual metal ion. The activators of PC synthase were determined to be exclusively the 15 metals that belong to groups 11 to 15 in the 4th, 5th, and 6th periods in the periodic system.

View this table: [in this window] [in a new window]   Table II Activation of GmhPCS1 and AtPCS1 by various metals Values are presented as the percentage of highest activity (determined for Cd2+: 2.7 nmol -SH groups ml1 min1 transferred). 5.7 pkat of GmhPCS1 or AtPCS1 were incubated in the 125-µl assay mixtures that contained 200 mM Tris-HCl, pH 8.0, 1 mM GSH, 10 mM -mercaptoethanol, and the individual metal ions.

Clearly, Mg²⁺, Ni²⁺, and Co²⁺ do not activate the synthases, which is in sharp contrast to the recently reported results (19). Our previous results demonstrated that no activation of purified S. cucubalus phytochelatin synthase could be observed when the following elements were tested at 0.5 mM concentration: LiCl, NaCl, RbBr, CsCl, Be(SO₄), MgCl₂, CaCl₂, SrCl₂, Ba(CO₂)₂, ScCl₃, Y(NO₃)₃, La(NO₃)₂, ZrOCl₂, HfOCl₂, VOSO₄, Cr(NO₃)₃, Na₂MoO₄, Mn(NO₃)₂, NH₄ReO₄, FeSO₄, OsCl₃, CoCl₂, RhCl₃, IrCl₂, Ni(NO₃)₃, Pd(NO₃)₂, K₂PtCl₄, Al(NO₃)₃, NaSeO₃, TeCl₄, NaI, Ce(SO₄)₂, Pr(NO₃)₃, Sm(NO₃)₃, Gd(NO₃)₃, TbCl₃, Ho(NO₃)₃, and UO₂(NO₃)₂.²

Role of Buffers in the Phytochelatin Synthase Assay-- It was reported recently that S-alkyl-GSH could activate PC synthase in the absence of heavy metals (19). The time dependence of S-alkyl-PCs synthesis in the metal-free assay was found to be comparable to that for the synthesis of PCs in an assay containing 50 µM Cd²⁺ and 10 mM GSH (19).

While investigating the synthesis of S-methyl-PCs in the assays where the addition of heavy metals was omitted, it could be observed that the choice of buffers had an important effect on the reaction kinetics. The experimental conditions as used by Vatamaniuk et al. (19) were performed in 200 mM HEPES-BTP, pH 8.0, whereas the assays by Grill et al. (10) were performed in 200 mM Tris-HCl, pH 8.0. The metal-independent induction of S-methyl-PCs synthesis could be observed when either of both buffers was used, however, the amount of S-methyl-PCs synthesized varied considerably. In an assay containing 10 mM S-methyl-GSH and no metals, 3.7 nmol·min¹· ml¹ -SCH₃ groups were transferred when the assay was performed in 200 mM HEPES-BTP, pH 8.0. In contrast, only 0.46 nmol·min¹·ml¹ -SCH₃ groups were transferred in the parallel assay performed in 200 mM Tris-HCl, pH 8.0. When 10 mM S-methyl-GSH was replaced with 10 mM GSH, and 0.5 mM Cd²⁺ was included in the PC synthase assay, the choice of buffers did not significantly effect the transfer of -SH groups: 26 nmol·min¹·ml¹ -SH groups were transferred when the assay was performed in 200 mM Tris-HCl, pH 8.0, and 29 nmol·min¹·ml¹ -SH groups were transferred when the PC synthase assay was performed in 200 mM HEPES-BTP, pH 8.0.

The role of HEPES-BTP or Tris-HCl on the observed differential activation of AtPCS1 by S-methyl-GSH in a metal-free assay will be investigated separately. After it was shown that the choice of buffers has an important effect on the PC synthase activity, we omitted HEPES-BTP and performed the subsequent experiments in 200 mM Tris-HCl, pH 8.0. Under such conditions, the activation of PC synthase by 10 mM S-methyl-GSH alone was shown to be minimal, i.e. only 2%, when compared with the standard experimental conditions including 10 mM GSH and 0.5 mM Cd²⁺.

Role of Cd·GS₂ in Phytochelatin Synthesis-- The effect of Cd·GS₂ on the activation of PC synthase was studied in the next experiment. Constant amounts of GSH and S-methyl-GSH (each 1 mM) and various amounts of Cd²⁺ (0.01-0.5 mM) were applied in several PC synthase assay mixtures. Other thiols (e.g. -mercaptoethanol) were omitted from this experiment. It can be assumed that GSH and Cd²⁺ instantly react and form Cd·GS₂ (19). The ratio between Cd·GS₂, GSH, and S-methyl-GSH, i.e. the compounds that were suggested to be accepted as substrates for PC synthase (19), was therefore dependent on the Cd²⁺ concentration in the reaction mixture. The transfer of -SH and -SCH₃ groups was monitored in these experiments.

The only products of these reactions were PC₂, S-methyl-PC₂, or pentapeptide -Glu-Cys--Glu-Cys(SCH₃)-Gly as identified by HPLC and MS/MS analysis. The enzymatically catalyzed transfer of -SH or -SCH₃ groups was clearly dependent on the concentration of GSH and Cd·GS₂ in the reaction mixtures. It could be observed that, in the reaction mixtures containing low concentration Cd·GS₂, the enzyme-catalyzed transfer of -SH groups was as high as the transfer of -SCH₃ groups (Fig. 5). However, in those reaction mixtures in which the Cd·GS₂ concentration was high, the PC synthase-catalyzed transfer of -SCH₃ groups was significantly higher than the transfer of -SH groups. The observed results suggest that Cd·GS₂ is not a substrate for PC synthase.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Dose dependence of enzymatic sulfur-group transfer as a function of metal·thiolate concentration used in the PC synthase assay. Each reaction mixture (125 µl) contained 0.1 µg of purified AtPCS1, 200 mM Tris-HCl, pH 8.0, 1 mM GSH, 1 mM S-methyl-GSH, and various concentrations of CdCl2 (0-1 mM).

Catalytic Mechanism of Phytochelatin Synthase-- Recently, a mechanism was proposed (19) for PC synthase activity that can be represented by the formula:

This is in contrast to the reaction catalyzed by metal-activated PC synthase as formulated previously (10, 24),

According to the latter reaction, apoPCs are the reaction products that subsequently form a rather stable metal·chelate complex.

There is no doubt that thiols play an essential role in this reaction but not in the manner recently proposed in a previous study (19). These authors postulated the formation of a glutathione·metal association, such as bis(glutathionato)·metal, which serves as a substrate for PC synthase. To circumvent the formation of Cd·GS₂, we replaced GSH with 10 mM S-methyl-GSH in all subsequent assays. The metals used in the experiment were Cd²⁺ and Zn²⁺ (each 0.5 mM). The assay mixtures were incubated for a short time, and the reaction products were determined by HPLC.

We observed that, in the absence of any -SH compounds, metal ions could not bring about the synthesis of S-methyl-PCs. Furthermore, the presence of 0.5 mM Cd²⁺ led to the complete inhibition of the low synthesis of S-methyl-PCs that was provoked by S-methyl-GSH. The synthesis of S-methyl-PCs could, however, be completely recovered by including GSH, DTT, -mercaptoethanol, -Glu-Cys, L-cysteine, or D-cysteine in the reaction mixture. An effect of the addition of 10 mM L-cysteine on the enzymatic formation of S-methyl-PCs is shown in Fig. 6.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Time course of enzymatic sulfur-group transfer in the presence () or absence () of 10 M L-cysteine. 2-ml assay mixtures contained 200 mM Tris-HCl, pH 8.0, 0.5 mM CdCl2, 10 mM S-methyl-GSH, and 1.6 µg of AtPCS1. After 7.5 min, cysteine was added (final concentration: 10 mM). Every 2.5 min, a 100-µl sample was taken from the reaction mixture and analyzed by HPLC.

The presence of thiols in the assay mixtures was obviously decisive for the reaction to proceed (Table III). The exchange of thiols by ascorbic acid, NaBH₄, imidazole, and histidine did not result in the formation of S-methyl-PCs, which excludes a simple reduction of -SH groups of the enzyme. Interestingly, significant differences in the activation of PC synthase by Cd²⁺ and Zn²⁺ could be observed when L-cysteine or D-cysteine was tested. Although high synthesis of S-methyl-PCs could be observed in the assay mixtures containing L-cysteine or D-cysteine and Cd²⁺, the enzyme was only weakly activated in the assay mixtures containing L-cysteine or D-cysteine and Zn²⁺ (Table III). The PC synthase activation was significantly higher also in the assay mixtures containing -mercaptoethanol and Cd²⁺ than in the assays containing -mercaptoethanol and Zn²⁺.

View this table: [in this window] [in a new window]   Table III Role of thiols on the activation of AtPCS1 The results are presented as relative activation and are compared to the compound that has the strongest effect on PC synthase activation, i.e. DTT (100% relative activation represents transfer of 6.7 µmol of -SCH3 groups ml1 for Cd2+, and 7.1 µmol of -SCH3 groups ml1 for Zn2+). GSH could not be tested under these conditions because it acts potentially both as an activator and a substrate. All investigated compounds were used in 10 mM concentration except NaBH4, which was applied in the following concentrations: 0.1, 1, 5, and 10 mg/ml. 0.1 µg of AtPCS1 was incubated in the 125-µl assay mixtures containing 200 mM Tris-HCl, pH 8.0, 10 mM S-methyl-GSH, 0.5 mM CdCl2, or 0.5 mM ZnCl2, and the investigated compound.

The effect of thiol concentrations on the PC synthase activation was studied in the next experiment. S-Methyl-GSH was used as a substrate in the assay mixtures where Cd²⁺ concentration was held constant (0.5 mM) and concentrations of DTT, -mercaptoethanol, L-cysteine, and D-cysteine varied from 0 to 20 mM. The transfer of -SCH₃ groups to form S-methyl-PCs depended on thiol concentrations in the PC synthase assays (Fig. 7). Furthermore, significant differences could be observed when the effects of individual thiols were compared at low millimolar concentrations. For example, although high PC synthase activity was observed in an assay mixture containing 0.5 mM Cd²⁺ and 2 mM DTT (which contains two -SH groups per mole), the enzyme was not active in assay mixtures containing 0.5 mM Cd²⁺ and either 2 mM L-cysteine or 2 mM D-cysteine.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Dose dependence of enzymatic sulfur-group transfer as a function of thiol concentration used in the PC synthase assay. DTT (), -mercaptoethanol (), L-cysteine (), and D-cysteine () were incubated in 125-µl PC synthase assay mixtures containing 200 mM Tris-HCl, pH 8.0, 10 mM S-methyl-GSH, 0.5 mM CdCl2, and 0.1 µg of AtPCS1.

The "thiol saturation" of PC synthase occurred at 5 mM DTT, 10 mM -mercaptoethanol, and 20 mM L-cysteine or D-cysteine in the assay (Fig. 7). An equal transfer of -SCH₃ groups (330 nmol µg¹ enzyme min¹) occurred at 1.7 mM DTT, 7.2 mM -mercaptoethanol, 9.7 mM L-cysteine, or 10.2 mM D-cysteine in the assay.

S-Methyl-PCs were the only reaction products of the experiments where the PC synthase activation by various metal·thiolate complexes was studied. The absence of any other reaction products could be confirmed by both HPLC and MS analysis. The metal·thiolate complexes that contributed to enzyme activation were obviously not used as substrates for PC synthase but rather to activate the enzyme. A recent model for the mechanism of PC synthesis (19), suggesting that the compound that activates PC synthase such as the metal·GS₂ complex is also the substrate for the enzyme is, in our opinion, not tenable.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report herein a comparison of the molecular characteristics of the recombinant PC synthase from A. thaliana and the recombinant hPC synthase from G. max. Significant differences in PC-synthesizing and hPC-synthesizing activities of recombinant AtPCS1 and GmhPCS1 were observed. While the PC synthase activity of GmhPCS1 was 1210 pkat/µg of enzyme, the hPC synthase activity of this enzyme was only 2.2 pkat/µg of enzyme when GSH was replaced by hGSH in the reaction mixture. hPC synthase activity of AtPCS1 was not detectable under such experimental conditions. However, the addition of GSH to hPC synthase assay containing hGSH and Cd²⁺ ions resulted in an increase in both GmhPCS1 and AtPCS1 hPC synthase activity. When GSH was replaced by S-methyl-GSH in hPC synthase assay, the formation of a pentapeptide -Glu-Cys(SCH₃)--Glu-Cys--Ala was unequivocally determined. The incorporation of -Glu-Cys(SCH₃) and -Glu-Cys--Ala (hGSH) into this peptide indicated that both GSH and hGSH are the substrates for the synthesis of hPCs. The mechanism that we propose for the biosynthesis of hPC₂ is thus,

An effective synthesis of hPCs in plants would, therefore, depend on the availability of GSH. However, in several legumes only hGSH, not GSH, was found (8, 15-17). The biosynthesis of hPCs and the heavy metal detoxification itself would be severely hindered in these plants because of the unavailability of the co-substrate GSH. Two hypotheses could explain metal detoxification in these legumes. 1) Because PCs and hPCs are synthesized in response to physiologically essential elements such as zinc and copper (22), GSH might be synthesized in very low amounts and instantly be used for the formation of hPCs involved in metal homeostasis. The presence of trace amounts of GSH in these legumes would be difficult to demonstrate. The availability of GSH could be the limiting factor for synthesis of hPCs and would consequently also determine the metal tolerance of individual legumes. It was recently reported that a cell suspension culture of a legume plant Vigna angularis is "hypersensitive" toward cadmium ions when compared with a suspension cell culture of tomato (25). Although it could be shown that V. angularis synthesized hPCs upon the exposure to Cd²⁺, GSH could not be detected in this plant by standard analytical techniques (15). This observation suggests that GSH might indeed be the limiting compound for efficient heavy metal tolerance in that system. 2) The hPC-synthesizing activity of GmhPCS1 when only hGSH is available suggests that selected legumes might also be able to synthesize hPCs in the absence of GSH. Although the hPC-synthesizing activity was very low, it might be sufficient for the heavy metal detoxification in vivo.

Based on these and former results, several conclusions can be made about the synthesis of homo-phytochelatins. It was previously suggested that GSH and PCs are donor compounds that contribute -Glu-Cys units during the synthesis of phytochelatins (10). The results presented herein suggest that GSH is a donor compound also during synthesis of hPCs. In the synthesis of PCs, the compounds that can accept -Glu-Cys units were shown to be GSH, PCs, des-Gly-PCs, -Glu-Cys, and water.² By showing that AtPCS1 effectively synthesizes hPCs when both GSH and hGSH are present, it was demonstrated that hGSH is another compound that can be bound at the PC synthase acceptor domain. It can be concluded that the presence of the substrate (GSH and its isoforms) and not the specificity of the enzyme determines the nature of PCs synthesized in any given plant.

We confirmed that S-methyl-GSH activates PC synthase to a very limited extent. Cd²⁺ ions present in the reaction mixture could completely inhibit this activation. The presence of thiols in the metal-containing PC synthase reaction mixture was recognized as decisive for PC synthase activation. In the absence of thiols, free metal ions cannot activate PC synthase, even if the substrate S-methyl-GSH is present. Under experimental conditions containing heavy metal ions and thiols, metal·thiol interaction and the formation of metal·thiolate complexes is essential (26-28).

The recognition of metal·thiolate complexes as essential to PC synthase activation raises doubts about recently published results (19) that suggest that PC synthase is activated by Mg²⁺. Applying Pearson's hard-soft acid-base concept, Mg²⁺ is a typical hard metal that does not show preference for soft ligands such as -SH groups and, thereby, would not be able to form a thiolate complex (29). Although metal·thiolate complexes appear critical to PC synthase activation, the precise mechanism of enzyme activation by free heavy metal ions or metal·thiolate complexes has not yet been elucidated.

The efficiency of metal activation of PC synthase strongly depends on the characteristics and concentration of thiols and metals used in the assay. For example, equivalent PC synthase-catalyzed transfer of -SCH₃ groups could be observed in assay mixtures containing S-methyl-GSH, DTT, and either Zn²⁺ or Cd²⁺. When DTT was replaced, however, with L-cysteine or D-cysteine, very little PC synthase activation could be observed in an assay containing Zn²⁺, although a significantly higher activation was observed in an assay containing Cd²⁺. The conclusion that the characteristics of metal·thiolate complexes are important for the enzyme activation may partially explain the high in vivo induction of accumulation of PCs by Cd²⁺ but not by Zn²⁺. Although Zn²⁺ was clearly recognized as a strong activator of PC synthase in vitro, in vivo induction of PCs by Zn²⁺ can be significantly lower.

Our results further contrast a recently proposed mechanism for PC synthase (19). We tested this proposed mechanism using S-methyl-GSH as substrate in our experiments. Effective PC synthesis resulting in S-methyl-PC formation could only be observed in the presence of metal ion and thiol. In addition, regardless of which metal·thiolate was used to activate the enzyme, only S-methyl-PC was formed, suggesting that the thiol that contributes to enzyme activation is not necessarily a substrate for PC synthesis. The previous claim that Cd·GS₂ is a substrate for PC synthase resulted from the observation that PC synthase catalyzed the synthesis of S-methyl-PCs from S-methyl-GSH even in the absence of heavy metal ions in the PC synthase assay (19). However, the results of our work presented herein show that the metal-free activation of PC synthase by S-methyl-GSH is a buffer-dependent observation and is not true for all reaction conditions.

We conclude, therefore, that PCs are synthesized in a reaction as proposed previously by our group (10),


Our results suggest that the direct products of the PC synthase-catalyzed reaction are apoPCs. The phytochelatin synthase-catalyzed reaction is terminated immediately after the metal ions are chelated by the apoPCs. The increased stability, of complexes containing chelating ligands such as phytochelatins over those containing comparable monodentate ligands such as cysteine or GSH, is well known (30), and the chelation of metal ions by apoPCs is suggested herein to be the final reaction step. The activity of phytochelatin synthase is, therefore, ultimately regulated by its reaction products the PCs.

	ACKNOWLEDGEMENT

We thank Dr. Klaus Raith from Martin-Luther-Universität Halle for performing MS and MS/MS analysis.

	FOOTNOTES

^* This work was supported by grant Sonderforschungsbereich 369 from the Deutsche Forschungsgemeinschaft, Bonn, and Fonds der Chemischen Industrie, Frankfurt.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF411075.

^¶ To whom correspondence should be addressed. Tel.: 49-345-5582-1200; Fax: 49-345-5582-1209; E-mail: kutch@ipb-halle.de.

Published, JBC Papers in Press, November 12, 2001, DOI 10.1074/jbc.M108254200

² A. Hochberger and M. H. Zenk, unpublished.

	ABBREVIATIONS

The abbreviations used are: PC, phytochelatin; PC_n, phytochelatin of chain length n; hPC, homo-phytochelatin of chain length n; GSH, glutathione; hGSH, homoglutathione, AtPCS1, A. thaliana phytochelatin synthase; GmhPCS1, G. max homo-phytochelatin synthase; BTP, 1,3-bis(tris-[hydroxymethyl]-methylaminopropane); Cd·GS₂, bis(glutathionato)cadmium; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); IPTG, isopropyl-1-thio--D-galactopyranoside; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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