Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific gamma-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase)

Overview

Grill and colleagues at Universität München (Lehrstuhl für Pharmazeutische Biologie and Genzentrum) report the isolation and biochemical characterisation of phytochelatin synthase from cell suspension cultures of the campion Silene cucubalus, the enzyme responsible for assembling the heavy-metal-binding peptides known as phytochelatins ([Glu(-Cys)]n-Gly oligomers, n = 2 to 11) from glutathione in higher plants. The enzyme is a gamma-glutamylcysteine dipeptidyl transpeptidase that polymerises gamma-Glu-Cys units onto a glutathione or phytochelatin acceptor, requires divalent or monovalent heavy-metal cations for activity (Cd2+ is the most effective activator, followed by Ag+, Bi3+, Pb2+, Zn2+, Cu2+, Hg2+, and Au+), and exhibits self-regulating product inhibition because the synthesised phytochelatin chelates the activating metal and removes it from the enzyme. The paper is the foundational primary citation for phytochelatin biosynthesis in the plant heavy-metal-detoxification literature; in the wiki its role is mechanistic background for metals/cadmium and any future page on plant-based mitigation or remediation of cadmium exposure, not occurrence data for food or supply-chain pages.

Why this matters

• It is the primary identification and characterisation of the enzyme (phytochelatin synthase) that builds the principal Cd-detoxifying peptide in plants. Every subsequent review of plant heavy-metal homeostasis (including the contemporary peer-reviewed reviews in this same source-retrieval batch) anchors its mechanism narrative on this 1989 PNAS paper.
• It establishes the molar stoichiometry between the synthesised phytochelatin and the activating Cd2+ ion (a 2:1 ratio of gamma-Glu-Cys dipeptide to Cd2+ in the newly formed complex), which sets the upper-bound expectation for how much cadmium a plant can sequester per unit of phytochelatin biosynthesis. This is the quantitative anchor downstream papers cite when discussing the capacity limits of plant cadmium (Cd) tolerance.
• It documents that the enzyme is constitutively present in plant cell cultures and not noticeably induced by heavy-metal exposure, locating the regulatory point in the activity of an existing enzyme pool (via heavy-metal activation) rather than in transcriptional induction. This contrasts the plant strategy with the animal strategy (metallothionein induction) and is the framing that subsequent comparative-mechanism reviews repeat.
• It establishes that the enzyme is taxonomically widespread among higher plants: phytochelatin synthase activity was detected in 5 of 21 cell-culture extracts tested, including representatives of Caryophyllaceae (Silene cucubalus), Berberidaceae (Podophyllum peltatum), Papaveraceae (Eschscholtzia californica), Chenopodiaceae (Beta vulgaris, i.e., chard/beet/spinach lineage), and the more primitive pteridophyte family Equisetaceae (Equisetum giganteum). The 21-extracts-tested figure includes plants where the enzyme could not be detected only because glutathione-degrading activities in the crude extracts masked it, not because the enzyme is absent.

Key concepts and structure

The paper is short (5 pages, including figures and references) and is organised into an abstract, a brief introduction reviewing the prior identification of phytochelatins as [Glu(-Cys)]n-Gly peptides (n = 2 to 11) in plants, a Materials and Methods section detailing the seven-step enzyme purification, a Results section with five subsections (characterisation of phytochelatin synthase, properties of the enzyme, substrate specificity, metal specificity, and kinetic analysis of the transpeptidase reaction), and a discussion that develops the dipeptidyl-transpeptidase mechanism and proposes the self-regulating loop.

Enzyme purification and physical properties

The enzyme was purified from 1 kg of Silene cucubalus cells (frozen 8 days after suspension transfer) by a multi-column procedure: cheesecloth filtration, 15 % (wt/vol) ammonium sulfate precipitation, Phenyl Sepharose CL-4B chromatography (10 mM 2-mercaptoethanol + 10 % vol/vol ethylene glycol), hydroxylapatite chromatography, AcA 34-Ultrogel chromatography (with 10 % glycerol throughout to stabilise the enzyme), QAE Fast Flow ion exchange (150-400 mM KCl gradient), Phenyl Superose hydrophobic-interaction at 15 % ammonium sulfate saturation (15-0 % gradient elution), Zn-chelating Sepharose (50 mM imidazole + 25 mM potassium phosphate, pH 8.0), Sephadex G-25 desalting (PD-10), and a final Mono Q polish chromatography (0.2-0.4 M KCl gradient). The purified enzyme ran as a single silver-stained band on SDS/PAGE and had a specific activity of 463 pkat/mg protein, representing approximately 160-fold enrichment over the crude extract (Materials and Methods; Results, Characterisation of Phytochelatin Synthase). The molecular weight estimate from gel-permeation HPLC (TSK G 3000 SW column, calibrated with catalase 240 kDa, BSA 67 kDa, ovalbumin 45 kDa, chymotrypsinogen A 25 kDa, myoglobin 17.8 kDa, cytochrome c 12.3 kDa) yielded active species at Mr 95,000 and Mr 50,000, while the denaturing SDS/PAGE molecular weight was 25,000. The authors infer that the holoenzyme is an oligomer of four 25-kDa subunits whose tetramer dissociates to an active dimer during purification (Results, Characterisation of Phytochelatin Synthase, Fig. 1A).

The enzyme was stored in 10 mM Tris·HCl, pH 8.0, 10 mM 2-mercaptoethanol, 0.01 % NaN3 to prevent microbial growth. Half-lives at storage temperature were 6 hours at 35°C, 34 hours at 22°C, and 140 hours at 4°C; only a small decrease in activity was observed after 500 hours in 30 % glycerol. Under standard incubation conditions (11 pkat enzyme, 1 mM glutathione, 0.1 mM Cd(NO3)2), phytochelatin formation was linear for 120 minutes. The enzyme is functional over a relatively narrow pH range with maximal activity at pH 7.9 (Fig. 1B) and a broad temperature range with maximal conversion rate at 35°C. Half-maximal activity was observed at 20°C and at 47°C. The isoelectric point of the main enzyme activity is approximately pH 4.8, with minor activities (less than 20 % of the main peak) at pH 4.1 and pH 6.5, possibly due to multiple forms of the enzyme.

Kinetic constants (Results, Properties of the Enzyme)

• Km for glutathione (with 0.1 mM Cd2+ present): 6.7 mM (Fig. 1C).
• Km for S-monobromobimane-glutathione (MBB-GSH, a sulfhydryl-protected substrate): 1.5 mM (Fig. 1D).
• Minimum kcat (calculated using catalytically inactive enzyme preparations): 0.2 s^-1.

The fact that MBB-GSH (a thiol-blocked glutathione derivative) has a lower Km than glutathione itself is the kinetic argument the authors use to rule out a glutathione-S-Cd complex as the actual substrate: if the free thiol were required, blocking it should raise Km, not lower it.

Substrate specificity (Results, Substrate Specificity)

Incubation of the purified enzyme with [Glu(-Cys)]n-Gly oligomers (n = 2 to 4 or 5) plus glutathione consistently produced the next higher homologue (n+1) by HPLC analysis with sulfhydryl-specific detection. This established that the enzyme is a phytochelatin-chain-elongating dipeptidyl transpeptidase that acts on phytochelatins of various chain lengths by adding one gamma-Glu-Cys unit and concomitantly liberating one glycine residue. The reaction is summarised as:

[Glu(-Cys)]n-Gly + [Glu(-Cys)]n-Gly → [Glu(-Cys)]n+1-Gly + [Glu(-Cys)]n-1-Gly

where n = 1, 2, 3,… The reaction therefore proceeds by stepwise dipeptidyl transfer rather than en-bloc transfer of an extended peptide block.

Metal specificity (Results, Metal Specificity; Table 1)

Activation of the purified enzyme by single metal salts was tested under the standard HPLC assay. The activity values reported in Table 1 (relative to 0.1 mM Cd(NO3)2 = 100, where 100 % corresponds to 4.1 nmol phytochelatin formed in 70 min):

No enzyme activation was detected with Al3+, Ca2+, Fe2+, Mg2+, Mn2+, Na+, or K+, distinguishing heavy-metal activation from a general divalent-cation effect. The activation rank order for the heavy metals tested (Cd2+ > Ag+ > Bi3+ > Pb2+ > Zn2+ > Cu2+ > Hg2+ > Au+) matches the set of metal ions previously known to induce phytochelatin synthesis in intact plant cells.

Kinetic time-course and stoichiometry (Results, Kinetic Analysis of the Transpeptidase Reaction; Figs. 2 and 3)

In a 15-fold purified enzyme preparation supplemented with 0.1 mM Cd2+ ions and 1 mM glutathione, phytochelatin synthesis began immediately after Cd2+ administration with no detectable lag period. The dipeptide [Glu(-Cys)]2-Gly appeared without a noticeable lag; the heptapeptide [Glu(-Cys)]3-Gly appeared after about 15 minutes, and the nonapeptide [Glu(-Cys)]4-Gly after about 1 further hour. After 100 minutes the reaction reached a halt because the administered Cd2+ had been completely complexed by the synthesised peptides and was no longer available to the enzyme; a second addition of Cd2+ at the same level restarted phytochelatin synthesis. Computation of these results yielded 23.7 nmol of gamma-Glu-Cys units in phytochelatin from an input of 10 nmol of Cd2+ in the first experiment (a 2.37:1 ratio of gamma-Glu-Cys to Cd, approximating the 2:1 dipeptide-to-Cd target). A second experiment, computed similarly, yielded 19.8 nmol of dipeptide from 10 nmol of Cd2+. The authors state that if all Cd2+ in the system were chelated by the synthesised peptides, the molar ratio of Cd to cysteine in the phytochelatin complex is 1:2 — exactly the ratio determined for purified Cd-phytochelatin complexes isolated from exposed plants in earlier work. Chelation of Cd2+ by EDTA, or pre-saturation of phytochelatin with Cd, instantaneously inhibited phytochelatin formation (data not shown), supporting the self-regulating loop in which the product chelates the activator.

In the absence of glutathione (Fig. 3B), the heptapeptide and later the nonapeptide still formed but at the expense of the pentapeptide [Glu(-Cys)]2-Gly substrate, consistent with an autotranspeptidase reaction in which phytochelatin itself serves as both gamma-Glu-Cys donor and acceptor. After prolonged exposure, non-specific oxidation of thiol groups occurred and pushed the equilibrium back toward shorter chains.

Tissue and taxonomic distribution (Results, Occurrence of Phytochelatin Synthase in Plants; Table 2)

In 5 of 21 plant cell culture extracts (no phytochelatin synthase activity could be detected in the other 16, although the authors attribute the failures in most cases to the presence of glutathione-degrading activities in the crude extracts rather than to true absence of the enzyme), phytochelatin synthase activity could be identified. Activity values, in pkat per litre of culture medium, at the indicated cell-culture age:

In all 5 control incubations without glutathione or Cd2+, no phytochelatin formation was observed. The taxonomic span — from the basal pteridophyte Equisetum through dicotyledon families of differing affinities — supports the authors’ inference that phytochelatin biosynthesis is a generally distributed mechanism in vascular plants, not a Silene-specific adaptation.

Methods (brief)

The paper reports primary enzymological work. Plant cell suspension cultures of Silene cucubalus were cultivated by the previously published method (ref 4). Substrates: [glycine-2-3H]glutathione from NEN; all other materials of highest purity from Sigma, Merck, and Boehringer Mannheim; S-monobromobimane-glutathione synthesised as described (ref 8). Enzyme assay: 200 mM Tris·HCl pH 8.0, 10 mM 2-mercaptoethanol, 0.1 mM Cd(NO3)2, 1 mM glutathione, plus enzyme, total volume 100 µL, incubated at 35°C for the specified time, stopped by addition of NaOH/NaBH4, acidified with HCl, supernatant separated and analysed by HPLC. Enzyme isolation as summarised under Enzyme Isolation in the source and Section “Enzyme purification and physical properties” above. Molecular weight by HPLC gel permeation on TSK G 3000 SW (Pharmacia-LKB) calibrated against six standard proteins. Phytochelatin isolation and sequence determination per refs 2 and 4. Activity is monitored either by HPLC (for the Glu-Cys-monitoring assays in panels A-C of Fig. 1) or by measuring liberated labelled glycine (panel D of Fig. 1).

The paper does not declare a randomisation scheme, a blinding protocol, or a formal pre-registration; this is in line with the 1989 standards of plant-biochemistry literature and is mentioned here only as a methodological-context note rather than a defect. The authors thank Ms Chr. Schweiger and Ms K. Gerle for technical assistance and Dr T. M. Kutchan for linguistic help with manuscript preparation. Funding: Bundesminister für Forschung und Technologie (Bonn; Grant BCT 372-8) and Fonds der Chemischen Industrie. Communicated by H. A. Barker on 5 May 1989; received for review on 5 February 1989.

Implications

• Microbiome: Out of scope. The paper does not engage gut microbiome or environmental microbiome contexts. WikiBiome federation does not apply.

Limitations

The paper is a primary biochemistry study; its scope is the enzyme and its substrate, not the human-exposure or food-contamination question that the wiki ultimately serves. The cell-culture system (Silene cucubalus suspension cells grown in liquid medium) does not directly model field-grown crops or edible plant tissues; subsequent literature has had to extend the Silene characterisation to species and tissues that humans actually consume. The Km for glutathione (6.7 mM) is high compared to typical cytosolic glutathione concentrations in unstressed plant cells (millimolar range), which the authors do not directly address; this raises a kinetic question about how rapidly phytochelatin can be produced under physiological substrate availability that the paper does not resolve. The 21-extracts-screen is a positive-result list (the 5 detections) presented as evidence for ubiquity, but the 16 negatives are explained away as masked by glutathione-degrading activities rather than reported as true negatives, which limits the strength of the ubiquity claim from this paper alone — later studies that cleanly resolved the masking issue established broader taxonomic coverage. The activation table (Table 1) tests metal ions in isolation rather than in physiologically realistic mixtures, so the rank order should not be projected onto field-relevant multi-metal exposures.

Related evidence

• Cadmium
• Lead
• Mercury
• Remediation evidence — drivers and interventions

Update history

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