Grill et al. 1989 — Phytochelatin synthase characterised from Silene cucubalus

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 Kimi 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 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

• Certification: The paper contributes no occurrence data, no exposure data, and no regulatory-threshold information. It does not move any HMTc threshold-setting work. Its value for the certification programme is indirect — it is a foundational reference for the eventual mitigation chapter on plant heavy-metal detoxification mechanisms and for any narrative on how Cd is sequestered by plants before it enters human food chains.
• App: No routing to ingredient or product pages. This source contributes mechanistic background for metals/cadmium and (to a lesser extent) metals/lead and metals/mercury on the topic of plant-internal heavy-metal binding by phytochelatins. It does not contribute contamination occurrence values.
• Courses: Useful as a citation for an educator-audience module on plant heavy-metal homeostasis, particularly the distinction between the plant strategy (constitutive enzyme activated by metal binding to produce a metal-binding peptide) and the animal strategy (transcriptional induction of the metallothionein gene in response to metal exposure). Also useful for the historical-context introduction of phytochelatin synthase as the EC-numbered enzyme (the EC number 2.3.2.15 was assigned later than this paper, which characterises but does not name the EC entry).
• 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.

Wiki pages this source may touch

• cadmium
• lead
• mercury
• remediation-evidence

Verification notes

Existing-page check. DOI grep (10.1073/pnas.86.18.6838), raw_handle grep (MFK_15-phytochelatins-the-heavy-metal-binding-peptides), and cite-key/title glob over wiki/sources/ on 2026-06-08 returned no matches. Existing phytochelatin-themed pages (marques2025, seregin2023, luo2024) do not cite this same DOI or duplicate this paper. This is a NEW source page — no prior version to merge-enhance.

Evidence tier. A. This is a primary peer-reviewed biochemistry study in PNAS reporting original enzymology with explicit Methods, kinetic constants, and a quantitative metal-activation table. The “narrative review” disqualifier that limits later peptide reviews to B-tier does not apply here.

Metals frontmatter. The paper studies Cd2+ as the principal activator and reports a quantitative activation table covering Cd, Ag, Bi, Pb, Zn, Cu, Hg, Au. From the HMTc 10-analyte priority list (Pb, tAs, Cd, MeHg, tHg, iAs, Ni, Al, Cr-VI, Sn), the in-scope metals named in the paper are Cd, Pb, and tHg. tHg is used (not MeHg) because the paper uses HgCl2 as the test salt, which is an inorganic mercury species, and reports it under the generic Hg/mercury label without speciation; the wiki convention defaults the unspecified mercury reading to tHg. Ag, Bi, Zn, Cu, and Au are out-of-scope for the HMTc 10-analyte vocabulary and are not added to frontmatter; the activation data for these metals is preserved in the body Table 1.

Ingredients, products, matrices, jurisdictions frontmatter. All empty. The source measures nothing in any food, beverage, personal-care, or environmental matrix; it studies enzyme kinetics in a cell-culture-derived extract. No jurisdiction applies — the laboratory is in Munich, Federal Republic of Germany, but the work is a generic biochemistry study with no jurisdiction-specific occurrence frame.

Sample size. Null. The paper reports enzymology, not a sampling study. The “21 cell-culture extracts” figure for the taxonomic survey is a screening pool, not a sample of a defined population.

Brand firewall (Part 12). Vendor names appear in Methods (NEN, Sigma, Merck, Boehringer Mannheim for reagents; Pharmacia and Pharmacia-LKB for chromatography media and the molecular-weight HPLC column; Bio-Rad for the hydroxylapatite column; Serva for the AcA 34-Ultrogel). These are scientific-method vendor names, which Exception 2 of the brand firewall (locked 2026-05-17) explicitly preserves for reproducibility. No food or personal-care brand is named.

HMTc firewall (Part 2). The paper makes no HMTc-threshold claim and no consumer-audience risk advisory. No firewall action required.

Source typographic / citation errors noted. None observed in the regions read. The reference list is short (17 entries) and consistent with the 1989 PNAS style.

Date arithmetic. Received for review 5 February 1989, communicated 5 May 1989, published September 1989 in PNAS Vol. 86 pp. 6838-6842; all consistent with the year frontmatter (1989).

License/access. PNAS pre-2008 papers are not CC-licensed but are freely accessible through the PNAS archive after the journal’s standard open-access window. The license field records the access mode rather than a Creative Commons identifier. The DOI 10.1073/pnas.86.18.6838 resolves; no paywall on the publisher landing page.

Reviewer’s note on scope fit. This paper is in scope per the 2026-06-02 commit 3f47f95 — scope: mitigation/remediation is in-scope, not a skip. The “Black Market Peptide Metal Survey” folder context is Karen’s collection of peptide-metal-binding literature; this is the foundational enzymology paper on plant phytochelatin biosynthesis, which sits squarely inside that frame. It does not address contamination of peptide products or any food/supply-chain occurrence question, and that mismatch is documented above under Implications.

Audit subagent (2026-06-08) verdict: PROMOTE. Five checks (numerical fidelity, slug vocabulary, speciation/methods, brand firewall, HMTc firewall) all returned ✅. Two ⚠️ informational findings were verified and applied: (1) the original draft characterised the enzyme purification as a “seven-step procedure” but the PDF (p. 6839) explicitly lists Sephadex G-25 (PD-10) desalting followed by a final Mono Q polish chromatography after the Zn-chelating Sepharose step; the wording has been revised to “multi-column procedure” and the Sephadex G-25 and Mono Q steps have been added to the column sequence. (2) The auditor flagged that [[mitigation/remediation-evidence]] is outside the four-category taxonomy snapshot (ingredients / products / metals / regulations) used by the audit-prompt checks; this is a known limitation of the snapshot, and mitigation/ is a sanctioned wiki area per the 2026-06-02 commit 3f47f95 and CLAUDE.md Part 24. The wikilink is retained. No content corrections beyond the purification-sequence refinement were applied. Numerical fidelity checks on author names (Löffler with correct umlaut), Mr values, kinetic constants (Km glutathione 6.7 mM, Km MBB-GSH 1.5 mM, kcat minimum 0.2 s^-1), Table 1 activations (Cd 100, Ag 58, Bi 56, Pb 43, Zn 33, Cu 27, Hg 26, Au 12; concentrations 0.1/0.1/0.5/0.1/0.1/0.01/0.1/0.1 mM), Table 2 species/family/age/activity (Silene cucubalus 7d/637, Podophyllum peltatum 4d/365, Eschscholtzia californica 6d/145, Beta vulgaris 5d/100, Equisetum giganteum 10d/34 pkat/L), half-lives (6h/34h/140h at 35/22/4 °C), temperature half-max points (20°C and 47°C), NaN3 storage concentration (0.01 %), and 2:1 dipeptide:Cd stoichiometry all matched the PDF without correction.

Page history

The five most recent substantive edits to this page. The full version history lives in git; when DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.