Phytochelatin synthase (PCS) is well-known for its role in heavy metal detoxification in plants, yeasts and non-vertebrate animals. It is a protease-like enzyme that catalyzes glutathione (GSH) to form phytochelatins (PCs), a group of Cys-rich and non-translational polypeptides with a high affinity to heavy metals. In addition, PCS also functions in xenobiotic metabolism by processing GS-conjugates in the cytosol. Because PCS is involved in GSH metabolism and the degradation of GS-conjugates, it is one of the important components in GSH homeostasis and GSH-mediated biodegradation. This chapter reviews the biochemical mechanism of PCS, how the enzyme activity is regulated, and its roles in heavy metal detoxification as well as GS-S-conjugate metabolism. This chapter also highlights the potential applications of PCS in the improvement of plant performance under combined stresses.
- Phytochelatin synthase
- heavy metal stress
- GS-conjugate metabolism
- combined pollution
Phytochelatins (PCs, (γGlu-Cys)n-Gly, n = 2–11) are cysteine-rich polypeptides that are synthesized non-translationally from the tripeptide glutathione (GSH, γGlu-Cys-Gly); this process is catalyzed by phytochelatin synthase (PCS, EC 220.127.116.11) [1, 2, 3, 4]. PCs play essential roles in heavy metal detoxification because of their high affinities to a broad range of metal ions, e.g. cadmium (Cd), mercury (Hg), arsenic (As), zinc (Zn), lead (Pb), silver (Ag), nickel (Ni) and copper (Cu) [1, 2, 3]. Upon exposure to heavy metals, PCs are synthesized in the cytosol to chelate free metal ion and to prevent the generation of hydroxyl radicals [4, 5, 6] (Figure 1). These PC-metal complexes eventually are transferred into the vacuole through specific tonoplast ABCC-type transporters for sequestration [22, 23, 24, 25, 26] (Figure 1). In plants, PCS is constitutively expressed in the cytosol and can be activated by multiple types of metal ions [1, 3, 6]. For example, AtPCS1 isolated from
PCS can be found in plants, yeasts and non-vertebrate animals and plays a critical role in responding to heavy metal stress in these organisms [28, 29, 30, 31, 32]. It was first partially purified from the suspension cells of bladder campion (
PCS is a key component for the heavy metal tolerance in plants. Its importance was first confirmed in the Arabidopsis mutants locking AtPCS1 activity, as these mutants show severe growth defects when challenged by heavy metals such as Cd2+, Hg2+, Zn2+, Pb2+, and As3+ [28, 43, 44, 45]. The synthesis of PCs is crucial to the local response to heavy metal stress and is also involved in the roots-to-shoots translocation of heavy metals [26, 37, 43, 46]. The first evidence of the long-distance transfer of PC-metal complexes is that the PCs synthesized in the roots can be translocated to the shoots via phloem loading and
PCS is a well-known multitasker involved in different biological processes [21, 47]. Besides its significant role in synthesizing PCs from GSH, PCS can catalyze the deglycination of GSH-
2. The biochemical mechanism of phytochelatin synthase
2.1 The domain organization of phytochelatin synthase
The eukaryotic PCS has two domains with distinct functions: a conserved N-terminal domain that shows γ-glutamylcysteine dipeptidyl transpeptidase activity and a variable C-terminal domain involved in metal sensing [52, 53, 54]. Using AtPCS1 as a model, the molecular functions of the N- and C-domains as well as the catalytic mechanism of eukaryotic PCS were revealed [6, 53, 54, 55]. The N-terminal half AtPCS1 is sufficient for deglycination of GSH and elongating PC molecules, indicating that the N-terminal domain carries out the core catalysis [53, 56]. However, the truncated AtPCS1 without the C-terminal domain is less thermostable and has lower PC synthetic activity than the full-length enzyme [53, 55, 56]. Notably, the deletion of the C-terminal domain completely impairs the PC synthesis activity of the enzyme in the presence of Zn2+ and partially inactivates PC synthesis in the Cd- or Hg-containing reactions [53, 55]. These findings suggest that the C-terminal domain is essential for stabilizing the protein and functions as a metal sensor [53, 55]. More evidence has shown that the C-terminal end of AtPCS1 is required for the augmentation of PC synthetic activity. One example is that the residues from Asp373 to the C-terminal end of AtPCS1 contain multiple regions involved in Zn-dependent and As-dependent activation of PC synthesis [45, 57, 58]. (Also see Section 2.4: The activation of phytochelatin synthase through the chelation of heavy metal ions).
PCS-like sequences also exist in prokaryotes with moderate sequence homology to the N-domain of eukaryotic PCS [39, 40, 41]. However, prokaryotic PCS likely has unique functions apart from PC synthesis. For example, the PCS homolog found in cyanobacterium
2.2 The core catalytic mechanism
Vatamaniuk et al.  first confirmed that the synthesis of PCs occurs through a ping-pong mechanism and involves two substrates: one GSH as the low-affinity substrate for the first step of PC synthesis and one metal-GSH conjugate as the high-affinity substrate for the second step . In the standard PC synthesis reactions
Right after GSH enters the catalytic site of PCS, a Gly residue is removed to form the γGlu-Cys acyl-enzyme intermediate, and then a metal-GS2 accepts the γGlu-Cys unit to generate a PC2 ((γGlu-Cys)2-Gly) [6, 21, 54, 55]. Following the synthesis of PC2, the elongation of PCs occurs using previously synthesized PCs as acceptors to receive γGlu-Cys [6, 21, 54, 55, 60]. The whole process can be described as two equations:
PCS and Cys proteases share similar core catalytic mechanisms to hydrolyze a GSH molecule and form a γ-Glu-Cys-acyl–enzyme intermediate [41, 52]. The Cys protease-like catalytic triad of PCS was confirmed based on the mutagenic studies of AtPCS1 and the crystal structures of NsPCS [41, 54, 55]. Vatamaniuk et al. and Romanyuk et al. reported that Cys56, His162, and Asp180 of AtPCS1 are the three residues of the catalytic triad among divergent PCS sequences [54, 55]. The molecular interaction between these residues and GSH was further revealed by Vivares et al. with the crystal structures of native NsPCS and the γ-Glu-Cys-acyl–enzyme intermediate at a 2.0-Å resolution . Although NsPCS only shares 36% identity at the amino acid level with AtPCS1, it contains the conserved catalytic triad and can catalyze the deglycination of GSH [39, 40, 41, 42]. These crystal structures provide details about the hydrolysis of the peptide bond that involves Cys56 and the 3D structure of the Cys-His-Asp catalytic triad . It is worth mentioning that NsPCS formed homodimers in the crystallization experiments . This is in agreement with the dimerization of the partially purified PCS from
2.3 Critical amino acids contributing to the enzyme activity
Based on the high-resolution crystal structure of NsPCS, multiple research groups have simulated putative 3D structures of eukaryotic PCS using various programs, and these structure models provide valuable information that uncovers the conserved molecular mechanism of PCS [56, 61, 62, 63, 64, 65]. For example, the molecular models of AtPCS1 reveal the key amino acids that contribute to the mechanism of the second substrate recognition and the enzyme activation through Thr phosphorylation [56, 61]. Chia et al. first reported how AtPCS1 might attract and stabilize the second substrate, metal-GS2, after the γ-Glu-Cys-acyl–enzyme intermediate is formed . In this study, the modeled AtPCS1 structure revealed a pocket in proximity to the first substrate-binding site, consisting of three loops containing several conserved amino acids, including Arg152, Lys185, and Tyr55. Mutations on Arg152 or Lys185 (Arg-to-Lys or Lys-to-Arg substitutions) resulted in the complete abrogation of enzyme activity, indicating that the arrangement of these positive charges is crucial for the binding of the second substrate. Mutations at Tyr55 did not completely impair the enzyme activity, but the Tyr55 mutant AtPCS1 showed lower catalytic activities than the wild-type enzyme due to a reduced affinity to metal-GS2. In addition, the mutation at Tyr55 reduced Cd2+ binding ability of the AtPCS1 protein. It was therefore suggested that Tyr55 binds to the Cd ion of metal-GS2 through cation-π interaction and thus contributes to the recognition of the second substrate. Besides these three amino acids, other conserved residues on the loops constituting the second substrate-binding pocket, including Gln50, Glu52, Glen157, Phe184, and Tyr186, are also important for the PC synthesis activity [61, 62].
Wang et al. identified that Thr49 is the phosphorylation site related to the activation of AtPCS1 . The mutant AtPCS1 with Thr49-to-Ala49 substitution could not be phosphorylated, and its PC synthesis activity was significantly lower than that of the wild-type enzyme. According to the proposed 3D model of AtPCS1, Thr49 is within proximity to Arg183, which is also crucial for the catalytic activity of AtPCS1, and both residues are next to the catalytic site and substrate binding pockets. It was proposed that the phosphorylated Thr49 interacts with Arg183, and that this interaction serves as a “molecular clip” to give the active site a conformation appropriate for catalysis. Because Thr49 and Arg183 are both highly conserved among PCS sequences, the activity of eukaryotic PCS may as well be regulated by similar phosphorylation modifications [56, 66].
2.4 The activation of phytochelatin synthase through the chelation of heavy metal ions
As a key component of early response to heavy metal stress, PCS protein is constitutively expressed in the cytosol for rapid activation stimulated by heavy metals [2, 3, 6]. The heavy metal ions entering cytosol are essential for forming the second substrate [6, 60]. They can also bind to PCS, resulting in augmentative activation [6, 55, 67, 68]. Moreover, heavy metals could be a critical factor that triggers PCS phosphorylation [56, 69]. For example, AtPCS1 phosphorylation only occurred in the presence of Cd2+ in the
PCS, confirmed to be a metalloenzyme
3. Phytochelatin synthase-targeting genetic engineering approaches in phytoremediation of heavy metals
3.1 The effects of phytochelatin synthase overexpression on the accumulation of heavy metals in plants
PC synthesis plays a critical role in heavy metal tolerance and accumulation. It is therefore no surprise that the breeding and engineering approaches for phytoremediation requiring heavy metal hyperaccumulators have focused on strategies to enhance PC biosynthetic capacity [48, 70, 71, 72, 73, 74, 75, 76]. Studies have shown that the transgenic plants expressing functional PCS usually have a higher tolerance to heavy metal stress. For example, the overexpression of AtPCS1 in Arabidopsis, tobacco or Indian mustard (
Although PCS can be a molecular tool for phytoremediation of heavy metal-contaminated soils and waters, the overexpression of PCS promotes the catabolism of GSH, which also plays essential roles in redox reactions and heavy metal stress [87, 88]. If the metabolic pathways supplying GSH cannot maintain specific levels in the presence of highly expressed PCS, the consumption of GSH usually leads to changes in the GSH/GSSG ratio that exacerbate oxidative stress [62, 72]. The increased GSH demand driven by PC synthesis may also affect other metabolic pathways requiring GSH . In these regards, the use of functional PCS with diminished catalytic activity could reduce the depletion of GSH, maintain redox homeostasis and supporting PC synthesis during exposure to heavy metals at the same time . Indeed, the Arabidopsis and
Besides the imbalance of cellular redox state, PCS overexpression could result in an unknown disruption in cellular metal homeostasis under heavy metal stress because PCS itself is a metalloenzyme and can bind a wide range of metal ions [90, 91]. Expressing synthetic genes encoding peptide analogs of PCs with a general structure of Met(Glu-Cys)nGly (n = 16–20) could be an alternative way to enhance the accumulation of heavy metals in the plants without the overexpression of PCS . The Arabidopsis transgenic plants transformed with the artificial genes encoding these PC-like polypeptides resulted in hyperaccumulation of Cd2+ and As3+/5+ in the plants . However, the impact of accumulating synthetic PC-like polypeptides on the overall metal homeostasis is yet to be determined.
3.2 Phytochelatin synthase-involving pathway engineering for enhancing heavy metal accumulation
Pathway engineering involved in the co-expression of both GSH synthesis and PC synthesis pathways is another strategy to preserve the balance of GSH metabolism in the cells with constitutive PC synthesis [72, 74]. A kinetic model of GSH and phytochelatin synthesis in plants suggests that at least two enzymes, γ-glutamylcysteine synthetase (γ-ECS) and PCS, should be increased to enhance PC synthesis without depleting the GSH pool . In fact, the effects of modified GSH/PC synthesis pathways have been tested in
Overall, the genetic engineering approaches involved in manipulating PC synthesis have shown promising prospects for improving the performance of plants in the phytoremediation of heavy metals. However, there are also setbacks pointing at the complexity of the stress response induced by heavy metals . Thus, while enhanced PC synthesis can contribute to the heavy metal chelating, other factors, such as the subsequent vacuolar sequestration or the delicate balance of GSH metabolic pathways under heavy metal stress, should be considered in order to achieve heavy metal tolerance.
4. The role of phytochelatin synthase in glutathione-
4.1 The involvement of phytochelatin synthase in the catabolism of glutathione derivates
PCS has a broad substrate selectivity and can use GS-derivates as substrates. For example, PCS isolated from plant species can accept
Besides its significant role in heavy metal detoxification, PCS also participates in the biodegradation of xenobiotic compounds because of its capability to process GS-conjugates [17, 18, 19, 20]. Glutathione conjugation is a major pathway to inactivate xenobiotic compounds in plant cells . Glutathione transferase (GST) detoxifies xenobiotics in the cytosol by transferring these compounds to GSH [10, 11, 96, 97]. These GS-conjugates enter vacuoles rapidly for sequestration and further degradation [12, 13]. In Arabidopsis, the transport of GS-conjugates for vacuolar sequestration is facilitated by AtABCC1/AtMRP1 and AtABCC2/AtMRP2, which also transfer PC-metal complexes into vacuoles [12, 13, 22, 24]. Because of the high efficiency of this sequestration mechanism, the subsequent catabolism of GS-conjugates is presumably processed in the vacuoles . First, vacuolar γ-glutamyl-transpeptidase (GGT) initializes the degradation of GS-conjugates by removing the γ-Glu-residue from the GS-conjugates to form Cys-Gly-conjugates [14, 15], and then, carboxypeptidase cleaves the Gly residue and results in the accumulation of the Cys-conjugates . Alternatively, the GS-conjugate degradation can be initiated by PCS when vacuolar sequestration is not an available route [17, 18, 19, 20]. The pathways of GS-conjugate metabolism are summarized in Figure 1.
4.2 Phytochelatin synthase may participate in initiating the first step of glutathione-
S-conjugates degradation in the cytosol
Monochlorobimane (MCB) is widely used as a model xenobiotic for Arabidopsis to study the catabolism of GS conjugates [14, 15, 17, 18, 19, 20, 98]. The bimane-labeled thiols can be analyzed by high performance liquid chromatography . In addition, the fluorescent GS-bimane can be directly monitored
The direct evidence showing the involvement of PCS in GS-conjugate metabolism is the defects in the turnover of GS-bimane shown in the Arabidopsis AtPCS1-deficient mutants [21, 47]. The AtPCS1-deficient mutant,
Although the GS-bimane conversion is altered in the AtPCS1-deficient mutants, the GS-bimane in these mutants still can be degraded through the major detoxification pathway in the vacuoles [18, 19, 20]. Besides, the overall turnover of GS-bimane in the mutants is only slightly affected without blocking the vacuolar transport pathway [18, 19, 20]. These findings underline that the vacuolar GGT-initiated GS-conjugates degradation is the major pathway among two compensatory routes responsible for the turnover of the xenobiotics .
In plant cells, both the cytosolic PCS and the vacuolar carboxypeptidase can catalyze the formation of γ-Glu-Cys-bimane [16, 17, 18, 19]. However, the vacuolar carboxypeptidase tends to catalyze the Cys-Gly-conjugates following the cleavage of γ-Glu-residue initiated by GGT . In this regard, PCS is supposed to be the primary component responsible for the γ-Glu-Cys-bimane formation observed in the process of GS-conjugate conversion. Another example showing the importance of PCS in the initiation of the cytosolic xenobiotic compound is the metabolism of the herbicide safener fenclorim . Fenclorim enhances GST activity in Arabidopsis and is subsequently degraded via the GS-conjugation pathway [97, 100]. In the Arabidopsis suspension cells, GS-fenclorim was sequentially processed to γ-Glu-Cys-fenclorim and Cys-fenclorim, suggesting that deglycination is the initial step to the catabolism of fenclorim [21, 100]. However, more evidence is needed to confirm the direct involvement of PCS in this process.
4.3 The glutathione-
S-conjugate conversion via phytochelatin synthase is metal-dependent
The presence of metal ions is a critical requirement for PCS-dependent catalysis of GS-bimane [17, 18, 19, 20]. Intriguingly, the efficiency of GS-bimane hydrolysis activated by different metal ions is separate from that of metal-stimulated PC synthesis . For example, the PC formation of AtPCS1 activated by Cd2+ is usually 2–5 times more efficient than the PC synthesis rate measured in the presence of Cu+/2+ [4, 6, 53]. On the other hand, AtPCS1 could catalyze the deglycination of GS-bimane 60% more efficiently in Cu+/2+ solutions than in the presence of Cd2+ . It was suggested that
5. Can phytochelatin synthase play a part in the phytoremediation of combined pollutions?
With global industrialization and the development of modern cropping systems, massive amounts of toxic substances such as pesticides, heavy metals and inorganic fertilizers have been released into the environment and caused massive pollutions [97, 102]. Inevitably, the combined contaminations have damaged the ecosystems and become global issues [103, 104].
In the case of soil pollution, the primary sources of heavy metals include pesticides, fertilizers, mining, industrial processing and wastewater [102, 105]. One example of combined pollutants is glyphosate-based herbicides, which are highly toxic to the environment yet are the most-used pesticides in the world . Heavy metals such as As, Ni, and Pb, which activate the catalytic activity of PCS, can be found as contaminants in many commercial glyphosate-based herbicides [3, 6, 106]. Interestingly, the
GSH and its derivates are widely involved in plant development and stress response, and GSH itself serves as a hub for the mechanisms of heavy metal detoxification, xenobiotics biodegradation and oxidative stress response [7, 9]. Because PCS is a key enzyme in GSH metabolism, it is not surprising that PCS should be involved in both heavy metal stress and the turnover of xenobiotics. Other critical enzymes in the GSH metabolic pathway such as GST have been used in combating multiple stresses, including heavy metal and xenobiotics degradation, and biotic stress . However, the role of PCS is primarily emphasized in heavy metal stress response despite its contribution to the degradation of GS-metabolites and innate immunity. Based on the knowledge about the diverse functions of PCS, it is worth exploring whether PCS can be a useful tool in enhancing the tolerance and performance of the plants challenged by combined stresses.
Both heavy metals and pesticides significantly arrest plant growth and development. The co-contamination of soils by both heavy metals and pesticides has raised concerns regarding crop safety and productivity, and is therefore crucial to remediate. Phytoremediation presents the advantages of high efficiency, low cost, and sustainability. Thus, it has been one of the most common strategies for the remediation of polluted soils. This chapter summarizes the critical role of PCS in heavy metal detoxification and the involvement of PCS in GS-conjugate degradation. In the presence of heavy metals, PCS catalyzes the synthesis of PCs and the initiation of GS-conjugate metabolism. Despite a large body of literature illustrating the function of PCS in heavy metal resistance, there has been less emphasis on the participation of PCS in the detoxification of xenobiotic compounds and its potential application in biodegradation. Given that PCS has diverse functions in different types of stress, this chapter discusses the potential inclusion of PCS to achieve phytoremediation for combined pollutions.
The key question related to PCS overexpression in plant materials for phytoremediation is how GSH homeostasis can be balanced. Although pathway engineering enhancing GSH metabolism and PCS activity seems a promising approach, the consequences of manipulating these pathways may not directly lead to improving the performance of plants exposed to stress, due to the complexity of the cellular GSH network. Thus, the challenge for the future is not only to characterize the involvement of PCS in stress responses but also to broaden our knowledge in PCS as a factor that regulates GSH status and cellular redox homeostasis.
I sincerely thank Dr. Rong-Huay Juang (National Taiwan University of Science and Technology) for stimulating discussion and critical reading of the manuscript.