Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.     

Role of C-terminal Cys-rich Region of Phytochelatin Synthase in Tolerance to Cadmium Ion Toxicity

Phytochelatins (PCs) are Cys-rich peptides, synthesized by PC synthase in response to heavy metal ions. The C-terminal Cys-rich region of the PC synthase has homology with functional domains of metallochaperone, metallothionein and thioredoxin. To test the possibility that the C-terminal Cys-rich region of PC synthase has a role in regulating PC synthesis, we introduced point mutations into the PC synthase, replacing Cys³⁵⁸, Cys³⁵⁹ Cys³⁶³ and Cys³⁶⁶ residues with Ala. The mutant PC synthase had a lower PC synthesis ability than the wild-type enzyme. Further, oxidative conditions severely damaged mutant PC synthase whilst the wild-type enzyme suffered less damage, suggesting that the Cys-rich region of PC synthase may play an important role in anti-oxidation activity. Although the C-terminal of PC synthase is not conserved, our studies support the possibility that this region performs several important biological functions.     

Enhancing the tolerance of zebrafish (Danio rerio) to heavy metal toxicity by the expression of plant phytochelatin synthase

Phytochelatin synthase (PC synthase) catalyzes a biosynthesis of phytochelatins (PCs), which are small molecules and glutathione (GSH)-derived metal-binding peptides that are essential for the detoxification of heavy metal ions in plants, fungi and worms. In order to enhance tolerance to heavy metal cytotoxicity, mRNA coding for PC synthase from Arabidopsis thaliana (AtPCS1) was introduced into the early embryos of zebrafish. As a result, the heterogeneous expression of PC synthase and the synthesis of PCs from GSH in embryos could be detected. The developing embryos expressing PC synthase (PC-embryos) became more tolerant to Cd toxicity (500 microM exposure). PC-embryos had significantly longer apparent lethal times for 50% of the population (LT50) of 8.17+/-1.08 days, although control embryos had apparent LT50 of 5.43+/-0.66 days. These data suggest that PC synthase can function in developmental zebrafish, and that PCs are highly effective in detoxifying Cd toxicity even in the whole body of a vertebrate species.     

Brassica juncea Produces a Phytochelatin-Cadmium-Sulfide Complex

Phytochelatins (PCs) are enzymically synthesized peptides produced in higher plants and some fungi upon exposure to heavy metals. We have examined PC production in the Se-tolerant wild mustard Brassica juncea and found that it produces two types of PC-Cd complexes with the same characteristics as those from fission yeast Schizosaccharomyces pombe, including a high molecular weight PC-Cd-sulfide form.     

Comparative analysis of the two-step reaction catalyzed by prokaryotic and eukaryotic phytochelatin synthase by an ion-pair liquid chromatography assay

Genes encoding phytochelatin (PC) synthase have been found in higher plants, fission yeast and worm. Recently, kinetic and mutagenic analyses of recombinant PC synthase have been revealing the molecular mechanisms underlying PC synthesis, however, a conclusive model has not been established. To clarify the mechanism of PC synthase found in eukaryotes, we have compared the two-step reactions catalyzed by the prokaryotic Nostoc PC synthase (NsPCS) and the eukaryotic Arabidopsis PC synthase (AtPCS1). Comparative analysis shows that in the first step of PC synthesis corresponding to the cleavage of -glutamylcysteine (-EC) from glutathione (GSH), free GSH or PCs acts as a donor molecule to supply a -EC unit for elongation of the PC chain, and heavy metal ions are required to carry out the cleavage. Furthermore, functional analyses of various mutants of NsPCS and AtPCS1, selected by comparing the sequences of NsPCS and AtPCS1, indicate that the N-terminal region (residues 1–221) in AtPCS1 is the catalytic domain, and in this region, the Cys⁵⁶ residue is associated with the PC synthesis reaction. These results enable us to propose an advanced model of PC synthesis, describing substrate specificity, heavy metal requirement, and the active site in the enzyme.     

Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.     

HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators

Phytochelatins (PCs) are nonprotein peptides with the general structure (γ-Glu-Cys)_n-Gly (PC_n), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (γ-Glu-Cys-Gly) and/or previously synthesized PC_n as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5′-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions.     

Heavy metals toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants

Plants experience oxidative stress upon exposure to heavy metals that leads to cellular damage. In addition, plants accumulate metal ions that disturb cellular ionic homeostasis. To minimize the detrimental effects of heavy metal exposure and their accumulation, plants have evolved detoxification mechanisms. Such mechanisms are mainly based on chelation and subcellular compartmentalization. Chelation of heavy metals is a ubiquitous detoxification strategy described in wide variety of plants. A principal class of heavy metal chelator known in plants is phytochelatins (PCs), a family of Cys-rich peptides. PCs are synthesized non-translationally from reduced glutathione (GSH) in a transpeptidation reaction catalyzed by the enzyme phytochelatin synthase (PCS). Therefore, availability of glutathione is very essential for PCs synthesis in plants at least during their exposure to heavy metals. Here, I reviewed on effect of heavy metals exposure to plants and role of GSH and PCs in heavy metal stress tolerance. Further, genetic manipulations of GSH and PCs levels that help plants to ameliorate toxic effects of heavy metals have been presented.     

Heavy metal accumulation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.     

植物螯合肽及其在抗重金属胁迫中的作用

植物螯合肽(PCs)广泛存在于植物体中,与植物抗重金属胁迫关系密切。植物螯合肽及其复合物是一类富含半胱氨酸的低分子量化合物。现有研究证明,PCS由谷胱甘肽(GSH)为底物的酶促反应合成,其合成受相关基因的调控,从模式植物拟南芥的突变体中已分离到与PCS合成有关的几个基因。植物螯合肽首先与重金属离子结合形成低分子量(LMW)复合物,以此形态经由细胞质进入液泡后,再与一个分子的植物螯合肽结合,形成对植物组织毒性较小的高分子量(HMW)复合物,从而达到缓解重金属对植物的危害作用。就植物螯合肽及其复合物的结构、生物合成、基因调控及重金属解毒机理等进行了综述,并对今后的研究方向提出了一些看法。     

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris

In yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1?lro1? double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae.     

Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance

Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.     

Characterization of phytochelatin synthase from tomato

The enzyme that synthesizes Cd-binding phytochelatins (PCs), PC synthase, has been studied in tomato ( Lycopersicon esculentum ) cell cultures and plants. This enzyme transfers γ-GluCys from GSH or PC to either GSH or an existing polymer of (γ-GluCys)_nGly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ-GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd²⁺, Ag⁺, Cu²⁺, Au⁺, Zn²⁺, Fe²⁺, Hg²⁺ and Pb²⁺. In crude protein extracts from tomato cells the enzyme has an apparent K_m of 7.7 m M for GSH in the presence of 0.5 m M Cd²⁺, and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium-resistant tomato cells growing in medium containing 6 m M CdCl₂ have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd-PC complexes in response to cadmium.     

Dissection of glutathione conjugate turnover in yeast

Xenobiotics are widely used as pesticides. The detoxification of xenobiotics frequently involves conjugation to glutathione prior to compartmentalization and catabolism. In plants, degradation of glutathione-S-conjugates is initiated either by aminoterminal or carboxyterminal amino acid cleavage catalyzed by a γ-glutamyl transpeptidase and phytochelatin synthase, respectively. In order to establish yeast as a model system for the analysis of the plant pathway, we used monochlorobimane as a model xenobiotic in Saccharomyces cerevisiae and mutants thereof. The catabolism of monochlorobimane is initiated by conjugation to form glutathione-S-bimane, which is then turned over into a γ-GluCys-bimane conjugate by the vacuolar serine carboxypeptidases CPC and CPY. Alternatively, the glutathione-S-bimane conjugate is catabolized by the action of the γ-glutamyl transpeptidase Cis2p to a CysGly-conjugate. The turnover of glutathione-S-bimane was impaired in yeast cells deficient in Cis2p and completely abolished by the additional inactivation of CPC and CPY in the corresponding triple knockout. Inducible expression of the Arabidopsis phytochelatin synthase AtPCS1 in the triple knockout resulted in the turnover of glutathione-S-bimane to the γ-GluCys-bimane conjugate as observed in plants. Challenge of AtPCS1-expressing yeast cells with zinc, cadmium, and copper ions, which are known to activate AtPCS1, enhanced γ-GluCys-bimane accumulation. Thus, initial catabolism of glutathione-S-conjugates is similar in plants and yeast, and yeast is a suitable system for a study of enzymes of the plant pathway.     

Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.     

Phytochelatin synthase: of a protease a peptide polymerase made

Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.     

植物螯合肽的转运与功能研究进展

植物螯合肽（phytochelatins,PCs）是由植物螯合肽合酶催化谷胱甘肽合成的一类生物小分子,结构式为（γ-Glu-Cys）n-Gly（n=2-11）,在真菌和高等植物耐受重金属胁迫机制中具有重要作用。近年来,人们在Pc介导重金属脱毒害的分子机理研究上取得了重要进展,发JLSpHMT1和SpABC2是PC在裂殖酵母中介导重金属液泡区室化的主要转运蛋白,鉴定了拟南芥液泡膜PC转运蛋AtABCC1和AtABCC2。此外,PCs也可能在超积累植物细胞内对重金属脱毒害具有重要功能。     

Cd, Mn, Ni and Se toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P_5B ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.