Phytochelatin Synthase, a Dipeptidyltransferase That Undergoes Multisite Acylation with γ-Glutamylcysteine during Catalysis

Abstract

Phytochelatin (PC) synthase has been assumed to be a ␥-glutamylcysteine dipeptidyl transpeptidase (EC 2.3.2.15) and, more recently, as exemplified by analyses of the immunopurified recombinant enzyme from Arabidopsis thaliana (AtPCS1-FLAG), has been shown to catalyze a PC synthetic reaction with kinetics that approximates a bisubstrate-substituted enzyme mechanism in which millimolar concentrations of free GSH and micromolar concentrations of heavy metal⅐GSH thiolates (e.g. cadmium⅐GS 2 ) or millimolar concentrations of S-alkylglutathiones serve as cosubstrates. Here, we show, by direct analyses of the stoichiometry of AtPCS1-FLAG-catalyzed PC synthesis, the kinetics and stoichiometry of acylation of the enzyme and release of free glycine from ␥-Glu-Cys donors, and the effects of the Cys-to-Ser or -Ala and Serto-Ala substitution of conserved residues in the catalytic N-terminal half of the enzyme, that PC synthase is indeed a dipeptidyltransferase that undergoes ␥-Glu-Cys acylation at two sites during catalysis, one of which, in accord with a cysteine protease model, likely corresponds to or is at least tightly coupled with Cys 56. The identity of the second site of enzyme modification remains to be determined, but it is distinguishable from the first Cys 56 -dependent site, which is amenable to ␥-Glu-Cys acylation by free GSH, because its acylation not only depends on the provision of Cd 2؉ or GSH with a blocked, S-alkylated thiol group, but is also necessary for net PC synthesis. We conclude that des-Gly-PCs are not generated as an immediate by-product, but rather that the enzyme catalyzes a dipeptidyl transfer reaction in which some of the energy liberated upon cleavage of the Cys-Gly bonds of the ␥-Glu-Cys donors in the first phase of the catalytic cycle is conserved through the formation of a two site-substituted ␥-Glu-Cys acyl-enzyme intermediate whose hydrolysis provides the energy required for the formation of the new peptide bond required for the extension of PC chain length by one ␥-Glu-Cys repeat per catalytic cycle.

CeHMT-1, a Putative Phytochelatin Transporter, Is Required for Cadmium Tolerance in Caenorhabditis elegans

“…PCs, which constitute a family of short-chain heavy metalbinding peptides with the general structure (␥-EC) n Xaa, where n ϭ 2-11, are derived from GSH and related thiols in a ␥-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (EC 2.3.2.15) (1,5,6). It is now almost 15 years since the partial purification of the enzyme capable of catalyzing PC synthesis (5), yet it is only in the last several years that its molecular identity has been determined by the cloning and characterization of genes encoding PC synthases (PCSs).…”

“…These are the ubiquitous thiol tripeptide glutathione (GSH), a family of small (4 -8 kDa) cysteine-rich proteins termed metallothioneins, and several higher molecular mass albeit sequence-unrelated metal-binding proteins. In all of the plants studied and in some fungi (as exemplified by Schizosaccharomyces pombe and Candida glabrata) and some animals (as exemplified by the model nematode Caenorhabditis elegans), a third class of cysteine-rich peptides termed phytochelatins (PCs) 1 has also been shown to be involved in heavy metal detoxification.PCs, which constitute a family of short-chain heavy metalbinding peptides with the general structure (␥-EC) n Xaa, where n ϭ 2-11, are derived from GSH and related thiols in a ␥-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (EC 2.3.2.15) (1,5,6). It is now almost 15 years since the partial purification of the enzyme capable of catalyzing PC synthesis (5), yet it is only in the last several years that its molecular identity has been determined by the cloning and characterization of genes encoding PC synthases (PCSs).…”

Functional Characterization of an Unusual Phytochelatin Synthase, LjPCS3, of Lotus japonicus

“…Two major breakthroughs were the identification of Cys-56 as the first acylation (gGlu-Cys donor) site in AtPCS1 and the demonstration that free GSH and a metal-GS thiolate complex are cosubstrates of the enzyme (Vatamaniuk et al, 2004;Romanyuk et al, 2006). An important finding was also that prokaryotic PCS homologs lack both the variable C-terminal domain and the second acylation (gGlu-Cys acceptor) site that is present in the eukaryotic PCS (Harada et al, 2004;Tsuji et al, 2004;Vivares et al, 2005).…”

Phytochelatin synthase, papain's cousin, in stereo

“…One of the most sophisticated mechanisms known to confer protection from metal toxicity is the enzyme-catalyzed synthesis of shortchain peptides, phytochelatins (PCs), which serve as high-affinity, thiol-rich cellular chelators and thereby contribute to the detoxification of heavy metal ions, most notably Cd 2ϩ , in vascular plants and some algae, diatoms, fungi, and invertebrates (2). Derived from glutathione (GSH) and related thiols in a ␥-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15) (1,3,4), PCs have the general structure (␥-Glu-Cys) n -Xaa, where n ϭ 2-11 and Xaa is usually Gly (Fig. 1).…”