Phytochelatin synthases (PC synthases) are soluble enzymes that catalyse the synthesis of heavy metal‐binding peptides termed phytochelatins (PCs) from the tripeptide glutathione (GSH) and related peptides. Though originally discovered in plants and a few fungi and characterised in terms of their role in the detoxification of nonessential heavy metal ions such as As
3+
, Cd
2+
or Hg
2+
through the heavy metal‐activated synthesis of PCs, it is now apparent that PC synthases and PC synthase (PCS)‐like enzymes, both of which deploy a Clan CA Cys protease‐type catalytic mechanism, are far more widespread and functionally versatile than was once thought. Full‐length PC synthases, consisting of a sequence‐conserved
N
‐terminal core catalytic domain and sequence‐variable
C
‐terminal domain, are found sporadically in all of the major eukaryotic taxa where they not only synthesise PCs for the detoxification of nonessential heavy metals and, as exemplified by Zn
2+
, the homeostasis of essential heavy metals but also participate in the cytosolic deglycination of glutathione (GS)‐conjugates and in one or more of the terminal steps underlying the innate immunity of higher plants to pathogens. Half‐length PCS‐like polypeptides, consisting of the
N
‐terminal catalytic domain but lacking the
C
‐terminal domain, are found in some bacteria, and in the one example characterised in detail catalyse a reaction strongly biased in favour of the deglycination of GSH over the synthesis of PCs. The full extent and mechanistic basis of the roles played by PC synthases as multitasking enzymes remain to be determined, but what is apparent is that the combined protease and peptide polymerase activities of the eukaryotic enzymes versus the limited protease activities of their bacterial counterparts confer a metabolic versatility on the former that the latter lack.
Key Concepts
Eukaryotic PC synthases catalyse the heavy metal‐activated posttranslational synthesis of short‐chain thiol‐rich γEC polymers, phytochelatins (PCs) with the general structure (γEC)
n
X, typically (γEC)
n
G, from glutathione (GSH; γECG) or related γEC‐containing peptides. PCs bind heavy metal ions with high affinity to contribute to the detoxification of nonessential heavy metals such as As
3+
, Cd
2+
or Hg
2+
and the homeostasis of essential heavy metals such as Zn
2+
.
The PC synthetic reaction catalysed by eukaryotic PC synthases is a γEC dipeptidyl transferase reaction consisting of two main phases: the initial cleavage (deglycination) of GSH or related γEC‐containing peptides to yield γEC followed by transfer of the γEC unit to the
N
‐terminus of another GSH molecule or a preexistent PC.
Eukaryotic PC synthases are dimeric proteins made up of two identical 50–55 kDa subunits, each of which consists of a sequence‐conserved 220–240 amino acid residue
N
‐terminal domain and a sequence‐variable 200–270 amino acid residue
C
‐terminal domain. The
N
‐terminal domain, alone, is sufficient for heavy metal‐activated PC synthesis, but the exact role played by the
C
‐terminal domain, other than in influencing the specificity and extent of catalytic activation by heavy metals
in vitro
and processes related to the innate immunity of plants to microbial pathogens, is unknown.
Steady‐state PC synthesis by eukaryotic PC synthases is by a ping‐pong (substituted enzyme) mechanism. In the first phase of the reaction, a γEC‐enzyme covalent intermediate is formed when the first substrate, the γEC donor (e.g. GSH), undergoes cleavage (deglycination) with the release of free Gly. In the second phase of the reaction, the γEC unit on the enzyme is transferred to the second substrate, the acceptor (e.g.
bis
(glutathionato)cadmium, CdGS
2
), concomitant with the formation of a γ‐peptide bond between the γEC unit and the
N
‐terminal Glu residue of GSH. Activation of core catalysis is contingent on the provision of a substrate pair in which the thiol groups of at least one of the two substrates are blocked through the formation of a metal thiolate or
S
‐alkylation. Although GSH and its corresponding heavy metal thiolates (e.g. Cd.GS
2
) are the usual substrates for this reaction,
S
‐alkyl GSH derivatives can substitute for both substrates.
Members of the PC synthase family are not only found in representatives of all of the major eukaryotic taxa but also sporadically as PC synthase (PCS)‐like polypeptides in bacteria. Unlike all known eukaryotic PC synthases, which are full‐length molecules consisting of both the
N
‐terminal core catalytic and
C
‐terminal domains, their bacteria PCS‐like homologs are half‐molecules lacking the
C
‐terminal domain.
As exemplified by the PCS‐like polypeptide from the cyanobacterium
Nostoc
sp. PCC 7120 (NsPCS), the enzymes from bacteria, unlike their equivalents from eukaryotes, catalyse only the first phase of the PC synthetic reaction, the deglycination of GSH to yield γEC.
The
N
‐terminal domains of eukaryotic PC synthases and the PCS‐like polypeptides of bacteria deploy a catalytic mechanism similar to that of the Clan CA Cys proteases papain, staphopain and cruzain. All of these enzymes possess a Cys‐His‐Asp/Asn catalytic triad, the Cys residue of which through cooperative interactions with the His and Asp/Asn residues undergoes acylation to generate an enzyme thioester.
The essential difference between catalysis by Clan CA Cys proteases, NsPCS and eukaryotic PC synthases is the identity of the second substrate. Clan CA Cys proteases and NsPCS catalyse α‐peptide hydrolysis by nucleophilic attack on the enzyme thioester intermediate by water, so precluding downstream γ‐peptide bond formation, whereas eukaryotic PC synthases are capable of catalysing nucleophilic attack on the thioester enzyme intermediate by a second thiol derivative concomitant with condensation of the γEC unit from the first substrate with the second substrate through the formation of a γ‐peptide bond.
Eukaryotic PC synthases are not only responsible for the synthesis of PCs for the detoxification of nonessential and homeostasis of essential heavy metals but are also involved in the cytosolic processing of glutathione
S
‐conjugates generated through the action of glutathione
S
‐transferases (GSTs). This they do through deployment of the first of the two phases of the PC synthetic reaction – deglycination of the GS substituent of the conjugate. The full significance of this capability is unknown, but its potential is considerable in that the GST‐mediated synthesis of GS‐conjugates is a critical preparatory step for the metabolism and/or detoxification of a broad range of endogenous secondary metabolites and xenobiotics.
PC synthases are implicated in the innate immune response of higher plants to microbial pathogens. The steps in innate immunity in which the enzyme participates, for instance the processing of antimicrobial compounds, appear to map to the
C
‐terminal domain of the plant enzyme.
