Phytochelatin synthase, papain's cousin, in stereo

Abstract

Supraoptimal levels of essential heavy metals (e.g., Cu and Zn) and trace or higher levels of nonessential heavy metals (e.g., As, Cd, and Hg) pose a threat because they undergo aberrant capping reactions with the thiol groups of proteins, displace endogenous metal cofactors from their cellular binding sites, and promote the formation of active oxygen species (1). One of the most sophisticated mechanisms known to confer protection from metal toxicity is the enzyme-catalyzed synthesis of short-chain peptides, phytochelatins (PCs), which serve as high-affinity, thiol-rich cellular chelators and thereby contribute to the detoxification of heavy metal ions, most notably Cd2+, in vascular plants and some algae, diatoms, fungi, and invertebrates (2). Derived from glutathione (GSH) and related thiols in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15) (1, 3, 4), PCs have the general structure (γ-Glu-Cys)n-Xaa, where n = 2–11 and Xaa is usually Gly (Fig. 1). Although studies of PC synthases have largely been concerned with the enzymes from eukaryotes, recent database searches have disclosed PC synthase-like sequences in the genomes of several prokaryotes (1, 5). In and of itself, this finding might not be of particular interest except that all of the prokaryotic PC synthase homologs identified are half the length of their cognates from eukaryotes (220–237 residues compared with 421–506 residues) because they lack the more sequence-variable C-terminal domain characteristic of the latter. Moreover, the one prokaryotic PC synthase homolog to have been assayed for activity, the alr0975 protein from the cyanobacterium Nostoc sp. PCC 7120 (NsPCS), catalyzes the deglycylation of GSH to γ-Glu-Cys at a high rate and the synthesis of PC2 at a relatively low rate (Fig. 1) (5, 6). It is as if the prokaryotic enzymes, despite an overall sequence identity of only 22–36% with the N-terminal domains of the eukaryotic enzymes, consist solely of the core catalytic domain of a canonical eukaryotic PC synthase (Fig. 1) (1, 7, 8). Against this background, the stereo structure of NsPCS in its native and γ-Glu-Cys-acylated state reported by Vivares et al. in a recent issue of PNAS (9) is seminal. The results presented not only represent the first crystal structure for a PC synthase but they also establish, as had been inferred tentatively from kinetic, protein chemical, and site mutagenic analyses of the prototypical eukaryotic PC synthase, Arabidopsis thaliana PC synthase 1 (AtPCS1) (Fig. 1) (1, 8, 10), that these enzymes belong to the papain superfamily and deploy a cysteine protease-like catalytic mechanism.