Abstract
Phytochelatin synthase (PCS) uses the substrates glutathione (GSH, γGlu-Cys-Gly) and a cadmium (Cd)-bound GSH (Cd∙GS2) to produce the shortest phytochelatin product (PC2, (γGlu-Cys)2-Gly) through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12–218) by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS) as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation) resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W) for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.
Highlights
Phytochelatins (PC) aren-Gly (n = 2–11) polymers representing major detoxification components in plants, fungi, and other organisms [1,2,3,4]
The polymerase chain reaction (PCR) products were treated with Dpn I to digest the parental DNA template, and the mutation-containing synthesized DNA was transformed into Escherichia coli DH5α cells (Sigma-Aldrich)
We did not include the C-terminal domain in the AtPCS1 model because native NsPCS contains only the N-terminal half of the eukaryotic Phytochelatin synthase (PCS) (Figure 1A)
Summary
Phytochelatins (PC) are (γGlu-Cys)n-Gly (n = 2–11) polymers representing major detoxification components in plants, fungi, and other organisms [1,2,3,4]. Previous studies have identified and characterized the genes encoding PCS in various eukaryotic organisms, such as Arabidopsis thaliana (AtPCS1) [12,13], Triticum aestivum (TaPCS1) [14], Lotus japonicas (LjPCS1) [15], Schizosaccharomyces pombe (SpPCS) [12,14], the nematode Caenorhabditis elegans (CePCS1) [16], and the cadmium hyperaccumulator Thlaspi caerulescens (TcPCS1) [17]. PCS is constitutively expressed at a transcriptional level, and cadmium (Cd) treatment marginally upregulates this expression [20,21]
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