Characterization of the Second Substrate Binding Site and Post-Transpational Modification in Arabidopsis Phytochelatin Synthase
Date Issued
2014
Date
2014
Author(s)
Chia, Ju-Chen
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-πinteraction 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.
Besides the effects of metal ions on substrate binding mechanism, the post-translational activation of PCS via phosphorylation was also discussed in this study. Previous studies had revealed that Thr49 is the site for phosphorylation and play an important role in the formation of the active site through the interaction with Arg183. In addition, Ser13 and Ser352 were also identified as phosphorylation sites of AtPCS1 expressed in Pichia pastoris expression system. In contrast with the activity of T49A mutant, mutations on these Ser residues did not significantly affect PCn catalysis of PCS. Ser352 was located on the heavy metal binding motifs on C-terminal domain, and phosphorylation on this site might involve in the metal-binding ability but not directly influence the catalysis of the enzyme. Besides, these Ser phosphorylation sites showed their identity in Arabidopsis. In the modest cadmium concentration, wild type Arabidopsis and PCS1 complementary lines were more tolerant than S13A and S532A lines, indicating that the phosphorylation sites on AtPCS1 are important for cadmium resistance of Arabidopsis. In summary, our data demonstrated that PCS activity is regulated via post-translational modification, and phosphorylated AtPCS1 may play critical roles in heavy metal stress in Arabidopsis.
Subjects
植物螯合素
植物螯合素合成酶
酵素催化機制
阿拉伯芥
轉譯後修飾
重金屬逆境
Type
thesis
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