title: Phytochelatin synthase is regulated by protein phosphorylation at a threonine residue near its catalytic site.
abstract: Heavy metals are toxic to most living organisms and cause health problems by contaminating agricultural products. In plants, phytochelatin synthase (PCS, EC 2.3.2.15) uses glutathione (GSH) as its substrate to catalyze the synthesis of heavy metal-binding peptides, known as phytochelatins (PC). PCS has been described as a constitutive enzyme that may be controlled by post-translational modifications. However, the detailed mechanism of its catalytic activity is not clear. In this study, in vitro experiments demonstrate that PCS activity increased following phosphorylation by casein kinase 2 (CK2) and decreased following treatment with alkaline phosphatase. Site-directed mutagenesis experiments at amino acids on AtPCS1 indicate that Thr 49 is the site for phosphorylation. This is further supported by fact that the mutant AtPCS1(T49A) cannot be phosphorylated, and its activity is significantly lower than that of the wild-type enzyme. In the modeled three-dimensional structure of AtPCS1, Arg 183 is within close proximity to Thr 49. The mutant AtPCS1(R183A) can be phosphorylated, but it shows much lower catalytic activity than the wild-type protein. This result suggested that Arg 183 may play an important role in the catalytic mechanism of AtPCS1. The possibility of the presence of a second substrate-binding site as a result of the interaction of these two amino acids is discussed. In addition, the activity of AtPCS1 was also found to be modulated by the C-terminal domain. The N-terminal catalytic domain of AtPCS1 was expressed (AtPCS1-N), and its catalytic activity was found to be even more sensitive to Cd or phosphorylation status than was the full-length enzyme.
authors: Department of Biochemical Science and Technology and Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan.
authors: Hsin-Chieh
authors: HC
authors: Wang
authors: Jiann-Shing
authors: JS
authors: Wu
authors: Ju-Chen
authors: JC
authors: Chia
authors: Chien-Chih
authors: CC
authors: Yang
authors: Yu-Jen
authors: YJ
authors: Wu
authors: Rong-Huay
authors: RH
authors: Juang
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D015971:Gene Expression Regulation, Enzymologic; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D013912:Threonine; D019881:Aminoacyltransferases; C502365:AT5G44070 protein, Arabidopsis