8IX7

Functional significance of serine 13 in the active site of rice Phi-class glutathione S-transferase F3.

8IX7 の概要

• エントリーDOI: 10.2210/pdb8ix7/pdb
• 分子名称: glutathione transferase, GLUTATHIONE (3 entities in total)
• 機能のキーワード: glutathion-s-transferase, plant protein
• 由来する生物種: Oryza sativa subsp. indica
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 50075.27

構造登録者

Lee, W.C.,Kong, K.H. (登録日: 2023-03-31, 公開日: 2024-04-03, 最終更新日: 2025-03-19)

主引用文献

Jin, S.B.,Jang, S.W.,Shin, J.A.,Jung, N.H.,Kim, H.A.,Park, S.Y.,Lee, W.C.,Kong, K.H.
Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa.
Pestic Biochem Physiol, 194:105463-105463, 2023

PubMed Abstract: Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and k/K values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the K and I values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (k/K) plot was used to estimate the pK value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pK value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pK of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.

PubMed: 37532308
DOI: 10.1016/j.pestbp.2023.105463

実験手法

X-RAY DIFFRACTION (1.7 Å)