Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase

Kohji Yamamoto, Kazuhiro Usuda, Yoshimitsu Kakuta, Makoto Kimura, Akifumi Higashiura, Atsushi Nakagawa, Yoichi Aso, Mamoru Suzuki

研究成果: ジャーナルへの寄稿記事

15 引用 (Scopus)

抄録

Background: Glutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST from Bombyx mori (bmGSTD) to examine its catalytic residues. Methods: The three-dimensional structure of bmGSTD was resolved by the molecular replacement method and refined to a resolution of 2.0 Å. Results: Structural alignment with a Delta-class GST of Anopheles gambiae indicated that bmGSTD contains 2 distinct domains (an N-terminal domain and a C-terminal domain) connected by a linker. The bound glutathione localized at the N-terminal domain. Putative catalytic residues were changed to alanine by site-directed mutagenesis, and the resulting mutants were characterized in terms of catalytic activity using glutathione and 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTD mutants indicated that Ser11, Gln51, His52, Ser67, and Arg68 are important for enzyme function. General significance: These results provide structural insights into the catalysis of glutathione conjugation in B. mori by bmGSTD.

元の言語英語
ページ(範囲)1469-1474
ページ数6
ジャーナルBiochimica et Biophysica Acta - General Subjects
1820
発行部数10
DOI
出版物ステータス出版済み - 10 1 2012

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Bombyx
Glutathione Transferase
Glutathione
Catalyst activity
Anopheles gambiae
Dinitrochlorobenzene
Detoxification
Mutagenesis
Xenobiotics
Site-Directed Mutagenesis
Catalysis
Alanine
Crystal structure
Kinetics
Substrates
Enzymes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

これを引用

Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase. / Yamamoto, Kohji; Usuda, Kazuhiro; Kakuta, Yoshimitsu; Kimura, Makoto; Higashiura, Akifumi; Nakagawa, Atsushi; Aso, Yoichi; Suzuki, Mamoru.

:: Biochimica et Biophysica Acta - General Subjects, 巻 1820, 番号 10, 01.10.2012, p. 1469-1474.

研究成果: ジャーナルへの寄稿記事

Yamamoto, Kohji ; Usuda, Kazuhiro ; Kakuta, Yoshimitsu ; Kimura, Makoto ; Higashiura, Akifumi ; Nakagawa, Atsushi ; Aso, Yoichi ; Suzuki, Mamoru. / Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase. :: Biochimica et Biophysica Acta - General Subjects. 2012 ; 巻 1820, 番号 10. pp. 1469-1474.
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AU - Yamamoto, Kohji

AU - Usuda, Kazuhiro

AU - Kakuta, Yoshimitsu

AU - Kimura, Makoto

AU - Higashiura, Akifumi

AU - Nakagawa, Atsushi

AU - Aso, Yoichi

AU - Suzuki, Mamoru

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N2 - Background: Glutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST from Bombyx mori (bmGSTD) to examine its catalytic residues. Methods: The three-dimensional structure of bmGSTD was resolved by the molecular replacement method and refined to a resolution of 2.0 Å. Results: Structural alignment with a Delta-class GST of Anopheles gambiae indicated that bmGSTD contains 2 distinct domains (an N-terminal domain and a C-terminal domain) connected by a linker. The bound glutathione localized at the N-terminal domain. Putative catalytic residues were changed to alanine by site-directed mutagenesis, and the resulting mutants were characterized in terms of catalytic activity using glutathione and 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTD mutants indicated that Ser11, Gln51, His52, Ser67, and Arg68 are important for enzyme function. General significance: These results provide structural insights into the catalysis of glutathione conjugation in B. mori by bmGSTD.

AB - Background: Glutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST from Bombyx mori (bmGSTD) to examine its catalytic residues. Methods: The three-dimensional structure of bmGSTD was resolved by the molecular replacement method and refined to a resolution of 2.0 Å. Results: Structural alignment with a Delta-class GST of Anopheles gambiae indicated that bmGSTD contains 2 distinct domains (an N-terminal domain and a C-terminal domain) connected by a linker. The bound glutathione localized at the N-terminal domain. Putative catalytic residues were changed to alanine by site-directed mutagenesis, and the resulting mutants were characterized in terms of catalytic activity using glutathione and 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTD mutants indicated that Ser11, Gln51, His52, Ser67, and Arg68 are important for enzyme function. General significance: These results provide structural insights into the catalysis of glutathione conjugation in B. mori by bmGSTD.

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