Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori

Yoshimitsu Kakuta, Kazuhiro Usuda, Takashi Nakashima, Makoto Kimura, Yoichi Aso, Kohji Yamamoto

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Background: Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues. Methods: The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu. Results: We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features. Conclusions: This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function. General Significance: Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.

Original languageEnglish
Pages (from-to)1355-1360
Number of pages6
JournalBiochimica et Biophysica Acta - General Subjects
Volume1810
Issue number12
DOIs
Publication statusPublished - Dec 1 2011

Fingerprint

Bombyx
Insecticides
Glutathione Transferase
Catalytic Domain
Glutathione
Insecticide Resistance
Molecular Conformation
Synchrotrons
Xenobiotics
Site-Directed Mutagenesis
Catalysis
Alanine
Detoxification
Mutagenesis
Kinetics
Molecular modeling
Synchrotron radiation
Dimers
Radiation
Refining

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori. / Kakuta, Yoshimitsu; Usuda, Kazuhiro; Nakashima, Takashi; Kimura, Makoto; Aso, Yoichi; Yamamoto, Kohji.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1810, No. 12, 01.12.2011, p. 1355-1360.

Research output: Contribution to journalArticle

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