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

• Published in: Biochimica et biophysica acta Vol. 1810; no. 12; pp. 1355 - 1360
• Main Authors: Kakuta, Yoshimitsu, Usuda, Kazuhiro, Nakashima, Takashi, Kimura, Makoto, Aso, Yoichi, Yamamoto, Kohji
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2011
• Subjects: active sites; alanine; Amino Acid Sequence; Animals; Biocatalysis; Bombyx - enzymology; Bombyx mori; catalytic activity; Catalytic Domain; Crystal structure; Crystallography, X-Ray; Glutathione; Glutathione transferase; Glutathione Transferase - chemistry; Glutathione Transferase - metabolism; insecticide resistance; insecticides; Kinetics; Lepidoptera; Models, Molecular; molecular conformation; molecular models; Molecular Sequence Data; Sequence Homology, Amino Acid; silkworms; Site-directed mutagenesis; surveys; SDS-PAGE; Glutathione transferase; Site-directed mutagenesis; Lepidoptera; GST; CDNB; GTX; GSH; Crystal structure; Glutathione
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2011.06.022

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. 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. 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. 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. 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. ► The crystal structure of bmGSTu has been determined with a resolution of 2.1 Å. ► Bound glutathione could be localized at the G-site. ► Recombinant bmGSTu catalyzed transfer of glutathione to a synthetic GST substrate. ► Val55, Glu67, and Ser68 in the G-site are catalytically important. ► The H-site shows unique features that may have relevance to pest control.