BACKGROUND: The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST. METHODS: A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis. RESULTS AND CONCLUSIONS: Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function. GENERAL SIGNIFICANCE: These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori. 2011 Elsevier B.V. All rights reserved.
BACKGROUND: The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST. METHODS: A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis. RESULTS AND CONCLUSIONS: Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function. GENERAL SIGNIFICANCE: These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori. 2011 Elsevier B.V. All rights reserved.