Screening and characterization of variant Theta-class glutathione transferases catalyzing the activation of ethylene dibromide to a mutagen.
Ethylene dibromide (EDB) is a widespread environmental pollutant and mutagen/carcinogen. Certain Theta-class glutathione transferases (GSTs), enzymes that catalyze the reaction of reduced glutathione (GSH) with electrophiles, activate EDB to a mutagen. Previous studies have shown that human GST T1-1, but not rat GST T2-2, activates EDB. We have constructed an E. coli lacZ reversion mutagenicity assay system in which expression of recombinant GST supports activation of EDB to a mutagen. Hexa-histidine N-terminal tagging of GST T1-1 results in greatly enhanced expression of the recombinant enzyme and gives a lacZ strain that shows a mutagenic response to EDB at extremely low levels (approximately 1 ng EDB per plate). The hexa-histidine-tagged enzyme was purified in one step by Ni(2+)-affinity chromatography. We applied the lacZ mutagenicity assay to the rapid screening of a library of variant GST Theta enzymes. Sequence variants with altered catalytic activities were identified, purified, and characterized.
Josephy
P David
PD
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada. djosephy@uoguelph.ca
Taylor
Patricia L
PL
Vervaet
Gaby
G
Mannervik
Bengt
B
D000818:Animals; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D015946:Ethylene Dibromide / Q000633:toxicity*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D009153:Mutagens; D011994:Recombinant Proteins; D015946:Ethylene Dibromide; C091152:Gstt2 protein, rat; D005982:Glutathione Transferase; C495790:glutathione transferase T1-1, human