Screening and characterization of variant Theta‐class glutathione transferases catalyzing the activation of ethylene dibromide to a mutagen

Abstract

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.

Genetic Variations in Human Glutathione Transferase Enzymes: Significance for Pharmacology and Toxicology

“…Characterization of the functional consequences of these variants is required for a thorough understanding of the pharmacogenetics of GSTs [162] and can also provide insight into the structure-activity relationships of the enzyme proteins. The effects of coding-sequence SNPs affecting the human Theta class GSTs are under investigation [163, 164]. A GST expression system based on Escherichia coli strains bearing a lacZ reversion target has been constructed and is being applied in studies of the functional consequences of nonsynonymous SNPs in human GSTT1 [164].…”