Dichloromethane mediated in vivo selection and functional characterizationof rat glutathione S-transferase theta 1-1 variants

Methylobacterium dichloromethanictum DM4 is able to grow with dichlorometha ne as the sole carbon and energy source by using a dichloromethane dehaloge nase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacte ria. Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane. This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carryin g a plasmid-encoded rGSTT1 gene. Transconjugants that still expressed the G ST T1 protein after dichloromethane treatment included rGSTT1 mutants encod ing protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions. A structural mo del of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to c ap the active site. The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichl oromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propa ne. These results provide the first experimental evidence for the involveme nt of Gln102 and Arg107 in catalysis, and illustrate the potential of in vi vo approaches to identify catalytic residues in GSTs whose activity leads t o toxic effects.