Biochemical characterization of glutathione transferase YliJ from Escherichia coli

Abstract

Glutathione transferases, or GSTs, protect cellular components from the dangers posed by electrophiles by catalyzing the conjugation of the electrophilic molecules with glutathione. This makes GSTs very successful detoxifying agents in most organisms. Glutathione transferase YliJ, also known as GstB, has been postulated to catalyze the glutathione mediated dehalogenation of bromoacetate. The objective of this research is to develop a simple purification procedure for YliJ, design a safe method for assaying YliJ activity, characterize YliJ, and to search for other potential substrates for YliJ. YliJ was overexpressed in E. coli cells and purified using ammonium sulfate precipitation and ion exchange chromatography. The presence and purity of YliJ was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. YliJ has a K[subscript M] of 1.60 mM, a k[subscript cat] of 175.0 s[superscript -1] and a catalytic efficiency of 1.09 x 10[superscript 5] M[superscript -1]s[superscript -1] for GSH when conjugated to bromoacetate. In comparison, YliJ has a K[subscript M] of 0.1753 mM, a k[subscript cat] of 155.3 s[superscript -1], and a catalytic efficiency of 8.86 x 10[superscript 4] M[superscript -1]s[superscript -1] for GSH when conjugated to iodoacetate. E. coli cells expressing YliJ protein and the knockout strain BW25113EyliJ lacking YliJ were tested for sensitivity to halogenated compounds and antibiotics. The results strongly support the suggestion that a small molecule containing a carboxylate moiety could be the physiological substrate of YliJ.