Investigation of substrate specificity of wildtype and mutant BphKLB400 (a glutathione S-transferase) from Burkholderia LB400

Abstract

Abstract The bphK gene located in the bph operon of Burkholderia LB400 encodes a protein, BphK LB400 , with significant sequence similarity to glutathione-S-transferases (GST), a group of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Comparison of the amino acid sequence of BphK LB400 with GST from other polychlorinated biphenyl (PCB)-degrading bacteria identified a number of highly conserved amino acids in the C-terminal region of the protein that may be associated with substrate specificity. In this study, two of these conserved amino acids in BphK LB400 (amino acids 152 and 180) were selected for mutation, using site-directed mutagenesis, and substrate specificity assays. BphK LB400 (wildtype and mutant) was over-expressed in Escherichia coli where the bphK gene (wildtype and mutant) is under the expression of a lac promoter and is induced by isopropyl thiogalactoside, and bacterial cell extracts were prepared for GST activity assays. Mutations at amino acids 152 and 180 were shown to affect GST activity of BphK LB400 using 1-chloro-2,4-dinitrobenzene, the model substrate for GST activity assays; 4-chlorobenzoate and 3-chlorobenzoate, intermediates in the polychlorinated biphenyl (PCB) degradation pathway, and 2,4-dichlorophenoxyacetate and atrazine, commonly used herbicides; as substrates. A BphK LB400 mutant (Ala180Pro) is identified in this study as having increased activity towards all substrates tested. This mutant may have potential in bioremediation.