Bioinformatic analysis and in vitro site-directed mutagenesis of conserved amino acids in BphK LB400, a specific bacterial glutathione transferase

Abstract

Glutathione transferases [GSTs: EC 2.5.1.18)] are ubiquitous multifunctional prokaryotic and eukaryotic enzymes involved in the cellular detoxification and excretion of a large variety of compounds. However, our understanding of the role of bacterial GSTs in metabolism is still in its infancy. The association of bacterial GST DNA with other genes involved in degradation of toxic pollutants, including polychlorinated biphenyls (PCBs), indirectly suggests a role for bacterial GSTs in biodegradation. Previously, in this laboratory, a specific bacterial GST, BphK LB400 isolated from Burkholderia xenovorans LB400, was shown to be capable of dehalogenating chlorinated organic substrates rendering them less toxic. However, little is known about the specific amino acids in BphK LB400 involved in catalysis in vitro. In this study, bioinformatic analysis of BphK LB400 and other bacterial GSTs, including PCB degraders, identified a number of amino acids that were identical in all bacterial GST sequences analysed. Two amino acids, Cys10 and His106, were selected for in vitro site-directed mutagenesis studies. In vitro GST activity assay results suggest that these two amino acids play a role in determining the catalytic activity of BphK LB400. Studies of bacterial cell extracts expressing BphK LB400 (wildtype and mutant) identified a specific mutant, Cys10Phe, with increased GST activity towards 1-chloro-2,4-dinitrobenzene (the model substrate for GSTs). BphK LB400 (mutant) with increased activity towards toxic chlorinated organic compounds could have potential for bioremediation of contaminated soil in the environment.