Functional analysis of glutathione s-transferase from the extremophile Halothece sp. PCC7418

Abstract

Glutathione S-transferase (GST) are a set of multifunctional enzymes encoded by large gene families. It has been functionally demonstrated that GSTs play vital roles in cellular detoxification, regulation of redox-dependent process, and stress responses. Although the GST gene family has been extensively studied across taxa, the function of the GST genes in primordial oxygenic phototrophs such as cyanobacteria is poorly understood. In this thesis, GSTs from extremophilic cyanobacterium Halothece sp. PCC7418 (hereafter Halothece GSTs) were identified and functionally characterized. The genome-based analysis showed that there were four GSTs in Halothece 7418 (GST_0647, GST _0729, GST _1478, and GST _3557). Phylogenetic relationship revealed that these cyanobacterial GSTs were highly divergent. GST _0647 and GST _1478 are paralogous genes while other two GSTs (GST_0729 and GST _3557) are distinct. These Halothece GSTs were cloned and successfully expressed in E. coli BL21. Stress tolerance of expressing cells were evaluated under salt and oxidative stresses. Amongst four expressing cells, the GST _3557 performed the greatest tolerance to oxidative and salt stresses. Viable cell count of GST_3557 under salt stress was higher than empty vector control approximately 18 folds. These results support the protective role and vital function of GST_3557 against abiotic stress in a heterologous expression system. Recombinant GST_3557 exhibited GST activity toward 1-chloro-2, 4- dinitrobenzene (CDNB) and glutathione (GSH) with a broad range of activity at pH 6.5–10.5. Kinetic parameters showed the apparent Km for CDNB and GSH was 0.14±0.02 and 0.74±0.29 mM, respectively. Thus, GST _3557 had high affinity for electrophilic substrate, CDNB. In case of peroxidase activity, GST _3557 did not perform activity in all our conditions tested. Results from this study provided insight into the molecular and cellular functions of cyanobacterial GST, which is less understood compared to other counterparts. These results contribute toward understanding the mechanism behind physiological plasticity under a heterologous expression system.