A Trifunctional Enzyme With Glutathione S-transferase, Glutathione Peroxidase And Superoxide Dismutase Activity

Reactive oxygen species (ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states the production of ROS is enhanced and the excess ROS damage various biomacromolecules including RNA, DNA, protein, sugars and lipids, and therefore results in ROS-mediated diseases. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). GPX is a kind of selenium-containing enzyme. It distributes extensively in cell, blood and tissues. GPXs prevent the organism from the damage of free radicals by scavenging hydrogen peroxide and lipid hydroperoxide and maintain the normally physiological functions of the cells and tissues. And SODs are also a kind of metallic enzymes, which distribute extensively in the tissues of both aerobes and aero tolerant organisms. SODs are classified into CuZn-SOD, Mn-SOD and Fe-SOD according to the different metal-containing. All the SODs catalyse O2 - into dismutation reaction. Modern medicine studies on reactive oxygen species (ROS) showed that the metabolic disorder of free radicals is believed to cause many disease, such as cataract, diabetes, Keshan disease and some cardiovascular disease. Thus it is important for drug development studies on both GPX and SOD.Our group has successively synthesized a series of GPX mimics and SOD mimics since the end of 1980s'. We link the N-end of the 17-peptide SOD mimic and the C-end of the 15-peptide GPX mimic with a linker. A DNA sequence encoding the bifunctional enzyme mimic was deduced and synthesized based on the bias codons of E. coli and cloned into pGEX-2T plasmid vector just at the downstream side of the GST gene. The recombinant pGEX-2T plasmid vector was transformed into E. coli strain BL21 (DE3) to express the goal protein, then the GST fusion protein was purified by affinity chromatography on glutathione Sepharose 4B. The expressed fusion protein exhibited catalytic activities of GST, SOD and GPx after incorporation of copper ions and Selenocysteins.