A fused selenium-containing protein with both GPx and SOD activities Huijun Yu a , Yan Ge a , Ying Wang a , Chi-Tsai Lin b , Jing Li a , Xiaoman Liu a , Tianzhu Zang c , Jiayun Xu a , Junqiu Liu a, * , Guimin Luo c , Jiacong Shen a a Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin University, Changchun 130012, People’s Republic of China b Institute of Marine Biotechnology, National Taiwan Ocean University, 2Pei-Ning Road, Keelung 2024, Taiwan, ROC c Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, People’s Republic of China Received 28 April 2007 Available online 8 May 2007 Abstract As a safeguard against oxidative stress, the balance between the main antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) was believed to be more important than any single one, for example, dual-functional SOD/CAT enzyme has been proved to have better antioxidant ability than either single enzyme. By combining traditional fusion protein technology with amino acid auxotrophic expression system, we generated a bifunctional enzyme with both GPx and SOD activities. It displayed better antioxidant ability than GPx or SOD. Such dual-functional enzymes could facilitate further studies of the cooperation of GPx and SOD and generation of better therapeutic agents. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Bifunctional enzyme; Artificial enzymes; Fusion protein; Glutathione peroxidase; Selenium-containing glutathione transferase; Superoxide dismutase Reactive oxygen species (ROS), such as superoxide anion, H 2 O 2 , organic peroxide, and hydroxyl radical, are products of normal metabolic activities and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. Mammals have three enzymes to dismutate superoxide anion into O 2 and H 2 O 2 ; two Cu/Zn superoxide dismutases (SODs), one cytosolic and the other extracellu- lar, and a mitochondrial MnSOD. H 2 O 2 is then detoxified either to water and O 2 by catalase (CAT) or to water by glutathione peroxidase (GPx). Studies indicated that each enzyme has a specific as well as an irreplaceable function and they act in a cooperative or synergistic way to ensure a global cell protection. It seems that there is a balance between both the activities and the intracellular levels of these antioxidants that are essential for the survival of organisms and their health. This balance may be more important for protection against oxidative stress than the level of any single antioxidant enzyme. And the imbalance of them plays a role in the pathogenesis of a number of human diseases such as ischemia/reperfusion injury, ath- erosclerosis, neurodegenerative diseases, cancer, and allergy. Among the events induced by imbalance of these antiox- idant enzymes, the one, which is most frequently implicated in the pathogenesis and interests most scientists, is that Cu/ Zn-SOD could be inactivated by high concentration of H 2 O 2 , resulting in inactivation [1] and fragmentation of the enzyme [2], and in turn acquire some peroxidase, which might cause oxidative injury. It was first discovered by Fridovich in the mid-1970s that Cu/Zn-SOD possessed a secondary activity that utilized its own dismutation prod- uct, H 2 O 2 , as a substrate and formed a copper-bound  OH radical [1]. They referred to this function as peroxidase activity, because this ‘‘bound’’  OH was capable of oxidiz- ing organic compounds similar to the peroxidases. They proposed the following mechanism. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.007 * Corresponding author. Fax: +86 431 5193421. E-mail address: junqiuliu@jlu.edu.cn (J. Liu). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 358 (2007) 873–878