Construction Of High Efficient And Smart Antioxidant Enzyme By Genetic Engineering

Enzymes are proteins with special functions, which can catalyze almost allchemical reactions in organisms. In natural physiological condition, organisms obtainenergy by redox reaction. O₂,·OH and H2O2are produced in this process and theyare called Reactive Oxygen Species (ROS) together with the unstable intermediate oflipid peroxide. Under normal conditons, there is a balance between the production anddestruction of ROS, which is no harm to organism. However, once the balance isbroken, the accumulated ROS will harm various biomacromolecules including protein,sugars, lipids, DNA and even ultimately lead cell apoptosisi. A range of humandiseases are related to the accumulated ROS, including cataract, keshan disease andcardiovascular disease. Thus, in the evolutionary process, organisms set up twodefensive systems, including enzymatic and non-enzymatic antioxidant systems. Theenzymatic antioxidant system consists of superoxide dismutase (SOD), catalase (CAT)and glutathione peroxidase (GPx). As the important member of this system, GPx cancatalyze the decomposition of H2O2using GSH as the substrate and also interdict theSeCysond-class reaction of the radical which is produced by lipid peroxide. Therefore,if GPx can be applicated as a promising antioxidant drug, it has the extremely vitalsignificance. Unfortunately, GPx has some drawbacks such as difficult heterogenousexpression, short half-lives, limited cellular accessibility and proteolytic digestion,which limite the pharmacological application. Thus, enormous efforts have been madeto simulate the efficient functions of GPx and great achievements have been made inelucidating the relationship between structure and function of enzyme, and theconstruction of the highly active artificial enzyme.In order to simulate the biological functions of GPx, scientists have developed avariety of chemical and biological methods including semisynthesis, antibodyenzyme technique, biological imprinting technique and auxotrophic express system.Among these methods, the E.coli. auxotrophic expression system is the most simple and effective method to obtain selenium proteins. Using the strategy, a serious ofGPx mimics have been prepared and even some artificial enzymes havedemonstrated an apparent "superactivity" compared to the natural GPx. However,the catalytic center of these artificial enzymes is SeCys, which is easily hydrolysised.Furthermore, ROS are not always the foe for us. Conversely, they are friendly to us,for example, they play important roles as vital signaling molecules in metabolism,and will not arise various illness unless overproducted in the human body. Therefore,building a new GPx model, either directly or indirectly, response to theconcentration change of ROS in cells, will be a novel goal and a major challenge forus.In the 20 natural amino acids, we find that SeMet and SeCys have similarstructure and the difference between them is that Se in SeMet exists in the form ofR-Se-R, which is more stable than that of SeH in SeCys. Therefore, we redesign thecatalytical center and insert the SeMet into sjGST to construct a new high efficientartificial enzyme.To build a new GPx model, either directly or indirectly, response to theconcentration change of ROS in cells, recently, two kinds of temperature responsiveartificial GPx models were reported as the pioneering work. However, the materialshave certain cytotoxicity and most importantly, they are not sensitive to theconcentration change of ROS in cells. To our knowledge, the increase of ROS in cellswill induce calcium ion intake decrease in mitochondrial, further leading to increasethe concentration of calcium ion in cytoplasm. Therefore, if we can design a calciumion-switch artificial GPx model, it will indirectly response to the concentration changeof ROS and eventually remove the excess ROS. Thus, we looked back to the natureprotein database and found such a protein scaffold, recoverin. Therefore, we insert theSeCysys into recoverin to construct a smart artificial enzyme.1. Construction of GPx mimic by using seleno-sjGSTBoth GST and GPx belong to thioredoxin superfamily due to the thioredoxin foldin their structure. They have the similar glutathione-binding domain. In view of theprevious experience, the specific substrate binding site and the exact orientation of thecatalytic center are important for acquiring high efficient enzyme mimics. We insertSeMet into glutathione-binding domain of sjGST using E.coli. methionineauxotrophic expression system and obtain a highly efficient artificial enzyme. Theengineered seleno-sjGST exhibits a surprising level of GPx catalytic activity toward the reduction of H2O2with GSH, which can rival naturally occurring GPx. Kineticstudies of seleno-sjGST showed that its second rate constants (kcat/Km H2O2and kcat/KmGSH) were as high as 109M-1min-1, which were similar to those of naturallyoccurring GPxs. For the first time, a new selenoenzyme with SeMet at the activecenter and such remarkable GPx activity was generated using E.coli. methionineauxotrophic expression system. Detailed kinetic studies revealed the characteristicinterSeCystion lines of sequential mechanism.2. Construction of calcium ion-switch GPx mimic by using seleno-recoverinAn excellent antioxidant enzyme is not only expected to have high catalyticalacitivty, but also to adjust ROS in a smart way. However, by means of chemicalsynthesis, it is difficult to synthesis such a smart material response to the change ofROS in cells, directly or indirectly. To our knowledge, the increase of ROS in cellswill induce calcium ion intake decrease in mitochondrial, further leading to increasethe concentration of calcium ion in cytoplasm. Thus, we looked back to the natureprotein database and found such a protein scaffold, recoverin, which is an EF-handcalcium-binding protein and a member of the neuronal calcium-sensors (NCS) family.Through the crystal structure analysis, we found that Ala127 exposed on the surfaceof the protein in the Ca2+-bound conformation,while buried in the interior of theprotein in the Ca2+-free conformation. Base on this protein scaffold, we can design acalcium ion-switch artificial GPx model.We firstly enable the recoverin to process calcium ion-switch GPx enzymaticactivity by substituting Ala127 with the catalytic group, selenocysteine, in acysteine-auxotrophic system. In Ca2+-free form of seleno-recoverin, the SeCys issequestered in the interior of the protein and is therefore unavailable for interactionwith the substrate GSH, therefore it doesn't display on any GPx activity. While uponbinding Ca2+, seleno-recoverin undergoes a conformational change that allows SeCysexposed to the surface of the protein, which results the H2O2reduction by GSH.Additionally, as a smart GPx mimic, the reversibility of the catalytic activity of theseleno-recoverin was tested. The result shows that the catalytic activity of the enzymeis completely reversible after multiple Ca2+-bound and Ca2+-free cycles. The optimaltemperature and pH for Seleno-recoverin was close to natural GPxs, and detailedkinetic studies revealed the characteristic parallel lines of ping-pong mechanism.3. The biological effects of seleno-recoverin As a potential clinical drug, we hope the switch-performance of seleno-recoverincan be really in function within the cells. Therefore, we constructed Fenton/Rh-B andVc/Fe2+/MT systems and employed mitochondria as research object to evaluate thebiological effects of seleno-recoverin for the extent of swelling and MDA content ofmitochondria in vitro. The results showed that Ca2+-bound seleno-recoverin was ableto reduce the swelling of during damage and decrease the maximal level of MDAaccumulation in its rapid phase, while Ca2+-free seleno-recoverin could not carry outthis function because of the SeCys sequestered in the interior of the enzyme. Althoughthe complexity of this system is far from real cells, after all to some extent, thissystem stimulated the intracellular reox changes, which would provide a basic workfor further designing more intelligent artificial GPxs.