Construction Selenium-containing Glutaredoxin By Genetic Engineering

ROS, which includes a number of reactive molecules such as O2-?,?OH and H2O2 , is associated with the cellular turnover of oxygen by aerobic organisms. Under normal conditions, there is a balance between the production of ROS and their destruction. ROS can at low levels function as important redox signals, but excessive production can have hazardous effects and damage membrane lipids, DNA, and proteins, and therefore results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, age-related diseases, neuronal apoptosis, cancer and cataract. 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 Trx (Thioredoxin) system, Grx (Glutaredoxin) system and several antioxidant enzymes, including glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. Due to their pivotal role in scavenging ROS, the enzymatic antioxidant system could act as promising defensive system. So much stidues demonstrated that GPX was more efficient than any other antioxidant enzymes in removing hydroperoxides. However, GPX has some drawbacks such as solution instability, limited cellular accessibility, immunogenicity, short half-lives, costs of production, and proteolytic digestion. Therefore, designing new biocatalysts based on naturally occurring scaffolds makes it possible not only to understand how enzymes work and the interactions between enzymes through the evolution, but also to generate novel catalysts.Selenocysteine (Sec) is the catalytically active residue of both GPX and the various GPX mimetics. Sec is encoded by an opal codon UGA, usually as a stop codon, and a special element is involved in its incorporation into protein. Therefore it is difficult to express Sec by traditional recombinant DNA technology. Due to the limitations of the chemical methods used in their preparation, most of the selenium-containing mimics have low homogeneity and complex structures which are difficult to characterize clearly, and can not realize site-directed incorporation, which hampers the kinetic mechanism studies and the further structure-function characterization.The available information from structural biology indicates that most proteins arise by limited modifications of preexisting protein scaffolds acquiring novel functional properties by recombination of preexisting modules such as amino acid substitution, insertion or deletion of peptide segments, or fusion of different structural domains. This principle can be exploited in the redesign of existing enzymes for novel efficiently antioxidant functions. We developed an approach to enzyme redesign that capitalizes on introducing catalytic residues into a substrate-binding site to create an active site capable of catalyzing a chemical reaction. It has been determined that proteins of the same family have in general low sequence identity but all share a similar three-dimensional architecture. This kind of proteins is supposed to be good model for redesigning.GPX (glutathione peroxidase), GST (glutathione S-tranferase) and Grx (glutaredoxin) have been identified belong to a Thioredoxin-fold (Trx-fold) family. The sequence identity between them does not suggest a close structural similarity. Moreover, there is no catalytic or biological function common to them. But they share a well preserved three-dimensional architecture, the Trx-fold. Based on this principle, we produce high efficient GPX mimic with single catalytically active residue and well-characterized structure by the methods of genetic engineering and auxotrophic selenium-containing protein expression system, which was developed recently. And we further construct a GPX-GST-Grx trifunctional enzyme model by genetic engineering. We studied mutant proteins and found out the differences of catalytic reaction and the evolution significance of the catalytic group–OH–SH and–SeH. Meanwhile, we investigated the biological effects of these artificial enzymes and found that they exhibited good antioxidant ability.1. Engineering glutaredoxin into high efficient GPX mimic Seleno-GrxWe successfully reparation of selenoenzyme Seleno-Grx. The active residue Cys48 in the Grx domain of mouse thioredoxin-glutathione reductase (TGR) was substituted with Sec in an auxotrophic expression system. The engineered selenoprotein exhibits a surprising level of GPX catalytic activity toward the reduction of H2O2 with GSH, which can rival naturally occurring GPX. For the first time, a small selenoenzyme with such remarkable GPX activity was generated. Kinetic studies of Seleno-Grx showed that its catalytic properties were similar to those of naturally occurring GPXs. For example, its second rate constant (kcat /Km H2O2) was as high as 1.55×107 M-1min-1. Detailed kinetic studies revealed the characteristic parallel lines of ping-pong mechanism with at least one covalent intermediate. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPX. The enhanced high GPX activity of Seleno-Grx was assumed to result from the intrinsic chemical properties of the incorporated selenocysteine. We analyze sequence homology of the separated Grx domain and determined that it belongs to Trx-fold superfamily with a conserved sequence. For structural modeling, we used the PredictProtein Server of Columbia University in New York and obtained the mouse-Grx model. Obviously, separation of the mouse-Grx from the entire mouse TGR has little influence on the structure. Mouse-Grx has a Trx-fold that can be identified by the presence of a characteristicβ-sheet core surrounded byα-helixes, which is another power evidence of the evolution. We anticipate that studies of the Seleno-Grx has laid a solid foundation further understanding of the relationships between structure and function of GPX.2. Contruction a trifunctional enzyme Seleno-Grx2Based on former studies, we choose a dithiol Mouse Grx2 as a model for further redesigning. By genetic site-direct mutation technology and auxotrophic expression system, a trifunctional enzyme Seleno-Grx2 with GPX, GST and Grx activities was generated. It endowed a GPX activity with 1837μmol·min-1·μmol-1 , a GST activtity with 15.17μmol·min-1·μmol-1,and a Grx activity with 618μmol·min-1·μmol-1, which is 28 times, 1.4 times, and 22 times more efficient than that of the natural Grx2, respectively. We have assayed the GST properties of Seleno-Grx2 with CDNB. The optimal temperature and pH for Seleno-Grx2 catalyzed conjugation of GSH with CDNB was found to be 45℃and pH 7.5 respectively. The steady-state kinetic studies of the Seleno-Grx2 with GST activity showed a sequential mechanism, which accorded with both that of natural GST and natural dithiol Grx.Mutations Cys37→Ser or Tyr and Cys39→Ser or Tyr were introduced by genetic mutation technology in the active site, and subsequently activities of all the mutants were measured and analyzed. The results showed that different catalytic group Ser, Tyr, Cys and Sec in the active site leaded to a significant change in the catalytic reactions, further defined meaning of the catalytic group -OH, -SH and–SeH in the active site of Trx-fold proteins through the evolution. This study provides strong evidence for the hypothesis that Grx, GST, and GPX derived from the same ancestor. This result also supports the notion that chemistry, not binding specificity, is the dominant factor in the evolution of new enzymatic activities. GSTs are one kind of cellular detoxification enzymes by conjugating the GSH to a wide range of endobiotic and xenobiotic electrophilic compounds. Grxs are oxidoreductases that catalyze GSH-dependent thiol-disulfide exchange reactions, as a result Grx regenerate inactivation proteins by reducing the disufulde bonds intramolecularely. Therefore, this GPX/GST/Grx trifunctional enzyme displayed enormous advantages in antioxidant defense as well as its pharmacological development as an antioxidant.3. The biological effects of antioxidant enzymeAntioxidant enzymes play a pivotal role in protection cells against oxidative damage. Artificial enzymes with antioxidant activities attract great attentions for scientists. Toward GPX mimic Seleno-Grx and GPX/GST/Grx trifunctional enzyme, we construct two systems (Vc/Fe2+/MT and Xanthine/XOD/Fe2+/MT) from subcell level to evaluate their biological effects, respectively. We demonstrate the damaged mitochondria have great changes in morphology, structure and function. The extent of swelling and MDA content of mitochondria was chosen as standards to be used to determine the extent of injury and protection of mitochondria. Seleno-Grx and Seleno-Grx2 reduced the swelling of mitochondria during damage and decrease the maximal level of MDA accumulation in its rapid phase. The ability of Seleno-Grx to protect mitochondria against oxidative damage is stronger than that of Ebselen, while the ability of Seleno-Grx2 is stronger than either that of Seleno-Grx or that. These results show that these mimics have great antioxidant activity. Such mimics may result in better clinical therapies for diseases mediated by ROS produced by mitochondria.