Antioxidant Enzyme Mimics With Synergism

The antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferases (GST) and so on, contribute dominatingly to enhance cellular antioxidant defense against various ROS. These enzymes play crucial roles in balancing the production and decomposition of ROS in living organisms, which act cooperatively and synergistically to scavenge ROS, as none of them can singlehandedly remove all forms of ROS. In order to imitate the synergism of the antioxidant enzymes, we generated some multifunctional mimics by chemical synthesis and biosynthesis.1. Generation of a 35-mer Peptide with Superoxide Dismutase and Glutathione Peroxidase Activities by Genetic EngineeringThe 17-mer peptide with SOD activity was linked to the 15-mer peptide with GPx acivity by a linker of 3 amino acids to form a 35-mer peptide, which was then turned into a new 35-mer peptide through alignment of homology sequence of nature enzymes, two and three-dimensional structure prediction, and molecular dynamics simulation. We inserted the DNA sequence encoding the new 35-mer peptide into the pGEX-2T vector at the C-terminus of the gene encoding Schistosoma japonicum GST (SjGST). The fusion protein was expressed in cysteine auxotrophic expression system with copper ion and we cleavaged the fusion protein by using thrombin to obtain the new 35-mer peptide. Thus, as the SOD activity center, copper was coordinated by histidine residues in N-terminus of 35-mer peptide. Selenocysteine, the catalytic residue of GPx was incorporated instead of the cysteine of the 35-mer peptide C-terminus in cysteine auxotrophic expression system. This new peptide enzyme has many advantages: low molecular weight, low immunogenicity, longer half-life in the blood, good cellular permeability and clear structure-functional relationship. We anticipate that the antioxidant enzyme mimics with synergism may hold promise for the treatment of human diseases and possession of potential applications in medicine as potent antioxidants. Design and generation of multifunctional mimics will become main current for development of artificial enzyme field.2. A novel 65-mer peptide with both SOD and GPx activitiesIn order to imitate the synergism of SOD and GPx, a 65-mer peptide (65P), which contains the sequences that can not only form the domain of SOD active center, but the domain of GPx catalytic triad upon incorporating some metals, was designed on the basis of native enzyme structural models and expressed the 65P in the cysteine auxotrophic expression system to obtain Se-65P. The Se-65P was converted into the Se-CuZn-65P by incorporating Cu2+ and Zn2+ into it. This Se-CuZn-65P displayed significantly high activities of both SOD and GPx because it has a delicate dual active center. Their synergism was evaluated by using a model in vitro, a ferrous sulfate/ascorbate-induced mitochondrial damage model system. At last, we proposed a cooperative catalytic mechanism of this novel dual functional peptide mimic. We anticipate that the peptide enzyme mimic with synergism holds promise for the treatment of human diseases and possession of potential applications in medicine as potent antioxidants. 3. Incorporation of a catalytic triad into the selenium-containing single chain antibody scFv-2F3 to inhance its GPx activityScFv-2F3 was the first single-chain Fv fragment, which could be converted into the Sec-containing Se-scFv-2F3 with high GPx activity by chemical modification of the Ser. The result of homology and molecular dynamics simulation demonstrated that Sec52 may be the active site of Se-scFv2F3. Moreover, the active site of natural GPx includes not only a Sec residue but also Trp and Gln, which form a catalytic triad in a depression on protein's surface. So, based on homology models and molecular dynamic simulation, we introduced a catalytic triad into the Se-scFv-2F3 by using site-directed mutation, then, transformed the mutated plasmid into the cysteine auxotrophic expression system. The GPx activity of Se-scFv-2F3 with catalytic triad was demonstrated to obviously enhance. This work will propose a new strategy for generation of GPx mimics in artificial enzyme field.4. Cyclodextrin-derived mimic displays various homologous GST activities.The two most important factors, substrate recognition and intermolecular catalysis in enzyme catalysis play vital roles in enzyme imitation. Cyclodextrins have been widely investigated as enzyme models since their hydrophobic cavities are capable of binding small organic molecules. Thus, cyclodextrins have been widely used as scaffolds for constructing artificial selenium or tellurium-containing enzymes. Recently, the GPx mimic 6-TeOdiCD was prepared from 6-TediCD since the diorganyl telluride can be oxidized into the corresponding telluroxide by the mild oxidants. The 6-TeOdiCD showed a significant rate acceleration of 106000 times for the hydrolysis of DNNPC. We found it could exhibit various homologous GST activities using different substrates. This work provides essential concepts and indications for constructing highly efficient enzyme model as well as important information on the understanding of native enzyme.