Construction Of Selenium-containing Antioxidant Enzyme By Genetic Engineering

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. 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 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 antioxidant drug. However, GPX has some drawbacks such as solution instability, limited cellular accessibility, immunogenicity, short half-lives, costs of production, and proteolytic digestion. And elevation of some kinds of SOD could cause disorder in organisms when other antioxidant enzymes such as GPX and CAT keep constant. Those factors limit the pharmacological use of the naturally occurring enzymes. Scientists have made a great deal of efforts to study their catalytic mechanisms as well as their relationships between them, and prepared many mimics that have the antioxidant ability. 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. 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/SOD bifunctional enzyme model by genetic fusion technology. Whilst we studied the biological effects of these artificial enzymes and found that they exhibited good antioxidant ability. 1. Engineering glutathione transferase into high efficient GPX mimic—Seleno-GST It is chemistry, not binding specificity that is the dominant factor in the evolution of new enzymatic activities. As a consequence, enzymes with similarfolds can catalyze very different chemical reactions upon introducing new catalytic groups into the active sites of the enzymes. Based on this principle, by replacing the active site serine9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione transferase from Lucilia cuprina was converted to a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPX in the specific glutathione (GSH) binding sites and the geometrical conformation for the active selenocysteine (Sec) in their common GSH-binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPXs. It is the first high efficient GPX mimic with single catalytically active residue and well-characterized structure, which prepared thoroughly by genetic engineering. The incorporation of Sec into LuGST1-1 significantly enhanced the enzymatic efficiency for decomposing organic hydroperoxides by nearly three orders of magnitudes. Double-reciprocal plots of initial velocity versus substrate concentration gave the kinetic parameters for the enzymatic reactions between GSH and hydroperoxides. kcat/KmGSH and kcat/KmH2O2 of seleno-LuGST1-1 were both approximately 107 M-1·min-1. kcat/KmGSH of seleno-LuGST1-1 was in the same order of magnitude as that of natural GPX. And although kcat/KmH2O2 of seleno-LuGST1-1 was still one order of magnitude lower than that of natural GPX (108 M-1·min-1), it was much higher than those of most GPX mimics (for example, 4.5 ×103 M-1·min-1 for selenium-containing catalytic antibody Se-4A4). 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 selenium-dependent ping-pong mechanism as opposed to the sequential one of the wild type GST wasassumed to result from the intrinsic chemical properties of the incorporated selenocysteine. Engineering GST into an efficient GPX-like biocatalyst provided a new proof for the previous assumption on that both GPX and GST were evolved from a common thioredoxin-like ancestor to accommodate different function along with evolution. And it also supports that the dominant factor in the evolution of new enzymatic activities is chemistry instead of binding specificity. We expect that the seleno-GST would offer a more suitable enzymatic model for further understanding of the relationships between structure and function of GPX. And this novel engineered selenoenzyme is also a prospective catalyst as an excellent antioxidant especially useful for both industrial and medical applications. 2. Construction of selenium-containing GPX/SOD bifunctional enzyme By genetic fusion technology and auxotrophic expression system, high efficient GPX mimic—seleno-LuGST1-1 was fused to the naturally occurring SOD and a bifunctional enzyme Seleno-GST-SOD with both GPX and SOD activities was generated. Its GPX activity was 286 μmol·min-1·μmol-1, which is 280-fold more efficient than Ebselen and only one order of magnitude lower than that of native GPX. Its SOD activity was 357 μmol·min-1·mg-1, which is about one sixth of that of native SOD. Detailed kinetic studies revealed the characteristic parallel lines of ping-pong mechanism with at least one covalent intermediate, which is in analogy with that of native GPX. The decrease of the two enzyme activities after fusion might result from the changes of the two proteins'conformation and the presence of more monomer, which caused by fusion. Conjugation of seleno-LuGST1-1 and SOD to form a single molecule should further insure their cooperation for that the accumulating levels of H2O2 from the dismutation of superoxide and the inactivation of SOD by H2O2 are both minimized as the result of immediate access of GPX mimic at the site of H2O2 formation. Seleno-GST-SOD therefore has stronger antioxidant ability than either GPX mimics or SOD. This is the first GPX/SOD dual-functional enzyme prepared thoroughly by genetic engineering. Other than chemically artificial enzymes, Seleno-GST-SOD was generated withhigh efficiency and homogeneity by genetic fusion technology, and laid a steady basis both for detailed studies on the cooperation mechanism of these enzymes and for its pharmacological development as an antioxidant. 3. The biological effects of antioxidant enzymes Antioxidant enzymes play a pivotal role in protection cells against oxidative damage. Artificial enzymes with antioxidant activities attract great attentions for scientists. They are of significance in elucidating enzyme reaction mechanism as well as in production of enzyme mimics for antioxidant drug. Toward high efficient GPX mimic—seleno-LuGST1-1 and GPX/SOD bifunctional 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-LuGST1-1 and Seleno-GST-SOD reduced the swelling of mitochondria during damage and decrease the maximal level of MDA accumulation in its rapid phase. The ability of seleno-LuGST1-1 to protect mitochondria against oxidative damage is stronger than that of Ebselen, while the ability of Seleno-GST-SOD is stronger than either that of seleno-LuGST1-1 or that of SOD. These results show that the mimics have great antioxidant activity. Such mimics may result in better clinical therapies for diseases mediated by ROS produced by mitochondria. We construct two systems (RSH / Fe3+ / O2 and t-BuOOH / GSH) to evaluate the biological effects of the previously prepared GPX mimics, 2-TeCD and 2-SeCD, and demonstrate they could protect DNA against the damage induced by oxidative stress. The antioxidant ability of 2-TeCD is stronger than that of 2-SeCD. And acting as their reductive substrate, GSH is more proper than DTT. Therefore, this is a kind of promising antioxidant drug.