Construction Of Selenium-containing Artificial Enzyme By Genetic Engineering With High Efficiency

Enzymes catalyze almost all chemical reactions with stereo- selectivities and specificities under mild conditions. Nature has created a lot of antioxidant enzymes. They have the capacity to catalyze the discomposing of peroxide, ROOH,H2O2,O2- etc. They are very important for the metabolism in living cells. 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), which acts 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. Those factors limit the pharmacological use of the naturally occurring enzymes. Scientists have made a great deal of efforts to design new mimics with high antioxidant ability to study their catalytic mechanisms as well as develop new medications.Selenium is the catalytically active element of both GPx and the various GPX mimics. 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. Hitherto, the active center of all reported macromolecular GPx mimics is Sec. The main innovation of our work is to incorporate Se-met into protein to mimic GPx and study the enzymic effects of the artificial enzyme.The main achievements are listed as bellow:1. The synthesis of Acetylselenomethionine.We synthesized acetylselenomethionine(Ac-Se-Met) based on the"Open-Ring"procedure. Budisa and his co-workers found that Ac-Se-Met could be deacetylated by endogenous aminoacylase of E coli. and incorporated into protein. Compared with some former procedures, this way is simpler and more efficient. But it must be carried out in rigorous condition.α-Amino-γ-butyrolactone·HBr reacted with acetic anhydride in the presence of triethylamine. Then the production reacted with MeSeLi in low temperature, which experienced an open-ring reaction to form the target compound. MS, IR and NMR spectra were consistent with the target compound.2. The expression of the target protein.It is chemistry, not binding specificity that is the dominant factor in the evolution of new enzymatic activities. As a consequence, enzymes with similar folds 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 Tyr7 with Met and then substituting it with selenomethionine in a methionine auxotrophic system, catalytically essential residue selenomethionine was bioincorporated into GSH-specific binding scaffold, and thus, the sjGST was converted to a selenium-containing enzyme, Se-Met-sjGST(Y7M), by genetic engineering. To our surprise, its high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione is comparable with those of natural GPxs. It is the first artificial enzyme with Se-Met at the active center, which traditionally is Sec.3. The investigation of the enzymic property & catalytic mechanism. Nature sjGST can not display any GPx activity. However, the activity of Se-containing Se-Met-sjGST(Y7M) is in the same order of magnitude as that of natural GPx. Double-reciprocal plots of initial velocity versus substrate concentration gave the kinetic parameters for the enzymatic reactions between GSH and hydroperoxides. Kinetic data indicated that Se-Met-sjGST(Y7M) could not be saturated by H2O2 and the reaction velocity became higher along with the higher concentration of H2O2. Double-reciprocal plots were consistent with sequential mechanism. Due to the structural difference between Se-Met and Sec, Se-Met-sjGST(Y7M) might undergo the catalytic cycle similar to that of diorganotellurium rather than natural GPxs. But it needs further investigation to confirm this catalytic mechanism.Taking advantage of the important structure similarities between Se-Met-sjGST(Y7M) and naturally occurring GPx in the specific glutathione (GSH) binding sites and the geometrical conformation for the active selenomethionine (Se-Met) 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. 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. 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.Generally, Met usually acts as structural block in protein. We changed sulfur in natural Met into selenium and incorporated this new amino acid into the active center of GSH-binding protein. We endowed Met new function in protein and provided a new platform to study the catalytic activity of selenium.